Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
  • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
  • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

• the present invention relates to pyrrolobenzodiazepines (PBDs), and their inclusion in targeted conjugates.
• PBDs of the present invention are dimer where the group linking the two PBD moieties comprises a benzene or pyridine ring substituted by a carboxy or ester group.
• PBDs pyrrolobenzodiazepines
• Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et ai., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58- I SO 487; Thurston, et ai., Chem. Brit., 26, 767-772 (1990); Bose, et ai., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et ai., J.
• PBDs are of the general structure:
• WO 2007/085930 describes the preparation of dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody.
• the linker is present in the bridge linking the monomer PBD units of the dimer.
• Dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody are described in WO 201 1/130598.
• the linker in these compounds is attached to one of the available N10 positions, and are generally cleaved by action of an enzyme on the linker group. More recently, the warhead:
• WO 2014/057074 discloses:
• WO2015/052322 discloses
• the present invention provides PBD dimers where the group linking the two PBD moieties comprises a benzene or pyridine ring substituted by a carboxy or ester group, as drug linkers, and conjugates made from these drug linkers, as well as the released drugs.
• the presence of the carboxy group or ester group should reduce the activity of the drug linker and warhead compared to the analogous drug linkers and warheads being unsubstituted.
• the conjugate with an ester group shows good activity in vitro and in vivo of a level which is comparable to the analogous unsubstituted conjugates.
• the carboxy group may limit the ability of the warhead to enter the cell when not conjugated.
• a first aspect of the present invention comprises a compound with the formula I:
• R" is a group of formula II:
• n and m are independently selected from 1 , 2 and 3;
• R° is selected from the group consisting of H, methyl, ethyl, iso-propyl and benzyl;
• Q c is selected from N and CH;
• Y and Y’ are selected from O, S, or NH;
• R 2 is selected from the group consisting of:
• each of R 11 , R 12 and R 13 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 R 2 group is no more than 5;
• R 15a and R 15b are 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
• R 14 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,
• R 16a and R 16b are independently selected from H, F, Ci -4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from Ci -4 alkyl amido and Ci -4 alkyl ester; or, when one of R 16a and R 16b is H, the other is selected from nitrile and a Ci -4 alkyl ester;
• R 2 is selected from the group consisting of:
• R 25a and R 25b are 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
• R 24 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;
• R 2’ is H or , where R 26a and R 26b are independently selected from H, F, Ci -4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from Ci -4 alkyl amido and Ci -4 alkyl ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a Ci -4 alkyl ester;
• R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, nitro, MesSn and halo;
• R and R’ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
• R 7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR’, nitro, MesSn and halo;
• R 6 , R 7 , R 9 are selected from the same groups as R 6 , R 7 and R 9 respectively;
• R 11a is selected from OH, OR A , where R A is Ci -4 alkyl, and SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
• R 30 is H, and R 31 is OH, OR A , where R A is Ci -4 alkyl;
• R 30 and R 31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound;
• R 30 is H and R 31 is SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, wherein if R 11a and R 31 are SO z M, M may represent a divalent pharmaceutically acceptable cation
• R L is a linker for connection to a cell binding agent, which is selected from:
• Q x is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue
• X is:
• G L is a linker for connecting to a Ligand Unit; and (iiib): where R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group;
• a second aspect of the present invention provides a method of making a compound of the first aspect of the invention, comprising at least one of the method steps set out below.
• the present invention relates to Conjugates comprising dimers of PBDs linked to a Ligand unit/targeting agent, wherein the PBD dimer is a derivative of formula I, or a pharmaceutically acceptable salt or solvate thereof.
• the Conjugates having the following formula IV:
• L is a Ligand unit (i.e., a targeting agent)
• D L is a Drug Linker unit that is a PBD dimer of formula III:
• R 2 , R 6 , R 7 , R 9 , R 11a , Y, R”, Y’, R 2’ , R 6’ , R 7’ , R 9’ , R 30 and R 31 are as defined in the first aspect of the invention;
• R LL is a linker for connection to a cell binding agent, which is selected from:
• G LL is a linker connected to a Ligand
• p is an integer of from 1 to 20.
• the Conjugates comprise a Ligand unit covalently linked to at least one Drug unit by a Linker unit.
• the Ligand unit 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.
• 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 drug loading is represented by p, the number of drug molecules per Ligand unit (e.g., an antibody). Drug loading may range from 1 to 20 Drug units (D) per Ligand unit (e.g., Ab or mAb).
