Pyrrolobenzodiazepines as unsymmetrical dimeric PBD compounds for inclusion in targeted conjugates

- MEDIMMUNE LIMITED

A compound with the formula I wherein: R2 is of formula II where Q is selected from OH, SH and NRN, and RN is selected from H, methyl and ethyl, as well as drug-linkers and drug-conjugates made from this compound.

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Description
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/EP2012/068506 filed Sep 20, 2012, and claims the benefit of U.S. Provisional Application No. 61/536,669 filed Sep 20, 2011, which is incorporated by reference herein.

The present invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepine dimers having a C2-C3 double bond and a propargyl group at the C2 position on one 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 this 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 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, published as WO 2011/130613, discloses the inclusion of these compounds in targeted conjugates. Co-pending International application PCT/US2011/032668, filed 15 Apr. 2011, published as WO 2011/130616, discloses unsymmetrical dimeric PBD compound bearing an aryl group in the C2 position of one monomer bearing a substituent designed to provide an anchor for linking the compound to another moiety, the other monomer bearing a non-aromatic group in the C2 position. The inclusion of these compounds in targeted conjugates is also disclosed.

DISCLOSURE OF THE INVENTION

The present inventors have developed further unsymmetrical dimeric PBD compounds for inclusion in targeted conjugates, eliminating the need for an aryl group bearing an anchor. The C2 group bearing a substituent designed to provide an anchor for linking the compound to another moiety in the present invention is a propargyl group.

The present invention comprises a compound with the formula I:


wherein:

R2 is of formula II:


where Q is selected from OH, SH and NRN, and RN is selected from H, methyl and ethyl; when there is a double bond present between C2′ and C3′, R12 is selected from:

(ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;

(ib) C1-5 saturated aliphatic alkyl;

(ic) C3-6 saturated cycloalkyl;


wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;


wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and


where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2′ and C3′,

R12 is


where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester;

R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;

where R and R′ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;

R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo; either:

(a) R10 is H, and R11 is OH, ORA, where RA is C1-4 alkyl;

(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or

(c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;

R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;

Y and Y′ are selected from O, S, or NH;

R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11, wherein if R11 and R11′ are SOzM, 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:


wherein:

R2 is of formula II:


where Q is selected from OH, SH and NRN, and RN is selected from H, methyl and ethyl; when there is a double bond present between C2′ and C3′, R12 is selected from:

(ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;

(ib) C1-5 saturated aliphatic alkyl;

(ic) C3-6 saturated cycloalkyl;


wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;


wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and


where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2′ and C3′,

R12 is


where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester;

R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;

where R and R′ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;

R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo; either:

(a) R10 is carbamate nitrogen protecting group, and R11 is O-ProtO, wherein ProtO is an oxygen protecting group;

(b) R10 is a hemi-aminal nitrogen protecting group and R11 is an oxo group;

R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;

Y and Y′ are selected from O, S, or NH;

R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11.

A fourth aspect of the present invention comprises a method of making a compound of formula I from a compound of formula II 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 is a dimer of formula I (supra).

In some embodiments, the Conjugates have the following formula III:
L-(LU-D)p  (III)
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 an integer of 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.

The PBD dimer D is of formula I, except that Q is selected from the group comprising: O, S, NRN, wherein RN is selected from the group comprising H and C1-2 alkyl.

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.

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 Ca2+ and Mg2+. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). 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(CH3)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.

C1-12 alkyl: The term “C1-12 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). 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 (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C7).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7).

Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).

C2-12 Alkenyl: The term “C2-12 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═CH2), 1-propenyl (—CH═CH—CH3), 2-propenyl (allyl, —CH—CH═CH2), isopropenyl (1-methylvinyl, —C(CH3)═CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).

C2-12 alkynyl: The term “C2-12 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, —CH2—C≡CH).

C3-12 cycloalkyl: The term “C3-12 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 (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7);

    • unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and

    • saturated polycyclic hydrocarbon compounds:

norcarane (C7), norpinane (C7), norbornane (C7).

C3-20 heterocyclyl: The term “C3-20 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. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6heterocyclyl”, 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:

N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);

O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);

S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7);

O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);

O3: trioxane (C6);

N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);

N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);

N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);

N2O1: oxadiazine (C6);

O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and,

N1O1S1: oxathiazine (C6).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

C5-20 aryl: The term “C5-20 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. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C3-20, C5-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6 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) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).

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) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (C16).

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:

N1: pyrrole (azole) (C5), pyridine (azine) (C6);

O1: furan (oxole) (C5);

S1: thiophene (thiole) (C5);

N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);

N2O1: oxadiazole (furazan) (C5);

N3O1: oxatriazole (C5);

N1S1: thiazole (C5), isothiazole (C5);

N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6);

N3: triazole (C5), triazine (C6); and,

N4: tetrazole (C5).

Examples of heteroaryl which comprise fused rings, include, but are not limited to:

    • C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);
    • C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);
    • C11 (with 2 fused rings) derived from benzodiazepine (N2);
    • C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and,
    • C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxathiin (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).

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 C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 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(OR1)(OR2), wherein R1 and R2 are independently acetal substituents, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, or, in the case of a “cyclic” acetal group, R1 and R2, 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)(OR1), wherein R1 is a hemiacetal substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR1)(OR2), where R1 and R2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 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)(OR1), where R1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 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, C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 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 C1-7 alkyl group (also referred to as C1-7 alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH3 (acetyl), —C(═O)CH2CH3 (propionyl), —C(═O)C(CH3)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 (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH3, —C(═O)OCH2CH3, —C(═O)OC(CH3)3, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH3 (acetoxy), —OC(═O)CH2CH3, —OC(═O)C(CH3)3, —OC(═O)Ph, and —OC(═O)CH2Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH3, —OC(═O)OCH2CH3, —OC(═O)OC(CH3)3, and —OC(═O)OPh.

Amino: —NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7 alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a “cyclic” amino group, R1 and R2, 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 (—NH2), secondary (—NHR1), or tertiary (—NHR1R2), and in cationic form, may be quaternary (—+NR1R2R3). Examples of amino groups include, but are not limited to, —NH2, —NHCH3, —NHC(CH3)2, —N(CH3)2, —N(CH2CH3)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)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH2, —C(═O)NHCH3, —C(═O)N(CH3)2, —C(═O)NHCH2CH3, and —C(═O)N(CH2CH3)2, as well as amido groups in which R1 and R2, 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)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH2, —C(═S)NHCH3, —C(═S)N(CH3)2, and —C(═S)NHCH2CH3.

Acylamido (acylamino): —NR1C(═O)R2, wherein R1 is an amide substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group, and R2 is an acyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH3, —NHC(═O)CH2CH3, and —NHC(═O)Ph. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH2, —OC(═O)NHMe, —OC(═O)NMe2, and —OC(═O)NEt2.

Ureido: —N(R1)CONR2R3 wherein R2 and R3 are independently amino substituents, as defined for amino groups, and R1 is a ureido substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH2, —NHCONHMe, —NHCONHEt, —NHCONMe2, —NHCONEt2, —NMeCONH2, —NMeCONHMe, —NMeCONHEt, —NMeCONMe2, and —NMeCONEt2.

Guanidino: —NH—C(═NH)NH2.

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 C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt.

Amidine (amidino): —C(═NR)NR2, wherein each R is an amidine substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH2, —C(═NH)NMe2, and —C(═NMe)NMe2.

Nitro: —NO2.

Nitroso: —NO.

Azido: —N3.

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 C1-7 alkyl group (also referred to as a C1-7 alkylthio group), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of C1-7 alkylthio groups include, but are not limited to, —SCH3 and —SCH2CH3.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group (also referred to herein as C1-7 alkyl disulfide). Examples of C1-7 alkyl disulfide groups include, but are not limited to, —SSCH3 and —SSCH2CH3.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH3 and —S(═O)CH2CH3.

Sulfone (sulfonyl): —S(═O)2R, wherein R is a sulfone substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, including, for example, a fluorinated or perfluorinated C1-7 alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)2CH3 (methanesulfonyl, mesyl), —S(═O)2CF3 (triflyl), —S(═O)2CH2CH3 (esyl), —S(═O)2C4F9 (nonaflyl), —S(═O)2CH2CF3 (tresyl), —S(═O)2CH2CH2NH2 (tauryl), —S(═O)2Ph (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, —SO2H.

Sulfonic acid (sulfo): —S(═O)2OH, —SO3H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH3 (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)2OR, wherein R is a sulfonate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)2OCH3 (methoxysulfonyl; methyl sulfonate) and —S(═O)2OCH2CH3 (ethoxysulfonyl; ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH3 and —OS(═O)CH2CH3.

Sulfonyloxy: —OS(═O)2R, wherein R is a sulfonyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)2CH3 (mesylate) and —OS(═O)2CH2CH3 (esylate).

Sulfate: —OS(═O)2OR; wherein R is a sulfate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)2OCH3 and —SO(═O)2OCH2CH3.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH2, —S(═O)NH(CH3), —S(═O)N(CH3)2, —S(═O)NH(CH2CH3), —S(═O)N(CH2CH3)2, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)2NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O)2NH2, —S(═O)2NH(CH3), —S(═O)2N(CH3)2, —S(═O)2NH(CH2CH3), —S(═O)2N(CH2CH3)2, and —S(═O)2NHPh.

Sulfamino: —NR1S(═O)2OH, wherein R1 is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)2OH and —N(CH3)S(═O)2OH.

Sulfonamino: —NR1S(═O)2R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)2CH3 and —N(CH3)S(═O)2C6H5.

Sulfinamino: —NR1S(═O)R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH3 and —N(CH3)S(═O)C6H5.

Phosphino (phosphine): —PR2, wherein R is a phosphino substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphino groups include, but are not limited to, —PH2, —P(CH3)2, —P(CH2CH3)2, —P(t-Bu)2, and —P(Ph)2.

Phospho: —P(═O)2.

Phosphinyl (phosphine oxide): —P(═O)R2, wherein R is a phosphinyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group or a C5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH3)2, —P(═O)(CH2CH3)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 C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH3)2, —P(═O)(OCH2CH3)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 C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH3)2, —OP(═O)(OCH2CH3)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 C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH3)2, —OP(OCH2CH3)2, —OP(O-t-Bu)2, and —OP(OPh)2.

Phosphoramidite: —OP(OR1)—NR22, where R1 and R2 are phosphoramidite substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH2CH3)—N(CH3)2, —OP(OCH2CH3)—N(i-Pr)2, and —OP(OCH2CH2CN)—N(i-Pr)2.

Phosphoramidate: —OP(═O)(OR1)—NR22, where R1 and R2 are phosphoramidate substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH2CH3)—N(CH3)2, —OP(═O)(OCH2CH3)—N(i-Pr)2, and —OP(═O)(OCH2CH2CN)—N(i-Pr)2.

Alkylene

C3-12 alkylene: The term “C3-12 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 C3-12 alkylene groups include, but are not limited to, —(CH2)n— where n is an integer from 3 to 12, for example, —CH2CH2CH2— (propylene), —CH2CH2CH2CH2— (butylene), —CH2CH2CH2CH2CH2— (pentylene) and —CH2CH2CH2CH—2CH2CH2CH2— (heptylene).

Examples of branched saturated C3-12 alkylene groups include, but are not limited to, —CH(CH3)CH2—, —CH(CH3)CH2CH2—, —CH(CH3)CH2CH2CH2—, —CH2CH(CH3)CH2—, —CH2CH(CH3)CH2CH2—, —CH(CH2CH3)—, —CH(CH2CH3)CH2—, and —CH2CH(CH2CH3)CH2—.

Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH2—, —CH2—CH═CH2—, —CH═CH—CH2—CH2—, —CH═CH—CH2—CH2—CH2—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH2—, —CH═CH—CH═CH—CH2—CH2—, —CH═CH—CH2—CH═CH—, —CH═CH—CH2—CH2—CH═CH—, and —CH2—C≡C—CH2—.

Examples of branched partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, —C(CH3)═CH—, —C(CH3)═CH—CH2—, —CH═CH—CH(CH3)— and —C≡C—CH(CH3)—.

Examples of alicyclic saturated C3-12 alkylene groups (C3-12 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 C3-12 alkylene groups (C3-12 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, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. 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, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

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, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
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 (L1), and a Spacer unit (L2). The Linker unit is connected at one end to the Ligand unit and at the other end to the PBD dimer compound.