• D Drug units
• p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20.
• a fourth aspect of the present invention provides the use of a conjugate of the third aspect of the invention in the manufacture of a medicament for treating a proliferative disease.
• the fourth aspect also provides a conjugate of the third aspect of the invention for use in the treatment of a proliferative disease.
• Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + .
• Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca 2+ and Mg 2+ .
• Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH 4 + ) and substituted ammonium ions (e.g. NH 3 R + , NhhF ⁇ , NHR3 + , NR 4 + ).
• Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine,
• phenylbenzylamine choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
• amino acids such as lysine and arginine.
• An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
• substituted refers to a parent group which bears one or more substituents.
• substituted 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.
• substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
• C-i- 12 alkyl The term“C 1-12 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
• the term“Ci -n 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 n carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
• the term“alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
• saturated alkyl groups include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), propyl (C3), butyl (C 4 ), pentyl (C5), hexyl (OQ) and heptyl (C7).
• saturated linear alkyl groups include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), n-butyl (C 4 ), n-pentyl (amyl) (C 5 ), n-hexyl (O Q ) and n-heptyl (C 7 ).
• saturated branched alkyl groups include iso-propyl (C 3 ), iso-butyl (C 4 ), sec-butyl (C 4 ), tert-butyl (C 4 ), iso-pentyl (C 5 ), and neo-pentyl (C 5 ).
• C 2-12 Alkenyl The term“C 2-12 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
• C 2-12 alkynyl The term“C 2-12 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
• C3-12 cycloalkyl refers 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.
• cycloalkyl groups include, but are not limited to, those derived from:
• norcarane (C7) norpinane (C7), norbornane (C7).
• C3-20 heterocyclyl refers 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.
• each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
• the prefixes e.g. C3-20, C3-7, C5-6, etc.
• the term“Cs-eheterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
• monocyclic heterocyclyl groups include, but are not limited to, those derived from:
• N 2 imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline
• N 1 O 1 tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ),
• dihydroisoxazole C5
• morpholine Ce
• tetrahydrooxazine Ce
• dihydrooxazine Ce
• oxazine Ce
• N1S1 thiazoline (C5), thiazolidine (C5), thiomorpholine (Ce) ' ,
• O1S1 oxathiole (C5) and oxathiane (thioxane) (Ce) ' , and,
• N1O1S1 oxathiazine (O Q ).
• 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 (Ce), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose,
• C5-20 aryl refers 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.
• each ring has from 5 to 7 ring atoms.
• the prefixes e.g. C3-20, C5-7, C5-6, C5-10, etc.
• the term“Cs-e 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”.
• carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (Ce), naphthalene (C10), azulene (Cio), anthracene (CM), phenanthrene (CM), naphthacene (Cis), and pyrene (OIQ).
• aryl groups which comprise fused rings include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1 H-indene) (Cg), indene (Cg), isoindene (Cg), tetraline (1 ,2,3,4-tetrahydronaphthalene (Cio),
• indane e.g. 2,3-dihydro-1 H-indene
• indene Cg
• isoindene Cg
• tetraline (1 ,2,3,4-tetrahydronaphthalene (Cio)
• acenaphthene C12
• fluorene C13
• phenalene C13
• acephenanthrene C15
• OIQ aceanthrene
• the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”.
• monocyclic heteroaryl groups include, but are not limited to, those derived from:
• N1O1 oxazole (C5), isoxazole (C5), isoxazine (Ce);
• N2O1 oxadiazole (furazan) (C 5 );
• N3O1 oxatriazole (C5);
• N1S1 thiazole (C5), isothiazole (C5);
• N2 imidazole (1 ,3-diazole) (C5), pyrazole (1 ,2-diazole) (C5), pyridazine (1 ,2-diazine) (Ce), pyrimidine (1 ,3-diazine) (Ce) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (Ce);
• N3 triazole (C5), triazine (Ce); and,
• heteroaryl which comprise fused rings, include, but are not limited to:
• Cg (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N 4 ) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N 3 ), benzothiofuran (Si), benzothiazole (N1S1), benzothiadiazole (N2S);
• Cio (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 (N 4 );
• C11 (with 2 fused rings) derived from benzodiazepine (N2); Ci 3 (with 3 fused rings) derived from carbazole (Ni), dibenzofuran (Oi), dibenzothiophene (Si), carboline (N 2 ), perimidine (N 2 ), pyridoindole (N 2 ); and,
• Ci 4 (with 3 fused rings) derived from acridine (N 1 ), xanthene (O 1 ), thioxanthene (Si), oxanthrene (O 2 ), phenoxathiin (O 1 S 1 ), phenazine (N 2 ), phenoxazine (N 1 O 1 ), phenothiazine (N 1 S 1 ), thianthrene (S 2 ), phenanthridine (N 1 ), phenanthroline (N 2 ), phenazine (N 2 ).