In one aspect, such a Conjugate is shown below in formula IIIa:
L-(A1a-L1s-L2y-D)p  (IIIa)

    • wherein:
    • L is the Ligand unit; and
    • -A1a-L1s-L2y- is a Linker unit (LU), wherein:
    • -A1- is a Stretcher unit,
    • a is 1 or 2,
    • L1- is a Specificity unit,
    • s is an integer ranging from 1 to 12,
    • -L2- 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 IIIb:

    • Also illustrated as:
      L-(A1a-L2y(-L1s)-D)p  (Ib)
    • wherein:
    • L is the Ligand unit; and
    • -A1a-L1a(L2y)- is a Linker unit (LU), wherein:
    • -A1- is a Stretcher unit linked to a Stretcher unit (L2),
    • a is 1 or 2,
    • L1- is a Specificity unit linked to a Stretcher unit (L2),
    • s is an integer ranging from 0 to 12,
    • -L2- 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-(A1a-L1s-L2y-D)p

    • wherein L, A1, a, L1, s, L2, D 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, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit, which is a covalent bond, a self-immolative group or together with —OC(═O)— forms a self-immolative group, and L2 optional.

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-A1a-L1s-L2y-*

    • where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 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, L1 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) L2, when a self-immolative group(s) is present. When the Specificity unit L1 is cleaved, or the linkage (i.e., the covalent bond) between L1 and L2 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, L1 is a Specificity unit connected to L2, A1 is a Stretcher unit connecting L2 to the Cell Binding Agent, L2 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 L1 and L2 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 L1 is cleavable, the structure and/or sequence of L1 is selected such that it is cleaved by the action of enzymes present at the target site (e.g., the target cell). L1 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. L1 units that are cleavable under reducing or oxidising conditions may also find use in the Conjugates.

In some embodiments, L1 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, L1 is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L1 may be cleaved by a lysosomal protease, such as a cathepsin.

In one embodiment, L2 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 L1 is cleavable by the action of an enzyme and L2 is present, the enzyme cleaves the bond between L1 and L2, whereby the self-immolative group(s) release the Drug unit.

L1 and L2, 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 L1 that connects to L2 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 L1 that connects to L2 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 L1 that connects to L2 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 L2 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 L1, 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 L2 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 L2 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 L1 and L2 is a cathepsin labile (e.g., cleavable) bond.

In one embodiment, L1 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 —X1—X2— in dipeptide, —NH—X1—X2—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 X1, and CO is the carbonyl group of X2.

Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:

    • -Phe-Lys-,
    • -Val-Ala-,
    • -Val-Lys-,
    • -Ala-Lys-, and
    • -Val-Cit-.

Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—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 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—Cl, Fmoc, Z;
    • Ser: Bzl, TBDMS, TBDPS;
    • Thr: Bz;
    • Trp: Boc;
    • Tyr: Bzl, Z, Z—Br.

In one embodiment, —X2— is connected indirectly to the Drug unit. In such an embodiment, the Spacer unit L2 is present.

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 —X2—.

Where a self-immolative group is present, —X2— is connected directly to the self-immolative group. In one embodiment, —X2— is connected to the group Y of the self-immolative group. Preferably the group —X2—CO— is connected to Y, where Y is NH.

—X1— is connected directly to A1. In one embodiment, —X1— is connected directly to A1. Preferably the group NH—X1— (the amino terminus of X1) is connected to A1. A1 may comprise the functionality —CO— thereby to form an amide link with —X1—.

In one embodiment, L1 and L2 together with —OC(═O)— comprise the group —X1—X2—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 L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.

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′ and L2 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 A1, 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 —NO2 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)—,
    • SO2, and
    • —S—.

The group Y is preferably —NH—, —CH2—, —O—, and —S—.

In some embodiments, L1 and L2 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, L1 and L2 together represent:

    • where the asterisk indicates the point of attachment to the remainder of L2 or the Drug unit, the wavy line indicates the point of attachment to A1, 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—, —CH2—, —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 A1. 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 L1 and the Ligand unit are indirectly connected via the Stretcher unit.

L1 and A1 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 A1 is:

    • where the asterisk indicates the point of attachment to L1, 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 A1 is:

    • where the asterisk indicates the point of attachment to L1, 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 A1 is:

where the asterisk indicates the point of attachment to L1, 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 A1 is:

    • where the asterisk indicates the point of attachment to L1, 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 A1 is:

    • where the asterisk indicates the point of attachment to L1, 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 A1 is:

    • where the asterisk indicates the point of attachment to L1, 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 A1 is:

    • where the asterisk indicates the point of attachment to L1, 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 A1 is:

    • where the asterisk indicates the point of attachment to L1, 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 A1 is through a thiol residue of the Ligand unit and a maleimide group of A1.

In one embodiment, the connection between the Ligand unit and A1 is:

    • where the asterisk indicates the point of attachment to the remaining portion of A1, L1, L2 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 A1 group, or to L1, L2 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 A1 group, or to L1, L2 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—,
    • —CH2C(═O)—
    • —C(═O)CH2—,
    • ═N—NH—, and
    • —NH—N═.

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 A1 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 A1 group.

Other groups suitable for connecting L1 to the Cell Binding Agent are described in WO 2005/082023.

In one embodiment, the Stretcher unit A1 is present, the Specificity unit L1 is present and Spacer unit L2 is absent. Thus, L1 and the Drug unit are directly connected via a bond. Equivalently in this embodiment, L2 is a bond.

L1 and D 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, L1 and D are preferably connected by a bond selected from:

    • —C(═O)NH—, and
    • —NHC(═O)—.

In one embodiment, L1 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 —X1—X2— in dipeptide, —NH—X1—X2—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 X1, and CO is the carbonyl group of X2.

Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:

    • -Phe-Lys-,
    • -Val-Ala-,
    • -Val-Lys-,
    • -Ala-Lys-, and
    • -Val-Cit-.

Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—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, L1-D is:

    • where —NH—X1—X2—CO is the dipeptide, —NH— is part of the Drug unit, the asterisk indicates the point of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.

In one embodiment, the dipeptide is valine-alanine and L1-D is:

    • where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is phenylalnine-lysine and L1-D is:

    • where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is valine-citrulline.

In one embodiment, the groups A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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 A1-L1 are:

    • where the asterisk indicates the point of attachment to 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 A1-L1 are:

    • where the asterisk indicates the point of attachment to 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 A1-L1 are:

    • where the asterisk indicates the point of attachment to 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 A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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 A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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 A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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 A1-L1 is:

    • where the asterisk indicates the point of attachment to L2 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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-A1-L1 are:

    • where the asterisk indicates the point of attachment to L2 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, L1 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), G1 is a Stretcher group (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (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:
G1-L1-L2-*

    • where the asterisk indicates the point of attachment to the Drug unit (D), G1 is a Stretcher unit (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or a self-immolative group(s).

L1 and L2 are as defined above. References to connection to A1 can be construed here as referring to a connection to G1.

In one embodiment, where L1 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 L1 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 G1 forms a connecting group upon reaction with a Ligand unit (e.g., a cell binding agent.

In one embodiment, the functional group G1 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, G1 comprises a maleimide group.

In one embodiment, the group G1 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 G1 is:

    • where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G1 is:

    • where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G1 is:

    • where the asterisk indicates the point of attachment to L1, 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 G1 is:

    • where the asterisk indicates the point of attachment to L1, 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 G1 is:

    • where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G1 is:

    • where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G1 is:

    • where the asterisk indicates the point of attachment to L1, 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 G1 is:

    • where the asterisk indicates the point of attachment to L1, 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,
    • —NH2,
    • —SH,
    • —C(═O)CH2X, where X is Cl, Br or I,
    • —CHO,
    • —NHNH2
    • —C≡CH, and
    • —N3 (azide).

In one embodiment, L1 is present, and G1 is —NH2, —NHMe, —COOH, —OH or —SH.

In one embodiment, where L1 is present, G1 is —NH2 or —NHMe. Either group may be the N-terminal of an L1 amino acid sequence.

In one embodiment, L1 is present and G1 is —NH2, and L1 is an amino acid sequence —X1—X2—, as defined above.

In one embodiment, L1 is present and G1 is COOH. This group may be the C-terminal of an L1 amino acid sequence.

In one embodiment, L1 is present and G1 is OH.

In one embodiment, L1 is present and G1 is SH.

The group G1 may be convertable from one functional group to another. In one embodiment, L1 is present and G1 is —NH2. This group is convertable to another group G1 comprising a maleimide group. For example, the group —NH2 may be reacted with an acids or an activated acid (e.g., N-succinimide forms) of those G1 groups comprising maleimide shown above.

The group G1 may therefore be converted to a functional group that is more appropriate for reaction with a Ligand unit.

As noted above, in one embodiment, L1 is present and G1 is —NH2, —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, G1 is —NH2 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 G1 is —NH2, it is protected with an Alloc or Fmoc group.

In one embodiment, where G1 is —NH2, 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 G1. Preferably, the wavy line indicates the point of attachment to G1.

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 L1 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 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 1×107 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 herein.

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 herein. 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.

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 an 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 WO2008070593, which are incorporated herein.

Methods of Treatment

The compounds 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. 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 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, 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's Injection, Lactated Ringer'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'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' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud'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's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmune 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 administered ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered 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 (carboxylate) 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+HR1R2), 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 Ca2+ and Mg2+, and other cations such as Al+3. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). 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(CH3)4+.

If the compound is cationic, or has a functional group which may be cationic (e.g. —NH2 may be —NH3+), 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 (RAOH, where RA is C1-4 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.

Isolation of Compounds of Formula I

Without wishing to be bound by theory, it has been found that compounds of formula I where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, have a tendency to reversibly dimerise on isolation. This reversible dimerisation is thought to occur due to reversible addition of Q on one molecule with the imine bond of another compound. Treatment with methanol, e.g. for 1 hour, can assist with isolation of the monomeric form of compounds of formula I.

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, —OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH2OH. 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. C1-7 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 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; 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:

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 R10 and R11 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 R2, R6, R7, R9, R6′, R7′, R9′, R12, X, X′ and R″ are as defined for compounds of formula I, ProtN is a nitrogen protecting group for synthesis and ProtO 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 ProtN is Troc and ProtO 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 ProtN is SEM, or an analogous group, and ProtO 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, where there is double bond between C2′ and C3′, can be synthesised from a compound of formula 3a:


where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of formula 2, by coupling an organometallic derivative comprising R12, 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 R12, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of formula 2, by coupling an alkyne of formula IIa:


by a Sonogashira coupling.

Compounds of formulae 3a and 3b, where there is double bond between C2′ and C3′, can be synthesised from a compound of formula 4:


where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of formula 2, by either 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 R12 to make a compound of formula 3b, or by coupling about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) an alkyne of formula IIa:


by a Sonogashira coupling to make a compound of Formula 3a.

The couplings of an organometallic derivative, such as an organoboron derivative, described above are usually carried out in the presence of a palladium catalyst, for example Pd(PPh3)4, Pd(OCOCH3)2, PdCl2, Pd2(dba)3. The coupling may be carried out under standard conditions, or may also be carried out under microwave conditions.

The Sonogashira coupling is carried out using two catalysts: a zerovalent palladium complex and a halide salt of copper(I). Phosphine-palladium complexes such as tetrakis(triphenylphosphine)palladium(0) are used for this reaction, but palladium(II) complexes can also be used because they are reduced to the palladium(0) species by the consumption of the terminal alkynes in the reaction medium. The oxidation of triphenylphosphine to triphenylphosphine oxide can also lead to the formation of Pd(0) in situ when catalysts such as bis(triphenylphosphine)palladium(II) chloride are used. In contrast, copper(I) halides react with the terminal alkyne and produce copper(I) acetylide, which acts as an activated species for the coupling reactions.

The reaction medium must be basic to neutralize the hydrogen halide produced as the byproduct of this coupling reaction, so alkylamine compounds such as triethylamine, diethylamine or piperidine are sometimes used as solvents, but also DMF or ether can be used as solvent. Other bases such as potassium carbonate or cesium carbonate are occasionally used.

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 ProtO is an oxo group and ProtN is SEM are described in detail in WO 00/12508, which is incorporated herein by reference. 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.

The synthesis of compounds of formula 4 where ProtO is a protected oxygen group for synthesis are described in WO 2005/085251, which synthesis is herein incorporated by reference.

Compounds of Formula 3b, where there is single bond between C2′ and C3′ can be synthesised from a compound of Formula 5a:


by an adaption of the methods described in the above references for the synthesis of compounds of formula 4 by triflation of the C2 keto group.

Compounds of Formula 5a may be synthesised from a compound of Formula 6:


by reaction with about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an appropriate alkene forming reagent, for example Wittig reagents (such as ylides), Tebbe reagents and Horner-Emmons-Wadworth reagents. Reference is made to the discussion on page 16 of WO 2010/043877, which is incorporated herein by reference.