• Halo -F, -Cl, -Br, and -I.
• Ether -OR, wherein R is an ether substituent, for example, a C 1-7 alkyl group (also referred to as a Ci- 7 alkoxy group, discussed below), a C 3-20 heterocyclyl group (also referred to as a C 3-20 heterocyclyloxy group), or a Cs- 2 o aryl group (also referred to as a Cs- 2 o aryloxy group), preferably a C ⁇ alkyl group.
• R is an ether substituent, for example, a C 1-7 alkyl group (also referred to as a Ci- 7 alkoxy group, discussed below), a C 3-20 heterocyclyl group (also referred to as a C 3-20 heterocyclyloxy group), or a Cs- 2 o aryl group (also referred to as a Cs- 2 o aryloxy group), preferably a C ⁇ alkyl group.
• Alkoxy -OR, wherein R is an alkyl group, for example, a C 1-7 alkyl group.
• C 1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n- propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu)
• Acetal -CH(OR 1 )(OR 2 ), wherein R 1 and R 2 are independently acetal substituents, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a Cs- 2 o aryl group, preferably a C 1-7 alkyl group, or, in the case of a“cyclic” acetal group, R 1 and R 2 , taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
• acetal groups include, but are not limited to, -CH(OMe) 2 , -CH(OEt) 2 , and -CH(OMe)(OEt).
• Hemiacetal -CH(OH)(OR 1 ), wherein R 1 is a hemiacetal substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a Cs- 2 o aryl group, preferably a C 1-7 alkyl group.
• hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and - CH(OH)(OEt).
• Ketal -CR(OR 1 )(OR 2 ), where R 1 and R 2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a Cw alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably a C alkyl group.
• 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).
• R 1 is as defined for hemiacetals
• R is a hemiketal substituent other than hydrogen, for example, a Cw alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably a Cw alkyl group.
• hemiacetal groups include, but are not limited to, -C(Me)(OH)(OMe), -C(Et)(OH)(OMe), -C(Me)(OH)(OEt), and
• Imino (imine): NR, wherein R is an imino substituent, for example, hydrogen, Cw alkyl group, a C 3-20 heterocyclyl group, or a Cs- 2 o aryl group, preferably hydrogen or a Cw alkyl group.
• R is an acyl substituent, for example, a Cw alkyl group (also referred to as Ci- 7 alkylacyl or Ci- 7 alkanoyl), a C 3-20 heterocyclyl group (also referred to as C 3-20 heterocyclylacyl), or a Cs- 2 o aryl group (also referred to as Cs- 2 o arylacyl), preferably a C 1-7 alkyl group.
• a Cw alkyl group also referred to as Ci- 7 alkylacyl or Ci- 7 alkanoyl
• C 3-20 heterocyclylacyl also referred to as C 3-20 heterocyclylacyl
• Cs- 2 o aryl group also referred to as Cs- 2 o arylacyl
• Carboxy (carboxylic acid): -C( 0)0H.
• Thionocarboxy (thionocarboxylic acid): -C( S)OH.
• Imidic acid: -C( NH)OH.
• acyloxy (reverse ester): -0C( 0)R, wherein R is an acyloxy substituent, for example, a C alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably a C alkyl group.
• R is an acyloxy substituent, for example, a C alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably a C alkyl group.
• Oxycarboyloxy: -0C( 0)0R, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C 3-2 o heterocyclyl group, or a Cs-2o aryl group, preferably a C alkyl group.
• R 1 and R 2 are independently amino substituents, for example, hydrogen, a C alkyl group (also referred to as Ci-7 alkylamino or di-Ci-7 alkylamino), a C 3-2 o heterocyclyl group, or a Cs-2o aryl group, preferably H or a C alkyl group, or, in the case of a “cyclic” amino group, R 1 and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
• Amino groups may be primary (-NH 2 ), secondary (-NHR 1 ), or tertiary (-NHR 1 R 2 ), and in cationic form, may be quaternary (- + NR 1 R 2 R 3 ).
• Examples of amino groups include, but are not limited to, -NH 2 , -NHCH 3 , -NHC(CH 3 ) 2 , -N(CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , and -NHPh.
• Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
• Thioamido (thiocarbamyl): -C( S)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
• Acylamido (acylamino): -NR 1 C( 0)R 2 , wherein R 1 is an amide substituent, for example, hydrogen, a C alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably hydrogen or a C alkyl group, and R 2 is an acyl substituent, for example, a C alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2oaryl group, preferably hydrogen or a C alkyl group.
• R 1 and R 2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
• R 2 and R 3 are independently amino substituents, as defined for amino groups, and R 1 is a ureido substituent, for example, hydrogen, a C alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably hydrogen or a C alkyl group.
• ureido groups include, but are not limited to, -NHCONH2, -NHCONHMe, -NHCONHEt, -NHCONMe 2 , -NHCONEt 2 , -NMeCONhh, -NMeCONHMe, -NMeCONHEt, - NMeCONMe 2 , and -NMeCONEt 2 .
• Tetrazolyl a five membered aromatic ring having four nitrogen atoms and one carbon atom
• Imino: NR, wherein R is an imino substituent, for example, for example, hydrogen, a C alkyl group, a C 3-20 heterocyclyl group, or a Cs- 2 o aryl group, preferably H or a Ci- 7 alkyl group.
• C1-7 alkylthio groups include, but are not limited to, -SCHs and -SCH2CH3.
• Disulfide -SS-R, wherein R is a disulfide substituent, for example, a C alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C alkyl group (also referred to herein as C1-7 alkyl disulfide).
• C alkyl disulfide groups include, but are not limited to, -SSCHs and -SSCH2CH3.
• Sulfine (sulfinyl, sulfoxide): -S( 0)R, wherein R is a sulfine substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C1-7 alkyl group.
• R is a sulfine substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C1-7 alkyl group.
• R is a sulfinate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C1-7 alkyl group.
• R is a sulfonate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C1-7 alkyl group.
• R is a sulfinyloxy substituent, for example, a C alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C alkyl group.
• R is a sulfate substituent, for example, a C alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C alkyl group.
• R 1 and R 2 are independently amino substituents, as defined for amino groups.
• R 1 and R 2 are independently amino substituents, as defined for amino groups.
• R 1 is an amino substituent, as defined for amino groups.
• R 1 is an amino substituent, as defined for amino groups
• R is a sulfonamino substituent, for example, a C alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably a C alkyl group.
• R 1 is an amino substituent, as defined for amino groups
• R is a sulfinamino substituent, for example, a C alkyl group, a C3-20 heterocyclyl group, or a C5-2o aryl group, preferably a C alkyl group.
• R is a phosphino substituent, for example, -H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a Cs-2o aryl group, preferably -H, a C alkyl group, or a C5-2o aryl group.
• Examples of phosphino groups include, but are not limited to, -PH2, -P(CH 3 ) 2 , -P(CH 2 CH 3 )2, -P(t-Bu) 2 , and -P(Ph) 2 .
• R is a phosphinyl substituent, for example, a C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably a C1-7 alkyl group or a Cs-2o aryl group.
• Phosphonate (phosphono ester): -P( 0)(OR) 2 , where R is a phosphonate substituent, for example, -H, a C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably -H, a C1-7 alkyl group, or a Cs-2o aryl group.
• R is a phosphonate substituent, for example, -H, a C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably -H, a C1-7 alkyl group, or a Cs-2o aryl group.
• R is a phosphate substituent, for example, -H, a C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably -H, a C1-7 alkyl group, or a Cs-2o aryl group.
• Phosphorous acid -OP(OH)2.
• Phosphite -OP(OR)2, where R is a phosphite substituent, for example, -H, a C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably -H, a C1-7 alkyl group, or a Cs-2o aryl group.
• R is a phosphite substituent, for example, -H, a C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a Cs-2o aryl group, preferably -H, a C1-7 alkyl group, or a Cs-2o aryl group.
• Examples of phosphite groups include, but are not limited to, -OP(OCH 3 )2,
• Phosphoramidite -OP(OR 1 )-NR 2 2, where R 1 and R 2 are phosphoramidite substituents, for example, -H, a (optionally substituted) C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a C5-20 aryl group, preferably -H, a C 1-7 alkyl group, or a Cs- 2 o aryl group.
• R 1 and R 2 are phosphoramidite substituents, for example, -H, a (optionally substituted) C1-7 alkyl group, a C 3- 2o heterocyclyl group, or a C5-20 aryl group, preferably -H, a C 1-7 alkyl group, or a Cs- 2 o aryl group.