Compounds of Formula 3b, where there is single bond between C2′ and C3′ can also be synthesised from a compound of Formula 5b:


by reaction with an appropriate alkene forming reagent, as discussed above.

Compounds of Formula 5b can be synthesised from compounds of formula 6 by a selective triflation using a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of the triflating agent.

Compounds of Formula 5b can also be used to synthesise a compound of formula 3c:


by the Sonogashira coupling an alkyne of formula IIa. These compounds can then be used to synthesise a compound of Formula 2, where there is single bond between C2′ and C3′, by reaction with an appropriate alkene forming reagent, as discussed above.

Compounds of formula I where R10 and R10′ are H and R11 and R11′ are SON, can be synthesised from compounds of formula I where R10 and R11 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.

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, 3rd Edition, John Wiley & 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, 3rd Edition, John Wiley & 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, Cl3CCH2OCH2—.
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, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

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 can be prepared as previously described. Linkers having a maleimidyl group (A), a peptide group (L1) and self-immolative group (L2) can be prepared as described in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. Linkers having a maleimidyl group (A) and a peptide group (L1) can be prepared as described in WO 2009/0117531, which is incorporated herein by reference. 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. Linkage of amine-based X substituents (of the PDB 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.

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.

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′, R7′, R9′, R10′, R11′ and Y′ are preferably the same as R6, R7, R9, R10, R11 and Y respectively.

Dimer Link

Y and Y′ are preferably O.

R″ is preferably a C3-7 alkylene group with no substituents. More preferably R″ is a C3, C5 or C7 alkylene. Most preferably, R″ is a C3 or C5 alkylene.

R6 to R9

R9 is preferably H.

R6 is preferably selected from H, OH, OR, SH, NH2, nitro and halo, and is more preferably H or halo, and most preferably is H.

R7 is preferably selected from H, OH, OR, SH, SR, NH2, NHR, NRR′, and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C1-7 alkyl, C3-10 heterocyclyl and C5-10 aryl groups. R may be more preferably a C1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH2Ph. Other substituents of particular interest are dimethylamino (i.e. —NMe2); —(OC2H4)qOMe, where q is from 0 to 2; nitrogen-containing C6 heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl.

These preferences apply to R9′, R6′ and R7′ respectively.

R2

R2 is of formula II:


where Q is selected from OH, SH and NRN, and RN is selected from H, methyl and ethyl.

In some embodiments, it is preferred that Q is NRN. In other embodiments, Q is OH. In further embodiments, R is SH.

RN is preferably selected from H and methyl. In some embodiment, RN is H. In other embodiments, RN is methyl.

R12

When there is a double bond present between C2′ and C3′, R12 is selected from:

(a) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;

(b) C1-5 saturated aliphatic alkyl;

(c) C3-6 saturated cycloalkyl;


wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;


wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and


where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.

When R12 is a C5-10 aryl group, it may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R12 is preferably phenyl. In other embodiments, R12 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.

When R12 is a C5-10 aryl group, it may be a C8-10 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-4yl, 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-4yl, 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.

When R12 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.

Where R12 is C5-7 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 C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para-positions, and more preferably is in the para-position.

Where R12 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).

R12 Substituents, when R12 is a C5-10 Aryl Group

If a substituent on R12 when R12 is a C5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.

If a substituent on R12 when R12 is a C5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).

If a substituent on R12 when R12 is a C5-10 aryl group is C1-7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).

If a substituent on R12 when R12 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.

If a substituent on R12 when R12 is a C5-10 aryl group is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.

Particularly preferred substituents when R12 is a C5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methylthiophenyl. Another particularly preferred substituent for R12 is dimethylaminopropyloxy.

Particularly preferred substituted R12 groups when R12 is a C5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R12 group is 4-nitrophenyl. R12 groups of particular interest include 4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.

When R12 is C1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.

When R12 is C3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.

When R12 is


each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5. In some embodiments, the total number of carbon atoms in the R12 group is no more than 4 or no more than 3.

In some embodiments, one of R21, R22 and R23 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.

In other embodiments, two of R21, R22 and R23 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.

In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that re not H are methyl.

In some embodiments, R21 is H.

In some embodiments, R22 is H.

In some embodiments, R23 is H.

In some embodiments, R21 and R22 are H.

In some embodiments, R21 and R23 are H.

In some embodiments, R22 and R23 are H.

An R12 group of particular interest is:

When R12 is


one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.

When R12 is


R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.

In some embodiments, R24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R24 is selected from H and methyl.

When there is a single bond present between C2′ and C3′,

R12 is


where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester.

In some embodiments, it is preferred that R26a and R26b are both H.

In other embodiments, it is preferred that R26a and R26b are both methyl.

In further embodiments, it is preferred that one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl.

M and z

It is preferred that M and M′ are monovalent pharmaceutically acceptable cations, and are more preferably Na+.

z is preferably 3.

Particularly preferred compounds of the present invention are of formula Ia:


where

n is 1 or 3;

R1a is methyl or phenyl;

RN is H, methyl or ethyl;

R12a is selected from:

Further group for R12a may be:

Other particularly preferred compounds of the present invention are of formula Ib:


where

n is 1 or 3;

R1b is methyl or phenyl; and

RN is H, methyl or ethyl.

3rd Aspect

The preferences expressed above for the first aspect may apply to the compounds of this aspect, where appropriate.

When R10 is carbamate nitrogen protecting group, it may preferably be Teoc, Fmoc and Troc, and may more preferably be Troc.

When R11 is O-ProtO, wherein ProtO is an oxygen protecting group, ProtO may preferably be TBS or THP, and may more preferably be TBS.

When R10 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.

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. 1H and 13C 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, F254), 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.

Compound 1 was synthesised as described in WO 2010/043880 (Compound 17), which is herein incorporated by reference.

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)-7-methoxy-8-(3-(((S)-7-methoxy-2-(3-(methylamino)prop-1-yn-1-yl)-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)-2-(4-methoxyphenyl)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (2)

A mixture of 1 (0.433 g, 0.40 mmol), CuI (0.008 g, 0.04 mmol, 0.1 eq), N-methyl-N-(2-propynyl)amine (0.10 ml, 1.20 mmol, 3 eq) and PPh3 (0.021 g, 0.08 mmol, 0.2 eq) was dissolved in anhydrous piperidine (5 mL) in the presence of molecular sieves under an argon atmosphere. Pd(PPh3)4 (0.046 g, 0.04 mmol, 0.1 eq) was added to the mixture, and the reaction was warmed to 60° C. and stirred overnight. The solvent was removed by rotary evaporation under reduced pressure and the resulting brown solid purified by flash column chromatography (silica gel, gradient 96% DCM-4% methanol). Compound 2 was obtained as a brown solid (0.213 g, 53%); Rf 0.32 [10% methanol in DCM]; LC-MS (5 min) 3.35 min, ES+ 995.35.

(b) (S)-2-((Z)-(((E)-4-methoxy-3-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)but-2-en-1-yl)imino)methyl)-4-(3-(methylamino)prop-1-yn-1-yl)-2,3-dihydro-1H-pyrrole-1-carbaldehyde (3)

Compound 2 (0.041 g, 0.04 mmol) was dissolved in anhydrous THF (3.8 mL) under an argon atmosphere and cooled to −78° C. in an acetone-dry ice bath. LiEt3BH (0.25 mL of a 1M solution in THF, 0.35 mmol, 6 eq) was added to the mixture, and the reaction was stirred at −78° C. for 40 minutes. The reaction was quenched with H2O (3 mL) and after addition of brine (3 mL) the aqueous phase was extracted (DCM, 9 mL-methanol 1 mL), dried (MgSO4) and the solvent was removed by rotary evaporation under reduced pressure. The crude was dissolved in a mixture of ethanol (3 mL), DCM (1.5 mL) and H2O (1.5 mL) and silica gel was added to the mixture. The reaction was stirred for four days at room temperature under an atmosphere of Argon. The mixture was filtered over a sinter funnel washing with a mixture of DMC (140 mL) and methanol (15 mL). The organic phase was washed with brine (200 mL), dried (MgSO4) and the solvent was removed by rotary evaporation under reduced pressure. The resulting brown solid was purified by flash column chromatography (silica gel, gradient 90% DCM-10% methanol). Compound 3 was obtained as a brown solid (0.029 g, 59%); Rf 0.11 [8% methanol in DCM]; LC-MS (5 min) 2.47 min, ES+ 702.28.

General Experimental Methods for Following Examples

LCMS data were obtained using an Agilent 1200 series LC/MS with an Agilent 6110 quadrupole MS, with Electrospray ionisation. Mobile phase A—0.1% Acetic acid in water. Mobile Phase B—0.1% in acetonitrile. Flow rate of 1.00 ml/min. Gradient from 5% B rising up to 95% B over 3 minutes, remaining at 95% B for 1 minute and then back down to 5% B over 6 seconds. The total run time is 5 minutes. Column: Phenomenex Gemini-NX 3 μm C18, 30×2.00 mm. Chromatograms based on UV detection at 254 nm. Mass Spectra were achieved using the MS in positive mode. Proton NMR chemical shift values were measured on the delta scale at 400 MHz using a Bruker AV400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Coupling constants are reported in Hz. Unless otherwise stated, column chromatography (by the flash procedure) were performed on Merck Kieselgel silica (Art. 9385). Mass spectroscopy (MS) data were collected using a Waters Micromass LCT instrument coupled to a Waters 2795 HPLC separations module. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F254). All other chemicals and solvents were purchased from Sigma-Aldrich or Fisher Scientific and were used as supplied without further purification.

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. 1H and 13C 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, F254), 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

Synthesis of Key Intermediate

Compound 4 was synthesised as described in WO 2010/043880 (Compound 6a), which is herein incorporated by reference.

(a) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (5)

Diol 4 (25.60 g, 29.9 mmol, 1.0 eq.), NaOAc (6.90 g, 84.1 mmol, 2.8 eq.) and TEMPO (188 mg, 1.2 mmol, 0.04 eq.) were dissolved in DCM (326 mL) under nitrogen. This was cooled to −8° C. and TCCA (9.70 g, 41.7 mmol, 1.40 eq.) was added portionwise over 20 min during which the solution turned dark brown which lightened as reaction proceeded. After 30 mins cold DCM (200 mL) was added and mixture was filtered through celite and then washed with a solution of saturated sodium hydrogencarbonate/sodium thiosulphate (1:1 v/v; 200 mL×2). The organic layer was dried over magnesium sulphate, filtered and concentrated under reduced pressure to yield the bis ketone 5 as a yellow/orange sponge (25.5 g, 100%). LC/MS (3.173 min (ES+)). m/z: 854.20 [M]+. 1H NMR (400 MHz, CDCl3) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d, 2H, J=10.1 Hz), 4.60 (dd, 2H, J=9.9, 3.1 Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.3, 3.0 Hz), 2.76 (dd, 2H, J=18.6, 10.2 Hz), 2.42 (p, 2H, J=5.8 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H).

(b) 1,1′-[[(Propane-1,3-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] (6)

Anhydrous 2,6-lutidine (1.984 g, 17.864 mmol, 6.22 eq.) was injected in one portion to a vigorously stirred solution of bis-ketone 5 (2.45 g, 2.977 mmol, 1.00 eq.) in dry DCM (90 mL) at −45° C. (dry ice/acetonitrile) under a nitrogen atmosphere. Anhydrous triflic anhydride (5.040 g, 17.864 mmol, 6.0 eq.) taken from a freshly opened ampule was injected rapidly dropwise while maintaining the temperature no higher than −40° C. The reaction mixture was allowed to stir at −45° C. for 1 h at which point TLC (50/50 v/v n-hexane/EtOAc) and LCMS revealed complete consumption of the starting material. The cold reaction mixture was immediately diluted with DCM (100 mL) and, with vigorous shaking, washed with water (1×50 mL), 5% citric acid solution (1×100 mL), saturated NaHCO3 (100 mL), brine (50 mL) and dried over magnesium sulphate, filtered and concentrated under reduced pressure to give the crude product which was purified by flash chromatography (silica gel, gradient elution 90:10 n-hexane/EtOAc v/v to 60:40 n-hexane/EtOAc v/v) to afford the bis enol triflate 6 as a yellow foam (2.097 g, 63%). LC/MS (3.916 min (ES+)). m/z: 1117.24 [M]+. 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=2.0 Hz), 5.51 (d, 2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.6 Hz), 4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J=16.4, 11.1, 2.3 Hz), 2.43 (p, 2H, J=5.9 Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H).

Example 2

a b c d e R

(a) Compounds 7a-e (i) General preparation of compounds 7c-d

Boronic acid (1.0 eq), triethylamine (4.0 eq) and (11aS,11a′S)-8,8′-(propane-1,3-diylbis(oxy))bis(7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-8,2-diyl)bis(trifluoromethanesulfonate) (6)(1.0 eq) were dissolved in a mixture of ethanol, toluene and water [3:6:1] (0.01 M) under a nitrogen atmosphere. The reaction mixture was degassed under nitrogen for 5 mins when tetrakis(triphenylphosphine)palladium(0) (0.04 eq) was added. The reaction mixture stirred at between 60° C. and 70° C. for 1 hour. Dichloromethane was added and washed with water. The organic layer was dried with MgSO4, filtered, and concentrated under reduced pressure. The residue was adsorbed onto silica and purified by flash chromatography (silica gel, gradient elution, 80/20 to 20/80 v/v hexane/ethyl acetate) to yield a mixture of recovered starting material, product and bis-substituted impurity.

(S)-7-methoxy-8-(3-((S)-7-methoxy-5,11-dioxo-2-((E)-prop-1-enyl)-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl trifluoromethanesulfonate (7c)

2.06 g (1.84 mmol) of 6 gave the following mixture:

0.446 g (24%) of 7c as a brown solid. LC/MS (3.885 min (ES+)). m/z: 1009.31 [M]+.

0.057 g of 6. LC/MS (3.920 min (ES+)). m/z: 1117.24 [M]+.

0.504 g of bis-substituted impurity as a brown solid. LC/MS (3.854 min (ES+)). m/z: 901.41 [M]+.

(S)-8-(3-((S)-2-(benzo[d][1,3]dioxol-5-yl)-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-yloxy)propoxy)-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-2-yl trifluoromethanesulfonate (7d)

0.200 g (0.179 mmol) of 6 gave the following mixture:

0.075 g (38%) of 7d as a brown solid. LC/MS (3.851 min (ES+)). m/z: 1089.28 [M]+.

0.024 g of 6. LC/MS (3.920 min (ES+)). m/z: 1117.24 [M]+.

0.052 g of bis-substituted impurity as a brown solid. LC/MS (3.774 min (ES+)). m/z: 1061.38 [M]+.

(ii)(S)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl trifluoromethanesulfonate (7e)

(11aS,11a′S)-8,8′-(propane-1,3-diylbis(oxy))bis(7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-8,2-diyl)bis(trifluoromethanesulfonate) (6) (2.00 g, 1.790 mmol, 1.0 eq), (4-methylpiperazin-1-yl)phenylboronic acid (0.394 g, 1.790 mmol, 1.0 eq) and triethylamine (0.574 mL, 4.117 mmol, 2.3 eq) were solubilised in a mixture of Toluene/Ethanol/H2O 6:3:1 (100 mL) under a nitrogen atmosphere. The reaction was flushed with nitrogen and palladium-tetrakistriphenylphosphine (20.7 mg, 0.0179 mmol, 0.01 eq) was added. The reaction was flushed with nitrogen and stirred at 30° C. for 16 hours. All the volatiles were subsequently removed under reduced pressure and the solid residue was partitioned between H2O (200 mL) and ethyl acetate (200 mL). The aqueous layer was extracted two more times with ethyl acetate (2×200 mL) before the combined organics were washed with brine (200 mL), dried with MgSO4, filtered and the volatiles removed under reduced pressure. The crude product was purified by silica gel column chromatography (CHCl3 100 to 95:5 v/v CHCl3/MeOH) to yield a mixture of recovered starting material, product and bis-substituted impurity.

0.669 g (33%) of 7e as a brown solid. LC/MS (2.751 min (ES+)), m/z: 1144.2 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 7.37 (s, 1H), 7.33-7.28 (m, 4H), 7.12 (m, 1H), 6.88 (d, J=8.8 Hz, 2H), 5.51-5.49 (d, J=10.0 Hz, 2H), 4.76-4.73 (dd, J=10.0, 4.0 Hz, 2H), 4.62-4.56 (m, 2H), 4.30-4.22 (m, 4H), 3.94-3.86 (m, 9H), 3.81-3.71 (m, 3H), 3.70-3.62 (m, 2H), 3.26-3.22 (m, 4H), 3.17-3.07 (m, 2H), 2.66-2.55 (m, 4H), 2.46-2.38 (m, 2H), 2.35 (s, 3H), 0.99-0.93 (m, 4H), 0.00 (s, 18H).

1.093 g of 6. LC/MS (3.917 min (ES+)).

0.094 g of bis-substituted impurity as a brown solid. LC/MS (2.150 min (ES+)). m/z: 1061.38 [M]+.

(b) General Preparation of Compounds 8a-e

A mixture of the appropriate mono-triflate from step (a)(1.0 eq), and triphenylphosphine (0.2 eq) was dissolved in anhydrous piperidine (0.05 M) in the presence of 4 Å molecular sieves under a nitrogen atmosphere. Propargylamine (6.0 eq), copper Iodide (0.1 eq), and tetrakis(triphenylphosphine)palladium(0) (0.1 eq) was then added to the mixture, and the reaction was warmed to between 50° C. and 60° C. for 0.5 hour to 1 hour. The mixture was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. The resulting brown oil was purified by silica gel column chromatography (DCM 100 to 90:10 v/v DCM/MeOH to 85:15 v/v DCM/MeOH).

(i) (S)-2-(3-aminoprop-1-ynyl)-7-methoxy-8-(3-((S)-7-methoxy-5,11-dioxo-2-((E)-prop-1-enyl)-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (8c)

0.418 g (0.414 mmol) of 7c yielded 0.150 g (40%) of product as a brown solid. LC/MS (2.576 min (ES+)). m/z: 914.42 [M]+. 1H NMR (400 MHz, CDCl3) δ 7.35-7.32 (m, 3H), 6.84 (s, 1H), 6.25-6.21 (d, J=14.9 Hz, 1H), 5.72-5.65 (m, 1H), 5.50-5.48 (d, J=9.9 Hz, 2H), 4.73-4.71 (m, 2H), 4.51-4.48 (m, 2H), 4.31-4.23 (m, 4H), 3.93-3.86 (m, 7H), 3.81-3.72 (m, 3H), 3.70-3.60 (m, 5H), 3.49-3.42 (m, 1H), 2.95-2.83 (m, 2H), 2.46-2.38 (m, 2H), 1.83 (d, J=6.7 Hz, 3H), 1.76-1.69 (m, 2H), 1.00-0.92 (m, 4H), 0.00 (s, 18H).

(ii) (S)-2-(3-aminoprop-1-ynyl)-8-(3-((S)-2-(benzo[d][1,3]dioxol-5-yl)-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-yloxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (8d)

1.120 g (1.03 mmol) of 7d yielded 0.507 g (50%) of product as a brown solid. LC/MS (2.596 min (ES+)). m/z: 944.2 [M]+. 1H NMR (400 MHz, CDCl3) δ 7.38 (s, 1H), 7.34 (s, 1H), 7.30 (m, 1H), 7.09 (m, 1H), 6.94 (d, J=1.6 Hz, 1H), 6.89 (dd, J=8.1, 1.6 Hz, 1H), 6.78 (d, J=8.1 Hz, 1H), 5.97 (s, 2H), 5.52-5.49 (dd, J=10.0, 4.3 Hz, 2H), 4.77-4.73 (dd, J=10.1, 6.1 Hz, 2H), 4.61-4.58 (dd, J=10.6, 3.4 Hz, 1H), 4.53-4.49 (dd, J=10.6, 3.4 Hz, 1H), 4.31-4.24 (m, 4H), 3.91-3.86 (m, 9H), 3.81-3.74 (m, 2H), 3.73-3.57 (m, 5H), 3.14-3.07 (m, 1H), 2.94-2.87 (m, 1H), 2.46-2.39 (m, 2H), 1.85-1.68 (m, 2H), 1.02-0.93 (m, 4H), 0.00 (s, 18H).

(iii) (S)-2-(3-aminoprop-1-ynyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (8e)

0.812 g (0.710 mmol) of 7e yielded 0.227 g (30%) of product as a brown solid. LC/MS (2.109 min (ES+)). m/z: 1049.48 [M+H]+.

(c) General Preparation of Compounds 9a-e

The appropriate SEM dilactam from step (b)(1.0 eq.) was solubilised in THF (0.02 M) and cooled to −78° C. under a nitrogen atmosphere. Super hydride solution (1M in THF, 2.04 eq.) was added dropwise over 5 minutes. After 20 minutes an aliquot was washed with water for LCMS and TLC analysis. After 30 minutes water was added and the cold bath removed. The organic layer was extracted with EtOAc (2×) and the combined organic extracts washed with brine, dried with MgSO4, filtered and the solvent removed under reduced pressure. The crude product was dissolved in MeOH/DCM/water (6:3:1) (0.01 M) and enough silica gel to form a thick stirring suspension.

After 5 days the suspension was filtered through a sintered funnel and washed with DCM/MeOH 9:1 until complete elution of product. The organic layer was washed with brine (2×), dried with MgSO4, filtered and the solvent removed under reduced pressure. Purification by silica gel column chromatography (CHCl3 100 to 90:10 v/v CHCl3/MeOH) afforded the product.

(i) (S)-2-(3-aminoprop-1-ynyl)-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (9c)

0.150 g (0.164 mmol) of 8c yielded 0.29 g (28%) of product as a brown solid. LC/MS (2.759 min (ES+)). m/z: 1243.50 [2M]+.

(ii) (S)-2-(3-aminoprop-1-ynyl)-8-(3-((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (9d)

0.250 g (0.251 mmol) of 8d yielded 0.026 g (15%) of product as a dark brown solid. LC/MS (2.758 min (ES+)). m/z: 1402.79 [2M]+.

(iii) (S)-2-(3-aminoprop-1-ynyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (9e)

0.227 g (0.217 mmol) of 8e yielded 0.028 g (17%) of product. LC/MS (2.759 min (ES+)). m/z: 657.4 [M-piperazine]+

These compounds were generally found to be in dimeric form when isolated (see discussion above). Treatment in methanol, by stirring for 1 hour, enabled detection of the monomeric form of the compounds. 9c: LC/MS (1.52 min (ES+)). m/z: 622.4 [M+H]+; 9d: LC/MS (1.51 min (ES+)). m/z: 702.5 [M+H]+.

Example 3

a b c d e R

(a) Compounds 10a-e (i) (9H-fluoren-9-yl)methyl(S)-1-((S)-1-(3-((S)-8-(3-((S)-2-(benzo[d][1,3]dioxol-5-yl)-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-yloxy)propoxy)-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-2-yl)prop-2-ynylamino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate (10d)

1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (0.0193 g, 0.101 mmol, 1.0 eq) was added to a solution of 8d (0.100 g, 0.101 mmol, 1.0 eq) and HO-Ala-Val-Fmoc (0.0415 g, 0.101 mmol, 1.0 eq) in dry dichloromethane (6 mL) at room temperature. After 30 minutes the reaction was diluted with dichloromethane (20 mL) and washed sequentially with water (10 mL) and brine (10 mL). The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 70:1 v/v DCM/MeOH to 50:1 v/v DCM/MeOH). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give product as a yellow solid (0.043 g, 31%).

LC/MS (3.785 min (ES+)). m/z: 1385.01 [M−H]+.

(b) Compounds 11a-e (i) (9H-fluoren-9-yl)methyl(R)-1-((S)-1-(3-((S)-8-(3-((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)prop-2-ynylamino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate (11d)

The SEM dilactam 10d (0.063 g, 0.0455 mmol, 1.0 eq.) was solubilised in THF (2.7) and cooled to −78° C. under a nitrogen atmosphere. Super hydride solution (1M in THF, 0.093 mL, 2.04 eq.) was added dropwise over 5 minutes. After 20 minutes an aliquot was washed with water for LCMS and TLC analysis. After 30 minutes water was added and the cold bath removed. The organic layer was extracted with EtOAc (2×10 mL) and the combined organic extracts washed with brine (10 mL), dried with MgSO4, filtered and the solvent removed under reduced pressure. The crude product was dissolved in MeOH/DCM/water (6:3:1) (0.01 M) and enough silica gel to form a thick stirring suspension.

After 5 days the suspension was filtered through a sintered funnel and washed with DCM/MeOH 9:1 until complete elution of product. The organic layer was washed with brine (2×), dried with MgSO4, filtered and the solvent removed under reduced pressure. Purification by silica gel column chromatography (CHCl3 100 to 90:10 v/v CHCl3/MeOH) afforded the product.

(c) Compounds 12a-e (i) (S)-2-amino-N—((S)-1-(3-((S)-8-(3-((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)prop-2-ynylamino)-1-oxopropan-2-yl)-3-methylbutanamide (12d)

Excess piperidine was added (0.01 mL, 0.1 mmol) to a solution of SEM-dilactam 11d (1 mg, 0.0009 mmol) in DMF (0.1 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (monitored by LC/MS). The reaction mixture was diluted with CH2Cl2 (1 mL) and the organic phase was washed with H2O (3×1 mL) until complete piperidine removal. The organic phase was dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude material 12d. LC/MS (1.917 min (ES+)). m/z: 872.2 [M+H]+

Example 4 Determination of In Vitro Cytotoxicity

K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37° C. in a humidified atmosphere containing 5% CO2 and were incubated with a specified dose of drug for 96 hours at 37° C. in the dark. The incubation was terminated by centrifugation (5 min, 300 g) and the cells were washed once with drug-free medium. Following the appropriate drug treatment, the cells were transferred to 96-well microtiter plates (104 cells per well, 8 wells per sample). Plates were then kept in the dark at 37° C. in a humidified atmosphere containing 5% CO2. The assay is based on the ability of viable cells to reduce a yellow soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Aldrich-Sigma), to an insoluble purple formazan precipitate. Following incubation of the plates for 4 days (to allow control cells to increase in number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well and the plates further incubated for 5 hours. The plates were then centrifuged for 5 minutes at 300 g and the bulk of the medium pipetted from the cell pellet leaving 10-20 μL per well. DMSO (200 μL) was added to each well and the samples agitated to ensure complete mixing. The optical density was then read at a wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and a dose-response curve was constructed. For each curve, an IC50 value was read as the dose required to reduce the final optical density to 50% of the control value.

Compound 13 has an IC50 of 0.5 nM in this assay.

Claims

1. A compound with the formula I: wherein:

R2 is of formula II:
where Q is selected from OH, SH and NHRN, and RN is selected from H, methyl and ethyl;
when there is a double bond present between C2′ and C3′, R12 is selected from: (ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3alkylene; (ib) C1-5 saturated aliphatic alkyl; (ic) C3-6 saturated cycloalkyl;
 wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;
 wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;and
 where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2′ and C3′,
R12 is
 where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester;
R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo;
either: (a) R10 is H, and R11 is OH, ORA, where RA is C1-4 alkyl; (b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R10 is H and R11 is SOZM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, and/or aromatic rings;
Y and Y′ are selected from O, S, or NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11, wherein if R11 and R11′are SOZM, M may represent a divalent pharmaceutically acceptable cation.

2. A compound according to any claim 1, wherein R7 is selected from H, OH and OR.

3. A compound according to claim 2, wherein R7 is a C1-4 alkyloxy group.

4. A compound according to claim 1, wherein Y is O.

5. A compound according to claim 1, wherein R″ is C3-7 alkylene.

6. A compound according to claim 1, wherein R9 is H.

7. A compound according to claim 1, wherein R6 is selected from H and halo.

8. A compound according to claim 1, wherein Q is NHRN.

9. A compound according to claim 8, wherein RN is H or methyl.

10. A compound according to claim 1, wherein Q is OH or SH.

11. A compound according to claim 1, wherein there is a single bond between C2′ and C3′, R12 is and R26a and R26b are both H.

12. A compound according to claim 1, wherein there is a single bond between C2′ and C3′, R12 is and R26a and R26b are both methyl.

13. A compound according to claim 1, wherein there is a single bond between C2′ and C3′, R12 is one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.

14. A compound according to claim 1, wherein there is a double bond between C2′ and C3′, and R12 is a C5-7 aryl group.

15. A compound according to claim 14, wherein R12 is phenyl.

16. A compound according to claim 1, wherein there is a double bond between C2′ and C3′, and R12 is a C8-10 aryl group.

17. A compound according to claim 1, wherein R12 bears one to three substituent groups.

18. A compound according to claim 1, wherein there is a double bond between C2′ and C3′, and R12 is a C1-5 saturated aliphatic alkyl group.

19. A compound according to claim 18, wherein R12 is methyl, ethyl or propyl.

20. A compound according to claim 1, wherein there is a double bond between C2′ and C3′, and R12 is a C3-6 saturated cycloalkyl group.

21. A compound according to claim 20, wherein R12 is cyclopropyl.

22. A compound according to claim 1, wherein there is a double bond between C2′ and C3′, and R12 is a group of formula:

23. A compound according to claim 22, wherein the total number of carbon atoms in the R12 group is no more than 4.

24. A compound according to claim 23, wherein the total number of carbon atoms in the R12 group is no more than 3.

25. A compound according to claim 22, wherein one of R21, R22 and R23 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.

26. A compound according to claim 22, wherein two of R21, R22 and R23 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.

27. A compound according to claim 1, wherein there is a double bond between C2′ and C3′, and R12 is a group of formula:

28. A compound according to claim 27, wherein R12 is the group:

29. A compound according to claim 1, wherein there is a double bond between C2′ and C3′, and R12 is a group of formula:

30. A compound according to claim 29, wherein R24 is selected from H, methyl, ethyl, ethenyl and ethynyl.

31. A compound according to claim 30, wherein R24 is selected from H and methyl.

32. A compound according to claim 1, wherein R10 and R11 and form a nitrogen-carbon double bond.

33. A compound according to claim 1 wherein R6′, R7′, R9′, R10′, R11′ and Y′ are the same as R6, R7, R9, R10, R11 and y respectively.

34. The use of a compound according to claim 1 in the manufacture of a medicament for treating a proliferative disease.

35. A compound according to claim 1 for use in the treatment of a proliferative disease.

36. A compound of formula II: wherein:

R2 is of formula II:
where Q is selected from OH, SH and NHRN, and RN is selected from H, methyl and ethyl;
when there is a double bond present between C2′ and C3′, R12 is selected from: (ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (ib) C1-5 saturated aliphatic alkyl; (ic) C3-6 saturated cycloalkyl;
 wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;
 wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;and
 where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2′ and C3′,
R12 is
 where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester;
R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo;
either: (a) R10 is carbamate nitrogen protecting group, and R11 is O-ProtO, wherein ProtO is an oxygen protecting group; (b) R10 is a hemi-aminal nitrogen protecting group and R11 is an oxo group;
R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, and/or aromatic rings;
Y and Y′ are selected from O, S, or NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11.

37. A compound according to any claim 36, wherein R7 is selected from H, OH and OR.

38. A compound according to claim 37, wherein R7 is a C1-4 alkyloxy group.

39. A compound according to claim 3, wherein Y is O.

40. A compound according to claim 36, wherein R″ is C3-7 alkylene.

41. A compound according to claim 36, wherein R9 is H.

42. A compound according to claim 36, wherein R6 is selected from H and halo.

43. A compound according to claim 36, wherein Q is NHRN.

44. A compound according to claim 43, wherein RN is H or methyl.

45. A compound according to claim 36, wherein Q is OH or SH.

46. A compound according to claim 36, wherein there is a double bond between C2′ and C3′, and R12 is a C5-7 aryl group.

47. A compound according to claim 46, wherein R12 is phenyl.

48. A compound according to claim 36, wherein there is a double bond between C2′ and C3′, and R12 is a C8-10 aryl group.

49. A compound according to claim 46, wherein R12 bears one to three substituent groups.

50. A compound according to claim 36, wherein there is a single bond between C2′ and C3′, R12 is and R26a and R26b are both H.

51. A compound according to claim 36, wherein there is a single bond between C2′ and C3′, R12 is and R26a and R26b are both methyl.

52. A compound according to claim 36, wherein there is a single bond between C2′ and C3′, R12 is one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.

53. A compound according to claim 36, wherein there is a double bond between C2′ and C3′, and R12 is a C1-5 saturated aliphatic alkyl group.

54. A compound according to claim 53, wherein R12 is methyl, ethyl or propyl.

55. A compound according to claim 36, wherein there is a double bond between C2′ and C3′, and R12 is a C3-6 saturated cycloalkyl group.

56. A compound according to claim 55, wherein R12 is cyclopropyl.

57. A compound according to claim 36, wherein there is a double bond between C2′ and C3′, and R12 is a group of formula:

58. A compound according to claim 57, wherein the total number of carbon atoms in the R12 group is no more than 4.

59. A compound according to claim 58, wherein the total number of carbon atoms in the R12 group is no more than 3.

60. A compound according to claim 57, wherein one of R21, R22 and R23 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.

61. A compound according to claim 57, wherein two of R21, R22 and R23 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.

62. A compound according to claim 36, wherein there is a double bond between C2′ and C3′, and R12 is a group of formula:

63. A compound according to claim 62, wherein R12 is the group:

64. A compound according to claim 36, wherein there is a double bond between C2′ and C3′, and R12 is a group of formula:

65. A compound according to claim 64 wherein R24 is selected from H, methyl, ethyl, ethenyl and ethynyl.

66. A compound according to claim 65, wherein R24 is selected from H and methyl.

67. A compound according to claim 36, wherein R10 is Troc.

68. A compound according to claim 36, wherein R11 is OTBS.

69. A compound according to claim 36, wherein R11 is oxo and R10 is SEM.

70. A compound according to claim 36, wherein R6′, R7′, R9′, R10′, R11′ and Y′ are the same as R6, R7, R9,R10, R11 and Y respectively.

71. A Conjugate having formula III: wherein: where Q is selected from —O—, —S— and —NRN and RN is selected from H, methyl and ethyl;

L-(LU-D)p   (I)
wherein L is a Ligand unit which is a cell binding agent that specifically binds to a target molecule on the surface of a target cell, LU is a Linker unit of formula -A1-L1-, wherein L1 is a dipeptide and A1 is selected from:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6;
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6;
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1 and m is 0 to 30; or
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1 and m is 0 to 30,
p is 1 to 20; and
D is a Drug unit which is a compound with the formula I:
R2 is of formula II:
when there is a double bond present between C2′ and C3′, R12 is selected from: (ia) C5-10 aryl group optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (ib) C1-5 saturated aliphatic alkyl; (ic) C3-6 saturated cycloalkyl;
 wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl C2-3 alkenyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;
 wherein one of R25a and R25b is H and the other is selected from: phenyl; which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
 where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2′ and C3′,
R12 is
 where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester;
R6 and R9 are independently selected from H, R, OH,OR SH, SR, NH2 NHR, NRR′ nitro, Me3 Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo;
either: (a) R10 is H, and R11 is OH, ORA, where RA is C1-4 alkyl; (b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group which chain may be interrupted by one or more heteroatoms and/or aromatic rings;
Y and Y′ are selected from O, S, or NH; R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11 wherein if R11 and R11′ are SOzM, M may represent a divalent pharmaceutically acceptable cation,
wherein LU is connected to D via the Q substituent of R2.

72. The Conjugate of claim 71, wherein A1 is where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1 and m is 0 to 30.

73. The Conjugate of claim 71, wherein A1 is: where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6.

74. The Conjugate of claim 73, wherein n is 5.

75. The Conjugate of claim 71, wherein L1 is selected from the group consisting of valine-alanine, valine-citrulline and phenyalanine-lysine.

76. A method of treating a mammal having a proliferative disease or an autoimmune disease, comprising administering an effective amount of the Conjugate of claim 71.

Referenced Cited
U.S. Patent Documents
3361742 January 1968 Julius et al.
3523941 August 1970 Leimgruber et al.
3524849 August 1970 Batcho et al.
3794644 February 1974 Karlyone et al.
4185016 January 22, 1980 Takanabe et al.
4239683 December 16, 1980 Takanabe et al.
4309437 January 5, 1982 Ueda et al.
4353827 October 12, 1982 Hunkeler et al.
4382032 May 3, 1983 Hunkeler et al.
4386028 May 31, 1983 Hunkeler et al.
4405516 September 20, 1983 Hunkeler et al.
4405517 September 20, 1983 Hunkeler et al.
4407752 October 4, 1983 Hunkeler et al.
4427587 January 24, 1984 Kaneko et al.
4427588 January 24, 1984 Kaneko et al.
4701325 October 20, 1987 Ueda et al.
4923984 May 8, 1990 Matsumura et al.
5418241 May 23, 1995 Jegham et al.
6362331 March 26, 2002 Kamal et al.
6562806 May 13, 2003 Thurston et al.
6608192 August 19, 2003 Thurston et al.
6660856 December 9, 2003 Wang
6884799 April 26, 2005 Kamal et al.
7049311 May 23, 2006 Thurston et al.
7067511 June 27, 2006 Thurston et al.
7244724 July 17, 2007 Liu et al.
7265105 September 4, 2007 Thurston et al.
7407951 August 5, 2008 Thurston et al.
7429658 September 30, 2008 Howard et al.
7528126 May 5, 2009 Howard et al.
7557099 July 7, 2009 Howard et al.
7612062 November 3, 2009 Gregson et al.
7704924 April 27, 2010 Thurston et al.
7741319 June 22, 2010 Howard et al.
8034808 October 11, 2011 Delavault et al.
8163736 April 24, 2012 Gauzy et al.
8487092 July 16, 2013 Howard et al.
8501934 August 6, 2013 Howard et al.
8592576 November 26, 2013 Howard et al.
8633185 January 21, 2014 Howard et al.
8637664 January 28, 2014 Howard et al.
8697688 April 15, 2014 Howard et al.
20040138269 July 15, 2004 Sun et al.
20070092940 April 26, 2007 Eigenbrot et al.
20070185336 August 9, 2007 Rossen et al.
20070232592 October 4, 2007 Delavault et al.
20080090812 April 17, 2008 Pepper et al.
20080092940 April 24, 2008 Nakajima
20080213289 September 4, 2008 Francisco et al.
20090148942 June 11, 2009 Mcdonagh et al.
20110196148 August 11, 2011 Howard et al.
20110201803 August 18, 2011 Howard et al.
20110256157 October 20, 2011 Howard et al.
20130028919 January 31, 2013 Howard et al.
20140066435 March 6, 2014 Howard et al.
Foreign Patent Documents
1813614 August 2007 EP
2027356 December 1969 FR
2586683 March 1987 FR
1299198 December 1972 GB
2053894 February 1981 GB
53-82792 July 1978 JP
57131791 August 1982 JP
58180487 October 1983 JP
WO 92/19620 November 1992 WO
WO 93/18045 September 1993 WO
WO 95/04718 February 1995 WO
WO 00/03291 January 2000 WO
WO 00/12506 March 2000 WO
WO 00/12507 March 2000 WO
WO 00/12508 March 2000 WO
WO 00/12509 March 2000 WO
WO 2004/043963 May 2004 WO
WO 2005/023814 March 2005 WO
WO 2005/040170 May 2005 WO
WO 2005/042535 May 2005 WO
WO 2005/082023 September 2005 WO
WO 2005/085177 September 2005 WO
WO 2005/085250 September 2005 WO
WO 2005/085251 September 2005 WO
WO 2005/085259 September 2005 WO
WO 2005/085260 September 2005 WO
WO 2005/110423 November 2005 WO
WO 2006/111759 October 2006 WO
WO 2007/039752 April 2007 WO
WO 2007/085930 August 2007 WO
WO 2008/010101 January 2008 WO
WO 2008/047242 April 2008 WO
WO 2008/070593 June 2008 WO
WO 2009/016516 February 2009 WO
WO 2010/010347 January 2010 WO
WO 2010/043877 April 2010 WO
WO 2010/043880 April 2010 WO
WO 2010/091150 August 2010 WO
WO 2011/023883 March 2011 WO
WO 2011/038159 March 2011 WO
WO 2011/100227 August 2011 WO
WO 2011/130598 October 2011 WO
WO 2011/130616 October 2011 WO
WO 2012/112708 August 2012 WO
WO 2012/128868 September 2012 WO
WO 2013/041606 March 2013 WO
WO 2013/053871 April 2013 WO
WO 2013/053873 April 2013 WO
WO 2013/055987 April 2013 WO
WO 2013/055990 April 2013 WO
WO 2013/055993 April 2013 WO
WO 2014/011518 January 2014 WO
WO 2014/011519 January 2014 WO
WO 2014/057072 April 2014 WO
WO 2014/057073 April 2014 WO
WO 2014/057074 April 2014 WO
WO 2014/022679 June 2014 WO
Other references
  • Adair, J.R. et al., “Antibody-drug conjugates—a perfect synergy,” Exp. Opin. Biol. Ther. (2012) 16 pages.
  • Adams et al., “Molecular modelling of a sequence-specific DNA-binding agent based on the pyrrolo[2,1-c][1,4]benzodiazepines,” Pharm. Pharmacol. Commun. (1999) 5:555-560.
  • Aird, R.E. et al., “ABCB1 genetic polymorphism influences the pharmacology of the new pyrrolobenzodiazepine derivative SJG-136,” Pharmacogenomics Journal (2008) 8(4):289-296.
  • Alley, M.C. et al., “SJG-136 (NSC 694501), a novel rationally designed DNA minor groove interstrand crosslinking agent with potent and broad spectrum antitumor activity. Part 2: Efficacy evaluations,” Cancer Res. (2004) 64:6700-6706.
  • Alley, M.C., “Efficacy evaluations of SJG-136 (NSC 694501), a novel pyrrolobenzodiazepine dimer with broad spectrum antitumor activity,” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2002) 43:63.
  • Althius, T. H. and Hess, H. J., “Synthesis and Identification of the Major Metabolites of Prazosin Formed in Dog and Rat,” J. Medicinal Chem. (1977) 20(1):146-148.
  • Antonow, D. et al.,“Parallel synthesis of a novel C2-aryl pyrrolo[2,1-c][1,4]benzodiazepine (PBD) library,” J. Comb. Chem. (2007) 9:437-445.
  • Arima et al., “Studies on Tomaymycin, a New Antibiotic. I. Isolation and Properties of Tomaymycin,” J. Antibiotics (1972) 25:437-444.
  • Arnould, S., “Impact on the cell cycle and involvement of ATM, ATR, chk1 and chk2 kinases in the cytotoxicity of SJG-136, a new pyrrolobenzodiazepine dimer,” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2004) 45:1298, Abstract No. 5618.
  • Arnould, S., “Time-dependent cytotoxicity induced by SJG-136 (NSC 694501): influence of the rate of interstrand cross-link formation on DNA damage signaling,” Mol. Canc. Therap. (2006) 5(6):1602-1609.
  • Banker, G.S. et al., Modern Pharmaceutics, Third edition, Marcel Dekker, New York (1996) 451 and 596.
  • Berge et al., “Pharmaceutical Salts,” J. Pharm. Sci. (1977) 66:1-19.
  • Berry, J. M. et al., “Solid-phase synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines,” Tetrahedron Letters (2000) 41:6171-6174.
  • Bose et al., “New Approaches to Pyrrolo[2,1-c][1,4]benzodiazepines: Synthesis, DNA-binding and cytotoxicity of DC-81,” Tetrahedron, 48, 751-758 (1992).
  • Bose, D.S. et al., “Effect of linker length on DNA-binding affinity, cross-linking efficiency and cytotoxicity of C8 linked pyrrolobenzodiazepine dimers,” J. Chem. Soc. Chem. Commun. (1992) 20:1518-1520.
  • Bose, D.S. et al., “Rational Design of a Highly Efficient Irreversible DNA Interstrand Cross-Linking Agent Based on the Pyrrolobenzodiazepine Ring System,” J Am. Chem. Soc., 114, 4939-4941 (1992).
  • Buhrow, S.A., “LC-MS/MS assay and dog pharmacokinetics of the dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501),” J. Chromat. B: Anal. Tech. Biomed. Life Sci. (2006) 840(1):56-62.
  • Burke, P.J. et al., “Anti-CD70 antibody-drug conjugates containing pyrrolobenzodiazepine dimers demonstrate robust antitumor activity,” AACR National Meeting, Apr. 2012, Chicago, Illinois, Abstract No. 4631.
  • Burke, P.J. et al., “Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system,” Bioorg. Med. Chem. Lett., Apr. 2009, 19(10):2650-2653.
  • Calcutt, M.W., “Determination of chemically reduced pyrrolobenzodiazepine SJG-136 in human plasma by HPLC-MS/MS: application to an anticancer phase I dose escalation study,” J. Mass Spectrom. (2008) 43(1):42-52.
  • Chen, Z. et al., “A novel approach to the synthesis of cytotoxic C2-C3 unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) with conjugated acrylyl C2-substituents,” Biorg. Med. Chem. Lett. (2004) 14:1547-1549.
  • Cheung, A., “Direct liquid chromatography determination of the reactive imine SJG-136 (NSC 694501),” J. Chromat. B: Anal. Techn. Biomed. Life Sci. (2005) 822(1-2):10-20.
  • Cipolla, L., “Pyrrolo[2,1-c][1,4]benzodiazepine as a scaffold for the design and synthesis of anti-tumour drugs,” Anti-Cancer Agents in Medicinal Chemistry (Jan. 2009) 9(1):1-31.
  • Clingen, P.H., “The role of nucleotide excision repair and homologous recomination in the sensitivity of mammalian cells to the minor groove crosslinking pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC694501),” Proceedings of the American Association for Cancer Research Annual Meeting (Jul. 2003) 44:524.
  • Clingen, P.H., “the XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136,” Nucl. Acids Res. (2005) 33(10):3283-3291.
  • Cooper, N. et al., “Synthesis of novel PBDs as anti-tumour agents,” Chem. Commun. (2002) 16:1764-1765.
  • Cooper, N., “Design, Synthesis and Evaluation of Novel C2-Aryl Pyrrolobenzodiazepines as Potential Anticancer Agents,” Thesis submitted to School of Pharmacy, University of London, Dated Oct. 5, 2006.
  • Courtney, S. M. et al., “A new convenient procedure for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepines”, Tetrahedron Letters, vol. 34, No. 33, 5327-28 (1993).
  • Dattolo, G. et al., “Polycondensed nitrogen heterocycles. IX. 5,6-dihydro-7H-pyrrolo[1,2-d][1,4]benzodiazepin-6-one,” J. Heterocyclic. Chem. (1980) 17:701-703.
  • Dong, Q. et al., “Reductive cleavage of TROC groups under neutral conditions with cadmium-lead couple,” Tetrahedron Left. (1995) 36(32):5681-5682.
  • Doronina, S.O. et al., “Development of potent monoclonal antibody auristatin conjugates for cancer therapy,” Nature Biotech. (2003) 21:778-784.
  • Dorwald, F.Z., Side Reactions in Organic Synthesis: a Guide to Successful Synthesis Design, Weinheim: Wiley-VCH Verlag GmbH & Co., KGaA (2005) Preface.
  • Doyle, M., “Response of staphylococcus aureus to subinhibitory concentrations of a sequence-selective, DNA minor groove cross-linking pyrrolobenzodiazepine dimer,” J. Antimicrob. Chemo., Aug. 2009, 64(5):949-959.
  • Dupont, C. et al., “Synthesis of rhazinilam analogue acting as an inhibitor of tubulin assembly,” Tetrahedron Lett. (2000) 41:5853-5856.
  • Farmer, J.D. et al., “Synthesis and DNA crosslinking ability of a dimeric anthramycin analog,” Tetrahedron Letters (1988) 29(40):5105-5108, Abstract only.
  • Farmer, J.D. et al., “DNA binding properties of a new class of linked anthramycin analogs,”, Chemical Abstracts, Abstract No. 239940r, vol. 114, No. 25, 25 899-903 (1991).
  • Fey, T. et al., “Silica-supported TEMPO catalyst: synthesis and application in the anelli oxidation of alcohols,” J. Org. Chem. (2001) 66:8154-8159.
  • Firsching, A. et al., “Antiproliferative and angiostatic activity of suramin analogues,” Cancer Res. (1995) 55:4957-4961.
  • Foloppe, M.P. et al., “DNA-binding properties of pyrrolo[2,1-c][1,4]benzodiazephine N10-C11 amidines,” Eur. J Med. Chem., 31, 407-410 (1996).
  • Fujisawa Pharmaceutical Co., Ltd., Abstract No. 139983k, “Benzodiazepine derivatives”, Chemical Abstracts, vol. 99, No. 17, 603 (1983).
  • Fujisawa Pharmaceutical Co., Ltd., Abstract No. 72145x, “Benzodiazepine derivatives”, Chemical Abstracts, vol. 98, No. 9, 638 (1983).
  • Fukuyama, T. et al., “Total Synthesis of (+)-Porothramycin B,” Tetrahedron Letters, vol. 34, 16, 2577-2580 (1993).
  • Gallmeier, E., “Targeted disruption of FANCC and FANCG in human cancer provides a preclinical model for specific therapeutic options,” Gastroenterology (2006) 130(7):2145-2154.
  • Gavezzotti, a., “Are crystal structures predictable?” Acc. Chem. Res. (1994) 27:309-314.
  • Greene, T.W. and Wuts, P.G.M., Protective Groups in Organic Synthesis, John Wiley & Sons, 2nd ed., Ch 7, 315-345 (1991).
  • Greene, T.W. et al., Protective Groups in Organic Synthesis, John Wiley & Sons (1999) 3rd Edition, 23-200, 503-549, 633-647.
  • Gregson, S. et al., “Synthesis of a novel C2/C2'-exo unsaturated pyrrolobenzodiazepine cross-linking agent with remarkable DNA binding affinity and cytotoxicity,” Chemical Communications, 797-798 (1999).
  • Gregson, S.J. et al., “Effect of C2/C3-endo unsaturation on the cytotoxicity and DNA-binding reactivity of pyrrolo-[2,1-c][1,4]-benzodiazepines,” Bioorg. Med. Chem. Lett. (2000) 10(16):1849-1851.
  • Gregson, S.J. et al., “Linker length modulates DNA cross-linking reactivity and cytotoxic potency of C8/C8' ether-linked C2-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers,” J. Med. Chem. (2004) 11611174.
  • Gregson, S.J. et al., “Synthesis of the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1- c][1,4]benzodiazepine dimer,” Biorg. Med. Chem. Lett. (2001) 11:2859-2862.
  • Gregson, S.J. et al., “Synthesis of the first examples of A-C8/C-C2 amide-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers,” Biorg. Med. Chem. Lett. (2003) 13:2277-2280.
  • Gregson, S.J. et al., “Design, Synthesis and Evaluation of a Novel Pyrrolobenzodiazepine DNA-Interactive Agent with Highly Efficient Cross-Linking Ability and Potent Cytotoxicity”, J. Med. Chem., 44: 737-748 (2001).
  • Gregson, S.J. et al., “Effect of C2-exo Unsaturation on the Cytotoxicity and DNA-Binding Reactivity of Pyrrolo[2,1-c]1,4]benzodiazepines”, Bioorganic & Medicinal Chemistry Letters, 10: 1845-1847 (2000).
  • Guichard, S.M., “Influence of P-glycoprotein expression on in vitro cytotoxicity and in vivo antitumour activity of the novel pyrrolobenzodiazepine dimer SJG-136,” Eur. J. Cancer (2005) 41(12):1811-1818.
  • Guiotto, A. et al., “Synthesis of novel C7-aryl substituted pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) via Pro-N10-trot protection and suzuki coupling,” Bioorganic & Medicinal Chemistry Letters, 8, No. 21, 3017-3018 (1998).
  • Hadjivassileva, T., “Antibacterial activity of pyrrolobenzodiazepine dimers, a novel group of DNA-binding compounds,” Abstracts of the Interscience Conference on Antimicrobial Agents and Chemotherapy (Oct.-Nov. 2004) 44:202.
  • Hadjivassileva, T., “Interactions of pyrrolobenzodiazepine dimers and duplex DNA from methicillin-resistant staphylococcus aureus,” Int. J. Antimicrob. Agents (2007) 29(6):672-678.
  • Hadjivassileva, T., “Pyrrolobenzodiazepine dimers: novel sequence-selective, DNA-interactive, cross-linking agents with activity against Gram-positive bacteria,” J. Antimicrob. Chemo. (2005) 56(3):513-518.
  • Hamaguchi, A., “DNA cross-linking and in vivo antitumour activity of the extended pyrrolo[2,1-c][1,4]benzodiazepine dimer SG2057 (DRG-16),” EJC Supplements (Nov. 2006) 4(12):96.
  • Hara et al., “DC 102, a new glycosidic pyrrolo(1,4)benzodiazepine antibiotic produced by streptomyces sp.”, J. Antibiotics, 41, 702-704 (1988).
  • Hartley, J.A. et al., “SJG-136 (NSC 694501), a novel rationally designed DNA minor groove interstrand crosslinking agent with potent and broad spectrum antitumor activity. Part 1: Cellular pharmacology, in vitro and initial in vivo antitumor activity,” Cancer Res. (2004) 64:6693-6699.
  • Hartley, J.A., “In vitro antitumor activity and in vivo DNA interstrand crosslinking by the novel pyrrolobenzodiazepine dimer SJG-136 (NSC 694501),” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2002) 43:489.
  • Hartley, J.A., “SJG-136 (NSC-D694501)—a novel DNA sequence specific minor groove crosslinking agent with significant antitumor activity,” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2000) 41:425.
  • Hartley, J.A. et al., “SG2285, a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity,” Cancer Res., Sep. 2010, 70(17):6849-6858.
  • Hochhauser, D., “Phase I study of sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors,” Clin. Cancer Res., Mar. 2009, 15(6):2140-2147.
  • Hochlowski, J. et al., “Abbeymycin, a new anthramycin-type antibiotic produced by a streptomycete,” J. Antibiotics, 40, 145-148 (1987).
  • Howard, P.W. et al., “Design, synthesis and biological evaluation of ZC-423, a novel C2-aryl substituted pyrrolobenzodiazepine (PBD) dimer,” Clinical Cancer Research (2005) 11(23):9015S-9016S (A205).
  • Howard, P.W. et al., “Synthesis of a novel C2/C2'-aryl-substituted pyrrolo[2,1-c][1,4]benzodiazepine dimer prodrug with improved water solubility and reduced DNA reaction rate,” Bioorg. Med. Chem., Sep. 2009, in Press, 4 pages now: Sep. 2009, 19:6463-6466.
  • Howard, P.W. et al., “The design, synthesis and biological evaluation of a set of C2-aryl substituted pyrrolo[2,1-c][1,4]benzodiazepine dimers,” EJC Supplements (2006) 4(12):95—Poster Abstract 301.
  • Howard, P.W., “Design, synthesis and biological evaluation of novel C2-aryl-substituted pyrrolo[2,1-c][1,4]benzodiazepine monomers,” Proceedings of the American Association for Cancer Research Annual Meeting (Apr. 2006) 47:132.
  • Hurley, L. and Needham-Vandevanter, D., “Covalent Binding of Antitumor Antibiotics in the Minor Groove of DNA. Mechanism of Action of CC-1065 and the Pyrrolo(1,4)benzodiazepines,” Acc. Chem. Res., 19, 230-237 (1986).
  • Itoh et al., “Sibanomicin, a new pyrrolo(1,4)benzodiazepine antitumor antibiotic produced by a micromonospora sp.” J. Antibiotics, 41, 1281-1284 (1988).
  • Janjigian, Y.Y., “A phase I trial of SJG-136 (NSC#694501) in advanced solid tumors,” Cancer Chemotherapy and Pharmacology , Aug. 2009, 65(5):833-838.
  • Jeffrey, S.C., “Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates,” J. Med. Chem. (2005) 48(5):1344-1358.
  • Jia, L., “Interspecies differences in pharmacokinetics and time-dissociated toxicokinetics of SJG-136,” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2004) 45:487.
  • Jia, L., “Use of the comet assay as a surrogate biomarker for the in vivo measurement of DNA damage to lymphocytes,” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2004) 45:452-453.
  • Jordan, V.C., “Tamoxifen: a most unlikely pioneering medicine, ” Nature Reviews: Drug Discovery (2003) 2:205-213.
  • Kamal et al., “Synthesis and DNA-binding affinity of A-C8/C-C2 alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers” Biorg. Med. Chem. Left. (2003) 13(22):3955-3958.
  • Kamal, a. et al., “Design, synthesis and evaluation of new noncross-linking pyrrolobenzodiazepine dimers with efficient DNA binding ability and potent antitumor activity,” J. Med. Chem. (2002) 45:4679-4688.
  • Kamal, A., “Development of pyrrolo[2,1-c][1,4]benzodiazepine beta-galactoside prodrugs for selective therapy of cancer by ADEPT and PMT,” Chemmedchem (2008) 3:794-802.
  • Kamal, A., “Remarkable DNA binding affinity and potential anticancer activity of pyrrolo[2,1-c][1,4]benzodiazepine-naphthalamide conjugates linked through piperazine side-armed alkane spacers,” Bioorg. Med. Chem. (2008) 16(15):7218-7224.
  • Kamal, A., “Remarkable enhancement in the DNA-binding ability of C2-fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines and their anticancer potential,” Bioorg. Med. Chem., Jan. 2009, 17(4):1557-1572.
  • Kamal, A., “Synthesis of fluorinated analogues of SJG-136 and their DNA-binding potential,” Bioorg. Med. Chem. Lett (2004) 14(22):5699-5702.
  • Kamal, A., et al., “An Efficient Synthesis of Pyrrolo[2,1-c][1,4] Benzodiazepine Antibiotics via Reductive Cyclization,” Bioorg. Med. Chem. Ltrs, 7, No. 14, 1825-1828 (1997).
  • Kamal, A., et al., “Synthesis of Pyrrolo [2,1-c][1,4]-Benzodiazepene Antibiotics: Oxidation of Cyclic Secondary Amine with TPAP”, Tetrahedron, v. 53, No. 9, 3223-3230 (1997).
  • Kamal, et al., “Synthesis of pyrrolo[2,1-c][1,4]benzodiazepines via reductive cyclization of w-azido carbonyl compounds by TMSI: an efficient preparation of antibiotic DC-81 and its dimers,” Biorg. Med. Chem. Left. (2000) 10:2311-2313.
  • Kaneko, T. et al., “Bicyclic and tricyclic analogues of anthramycin,” J. Med. Chem. (1985) 28:388-392.
  • Kang, G.-D. et al., “Synthesis of a novel C2-aryl substituted 1,2-unsaturated pyrrolobenzodiazepine,” Chem. Commun. (2003) 1688-1689.
  • Kohn, K., “Anthramycin,” Antibiotics III, Springer-Verlag, NY, 3-11 (1975).
  • Konishi, M. et al., “Chicamycin, a new antitumor antibiotic II. Structure determination of chicamycins A and B,” J Antibiotics, 37, 200-206 (1984).
  • Kunimoto et al., “Mazethramycin, a new member of anthramycin group antibiotics,” J. Antibiotics, 33, 665-667 (1980).
  • Langley, D.R. and Thurston, D.E., “A versatile and efficient synthesis of carbinolamine-containing pyrrolo[1,4]benzodiazepines via the cyclization of N-92-aminobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thioacetals: total synthesis of prothracarcin,” J. Org. Chem., 52, 91-97 (1987).
  • Langlois, N. et al., “Synthesis and cytotoxicity on sensitive and doxorubicin-resistant cell lines of new pyrrolo[2,1-c][1,4]benzodiazepines related to anthramycin,” J. Med. Chem. (2001) 44:3754-3757.
  • Leber, J.D. et al., “A revised structure for sibiromycin,” J Am. Chem. Soc., 110, 2992-2993 (1988).
  • Leimgruber, W. et al., “Isolation and characterization of anthramycin, a new antitumor antibiotic,” J Am. Chem. Soc., 87, 5791-5793 (1965).
  • Leimgruber, W. et al., “The structure of anthramycin,” J Am. Chem. Soc., 87, 5793-5795 (1965).
  • Leimgruber, W. et al., “Total synthesis of anthramycin,” J Am. Chem. Soc., 90, 5641-5643 (1968).
  • Lown et al., “Molecular Mechanism of Binding of Pyrrolo(1,4)benzodiazepine antitumour agents to deoxyribonucleic acid—anthramycin and tomaymycin,” Biochem. Pharmacol. (1979), 28 (13), 2017-2026.
  • Marin, D., “Voltammetric studies of the interaction of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) monomers and dimers with DNA,” J. Electroanal. Chem. (2006) 593(1-2):241-246.
  • Martin, C. et al., “Sequence-selective interaction of the minor-groove interstrand cross-linking agent SJG-136 with naked and cellular DNA: footprinting and enzyme inhibition studies,” Biochem. (2005) 44(11):4135-4147.
  • Mori, M. et al., “Total syntheses of prothracarcin and tomaymycin by use of palladium catalyzed carbonylation,” Tetrahedron (1986) 42(14):3793-3806.
  • Mountzouris, J.A. et al., “Comparison of a DSB-120 DNA interstrand cross-linked adduct with the corresponding bis-Tomamycin adduct,” J. Med. Chem. (1994) 37:3132-3140.
  • Nagasaka, T. and Koseki, Y, “Stereoselective Synthesis of Tilivalline,” Journal of Organic Chemistry, vol. 63, No. 20, 6797-6801 (1998).
  • Nagasaka, T. et al., “Stereoselective Synthesis of Tilivalline,” Tetrahedron Letters, 30:14, 1871-1872 (1989).
  • Narayanaswamy, M., “A novel HPLC/MS assay to measure DNA interstrand cross-linking efficacy in oligonucleotides of varying sequence,” EJC Supplements (Nov. 2006) 4(12):92-93.
  • Narayanaswamy, M., “An assay combinding high-performance liquid chromatography and mass spectrometry to measure DNA interstrand cross-linking efficiency in oligonucleotides of varying sequences,” Anal. Biochem. (2008) 374(1):173-181.
  • Narayanaswamy, M., “Use of HPLC-MS to characterize novel mono and intrastrand cross-linked DNA adducts formed by the sequence-selective DNA-interactive agent SJG-136,” Proceedings of the American Association for Cancer Research Annual Meeting (Apr. 2007) 48:760-761.
  • Narukawa, Y., “General and efficient synthesis of 2-alkylcarbapenems synthesis of dethia carba analogs of clinically useful carbapenems via palladium-catalyzed cross-coupling reaction,” Tetrahedron (1997) 53:539-556.
  • O'Neil, Chemical Abstract No. 171573p, “The synthesis of Functionalized Pyrrolo-[2,1-c][1,4]-Benzodiazepines”, Chemical Abstracts, vol. 126, No. 13, 618 (1997).
  • O'Neil, I.A., et al., “The Synthesis of Functionalized Pyrrolo-[2,1-c][1,4]-Benzodiazepines,” Synlett, 75-78 (1997).
  • O'Neil, I.A. et al., “DPPE: A Convenient Replacement for Triphenylphosphine in the Staudinger and Mitsunobu Reactions”, Tetrahedron Letters, vol. 39, No. 42, 7787-7790 (1998).
  • Pepper, C., “Fludarabine-mediated suppression of the excision repair enzyme ERCC1 contributes to the cytotoxic synergy with the DNA minor groove crosslinking agent SJG-136 (NSC 694501) in chronic lymphocytic leukaemia cells,” Br. J. Cancer (2007) 97(2):253-259.
  • Pepper, C.J. et al., “The novel sequence-specific DNA cross-linking agent SJG-136 (NSC 694501) has potent and selective in vitro cytotoxicity in human B-cell chronic lymphocytic leukemia cells with evidence of a p53-independent mechanism of cell kill,” Cancer Res. (2004) 64:6750-6755.
  • Puzanov, I., “Phase I and pharmacokinetic trial of SJG-136 administered on a daily x5 schedule,” EJC Supplements (Nov. 2006) 4(12):93.
  • Quintas-Cardama, A., “Sequencing of subcloned PCR products facilitates earlier detetction of BCR-ABL1 and other mutants compared to direct sequencing of the ABL1 kinase domain,” Leukemia (2008) 22(4):877-878.
  • Rahman, K.M., “Effect of microwave irradiation on covalent ligand-DNA interactions,” Chem. Commun. (Cambridge, UK), Apr. 2009, 20:2875-2877.
  • Rahman, K.M., “Rules of DNA adduct formation for pyrrolobenzodiazepine (PBD) dimers,” Proceedings of the American Association for Cancer Research Annual Meeting (Apr. 2010) 51:851.
  • Rahman, K.M., “The pyrrolobenzodiazepine dimer SJG-136 forms sequence-dependent intrastrand DNA cross-lins and monoalkylated adducts in addition to interstrand cross-links,” J. Am. Chem. Soc., Sep. 2009, 131(38):13756-13766.
  • Reid, J.M., “LC-MS/MS assay and rat pharmacokinetics and metabolism of the dimeric pyrrolobenzodiazepine SJG-136,” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2004) 45:1248.
  • Rich, I.N., “Validation and development of a predictive paradigm for hemotoxicology using a multifunctional bioluminescence colony-forming proliferation assay,” Toxicological Sci. (2005) 87(2):427-441.
  • Sagnou, M.J. et al., “Design and Synthesis of Novel Pyrrolobenzodiazepine (PDB) Prodrugs for Adept and GDEPT,” Bioorganic & Medicinal Chemistry Letters, 10, 2083-2086 (2000).
  • Schweikart, K., “In vitro myelosuppression of SJG-136, a pyrrolobenzodiazepine dimer: comparison to bizelesin,” Proceedings of the American Association for Cancer Research Annual Meeting (Mar. 2004) 45:486.
  • Shimizu, K et al., “Prothracarcin, a Novel Antitumor Antibiotic,” J. Antibiotics, 35, 972-978 (1982).
  • Smellie, M. et al., “Cellular pharmacology of novel C8-linked anthramycin-based sequence-selective DNA minor groove cross-linking agents,” Br. J. Cancer (1994) 70:48-53.
  • Smellie, M. et al., “Sequence selective recognition of duplex DNA through covalent interstrand cross-linking,” Biochem. (2003) 42:8232-8239.
  • Souillac, P. et al., “Chracterization of delivery systems, differential scanning calorimetry,” Encyclopedia of Controlled Drug Delivery (1999) 212-227 (pp. 217-218).
  • Suggitt, M., “The hollow fibre model—facilitating anti-cancer pre-clinical pharmacodynamics and improving animal welfare,” Int. J. Oncol. (2006) 29(6):1493-1499.
  • Suggs, J.W. et al., “Synthesis and structure of anthramycin analogs via hydride reduction of dilactams,” Tetrahedron Letters, 26, No. 40, 4871-4874 (1985).
  • Sutherland, M.S.K. et al., “SGN-CD33A: a novel CD33-directed antibody-drug conjugate, utilizing pyrrolobenzodiazepine dimers, demonstrates preclinical anti-tumor activity against multi-drug resistant human AML,” American Society of Hematology (Dec. 8-12, 2012) Atlanta, Georgia, Abstract No. 3589.
  • Takeuchi, T. et al., “Neothramycins A and B, New Antitumor Antibiotics,” J. Antibiotics, 29, 93-96 (1976).
  • Tercel, M. et al., “Unsymmetrical DNA cross-linking agents: combination of the CBI and PBD pharmacophores,” J. Med. Chem. (2003) 46:2132-2151.
  • Thurston, D. E., “Advances in the study of Pyrrolo[2,1-c][1,4] benzodiazepine (PBD) Antitumour Antibiotics”, Molecular Aspects of Anticancer Drug-DNA Interaction, Neidle, S. and Waring, M.J., Eds.; Macmillan Press Ltd, 1:54-88 (1993).
  • Thurston, D.E. and Bose, D.S., “Synthesis of DNA-Interactive Pyrrolo[2,1-c][1,4]benzodiazepines,” Chem. Rev., 94:433-465 (1994).
  • Thurston, D.E. and Thompson, A.S., “The molecular recognition of DNA,” Chem. Brit., 26, 767-772 (1990).
  • Thurston, D.E. et al., “Effect of A-ring modifications on the DNA-binding behavior and cytotoxicity of pyrrolo[2,1-c][1,4]benzodiazepines”, Journal of Medicinal Chemistry, 42:1951-1964 (1999).
  • Thurston, D.E. et al., “Synthesis of Sequence-selective C8-linked Pyrrolo [2,1-c][1,4] Benzodiazepine DNA Interstrand Cross-linking Agent,” J. Org. Chem., 61:8141-8147 (1996).
  • Thurston, D.E. et al., “Synthesis of a novel GC-specific covalent-binding DNA affinity-cleavage agent based on pyrrolobenzodiazepines (PBDs),” Chemical Communications, 563-565 (1996).
  • Thurston, D.E., “Nucleic acid targeting: therapeutic strategies for the 21st century,” Brit. J. Cancer (1999) 80(1):65-85.
  • Tiberghien, A.C. et al., “Application of the stille coupling reaction to the synthesis of C2-substituted endo-exo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs),” Biorg. Med. Chem. Lett. (2004) 14:5041-5044.
  • Tiberghien, A.C., “Application of the stille coupling reaction to the synthesis of C2-substituted endo-exo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs),” Bioorg. Med. Chem. Lett. (2008) 18(6):2073-2077.
  • Tozuka et al., “Studies on tomaymycin. II. Total synthesis of the antitumor antibiotics, E-and Z-tomaymycins,” J. Antibiotics (Tokyo) (1983) 36:276-282.
  • Tsunakawa, M. et al., “Porothramycin, a new antibiotic of the anthramycin group: Production, isolation, structure and biological activity,” J. Antibiotics, 41:1366-1373 (1988).
  • Umezawa, H. et al., Chemical Abstract No. 4427a, “Mazethramycins” Chemical Abstracts, vol. 90, No. 1, 428 (1979).
  • Vippagunta, S.R. et al., “Crystalline solids,” Adv. Drug Delivery Rev. (2001) 48:3-26.
  • Wang, J.H., “Determination of antitumor agent AJG-136 in human serum by HPLC with tandem mass spectrometric detection (HPLC-MS/MS),” Abstracts of Papers American Chemical Society (Mar. 13, 2005) 229(1):U119.
  • Weidner-Wells, M.A. et al., “Photochemical approach to the synthesis of the pyrrolo[1,4]benzodiazepine antibiotics,” J. Org. Chem. (1989) 54:5746-5758.
  • Wells, G. et al., “Design, synthesis and biophysical and biological evaluation of a series of pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugates,” J. Med. Chem. (2006) 49:5442-5461.
  • Wilkinson, G.P., “Pharmacokinetics and intracellular pharmacological characteristics of the novel pyrrolobenzodiazepine (PBD) dimer SJG-136,” Proceedings of the American Association for Cancer Research Annual Meeting (Jul. 2003) 44:320.
  • Wilkinson, G.P., “Pharmacokinetics, metabolism and glutathione reactivity of SJG-136,” Br. J. Cancer (2003) 88(Supp. 1):S29.
  • Wilkinson, G.P., “Preliminary pharmacokinetic and bioanalytical studies of SJG-136 (NSC 694501), a sequence-selective pyrrolobenzodiazepine dimer DNA-cross-linking agent,” Investigational New Drugs (2004) 22(3):231-240.
  • Wilson, S.C. et al., “Design and Synthesis of a Novel Epoxide-Containing Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) via a New Cyclization Procedure,” Tetrahedron Letters, 36, No. 35, 6333-6336 (1995).
  • Wilson, S.C. et al., “Design, Synthesis, and Evaluation of a Novel Sequence-Selective Epoxide-Containing DNA Cross-Linking Agent Based on the Pyrrolo[2,1-c][1,4]benzodiazepine System”, J. Med. Chem. 42:4028-4041 (1999).
  • Wolff, M.E., Burger's Medicinal Chemistry, 4th Edition, Part I, Wiley: New York (1979) 336-337.
  • Wolff, M.E., Burger's Medicinal Chemistry, 5th Edition, Part I, John Wiley & Sons (1995) 975-977.
  • Workman, P. et al., “United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) guidelines for the welfare of animals in experimental neoplasia (second edition),” Br. J. Cancer (1998) 77(1):1-10.
  • Xie, G. et al., “Bisindolylmaleimides linked to DNA minor groove binding lexitropsins: synthesis, inhibitory activity against topoisomerasel, and biological evaluation,” J. Med. Chem. (1996) 39:1049-1055.
  • Antonow, D. et al., “Synthesis of DNA-Interactive Pyrrolo [2,1-c][1,4] benzodiazepines (PBDs)” Chemical Reviews, 2011, 111(4):2815-2864.
  • Shimizu et al., “Prothracarcin, a novel antitumor antibiotic,” The Journal of Antibiotics (1982) 29, 2492-2503.
  • Dubowchik et al., “Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific in Vitro Anticancer Activity,” Bioconjugate Chem. (2002) 13, 855-869.
  • International Search Report and Written Opinion for Application No. PCT/US2011/032664 dated Aug. 19, 2011 (13 pages).
  • International Search Report and Written Opinion for Application No. PCT/EP2012/068506 dated Dec. 20, 2012 (9 pages).
Patent History
Patent number: 9266894
Type: Grant
Filed: Sep 20, 2012
Date of Patent: Feb 23, 2016
Patent Publication Number: 20150158869
Assignee: MEDIMMUNE LIMITED (Cambridge)
Inventor: Philip Wilson Howard (London)
Primary Examiner: Taofiq A Solola
Application Number: 14/346,006
Classifications
Current U.S. Class: Tricyclo Ring System Which Contains The Hetero Ring As One Of The Cyclos (540/586)
International Classification: C07D 487/04 (20060101); C07D 519/00 (20060101); A61K 47/48 (20060101); C07K 5/062 (20060101); C07K 5/065 (20060101); C07K 5/078 (20060101);