• Examples of phosphoramidite groups include, but are not limited to, -OP(OCH2CH3)-N(CH3)2,
• C 3-12 alkylene refers 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.
• alkylene includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
• linear saturated C 3-12 alkylene groups include, but are not limited to, -(CH 2 ) n - where n is an integer from 3 to 12, for example, -CH 2 CH 2 CH 2 - (propylene),
• branched saturated C 3-12 alkylene groups include, but are not limited to, -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH3)CH 2 CH 2 CH2-, -CH 2 CH(CH 3 )CH 2 -,
• alicyclic saturated C3-i2 alkylene groups include, but are not limited to, cyclopentylene (e.g. cyclopent-1 ,3-ylene), and cyclohexylene
• C3-12 cycloalkylenes examples 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).
• Bis-oxy-Ci-3 alkylene The term“Bis-oxy-C- 1-3 alkylene”, as used herein, pertains to a bidentate moiety of formula -0-(CH 2 ) q -0-, where q is from 1 to 3, i.e. -OCH2CH2CH2O-, - OCH2CH2O-, -OCH2O-.
• 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:
• R’ 10 is R as defined above.
• suitable groups are described on pages 503 to 549 of Greene, T.W. and Wuts, G.M., Protective Groups in Organic Synthesis, 3 rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
• Hemi-aminal nitrogen protecting group pertains to a group having the following structure:
• R’ 10 is R as defined above.
• suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T.W. and Wuts, G.M., Protective Groups in Organic Synthesis, 3 rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
• the groups Carbamate nitrogen protecting group and Hemi-aminal nitrogen protecting group may be jointly termed a“nitrogen protecting group for synthesis”.
• a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO ), a salt or solvate thereof, as well as conventional protected forms.
• a reference to an amino group includes the protonated form (-N + HR 1 R 2 ), a salt or solvate of the amino group, for example, a
• hydrochloride salt as well as conventional protected forms of an amino group.
• a reference to a hydroxyl group also includes the anionic form (-0 ), a salt or solvate thereof, as well as conventional protected forms.
• a corresponding salt of the active compound for example, a pharmaceutically-acceptable salt.
• a pharmaceutically-acceptable salt examples are discussed in Berge, et al., J. Pharm. Sci., 66, 1 -19 (1977).
• a salt may be formed with a suitable cation.
• suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as AG 3 .
• suitable organic cations include, but are not limited to, ammonium ion (i.e. NH 4 + ) and substituted ammonium ions (e.g. NH 3 R + , NH2R2 + , NHR3 + , NR 4 + ).
• suitable substituted ammonium ions are those derived from: ethylamine, diethylamine,
• diethanolamine piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
• amino acids such as lysine and arginine.
• An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
• a salt may be formed with a suitable anion.
• 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.
• suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,
• Suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
• 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.
• the invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R A OH, where R A is C1-4 alkyl):
• carbinolamine and carbinolamine ether forms of the PBD can be called the carbinolamine and carbinolamine ether forms of the PBD (as described in the section relating to R 10 above).
• the balance of these equilibria depends on the conditions in which the compounds are found, as well as the nature of the moiety itself.
• Certain compounds of the invention 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 l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and b-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or“isomeric forms”).
• stereoisomers refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
• Diastereomer refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
• Enantiomers refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.
• the compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention.
• a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
• the terms“racemic mixture” and“racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
• isomers 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).
• a reference to a methoxy group, -OCH 3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH.
• a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta- chlorophenyl.
• 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).
• keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
• tautomer or“tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier.
• proton tautomers also known as prototropic tautomers
• Valence tautomers include interconversions by reorganization of some of the bonding electrons.
• H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 0 and 18 0; and the like.
• isotopes examples include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to 2 H (deuterium, D), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 CI, and 125 l.
• isotopically labeled compounds of the present invention for example those into which radioactive isotopes such as 3H, 13C, and 14C are incorporated.
• Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single- photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
• Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.
• An 18F labeled compound may be useful for PET or SPECT studies.
• Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
• substitution with heavier isotopes, particularly deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index.
• deuterium in this context is regarded as a substituent.
• the concentration of such a heavier isotope, specifically deuterium may be defined by an isotopic enrichment factor.
• any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.
• 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.
• 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.
• the Ligand unit may be a protein, polypeptide or peptide.
• 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).
• the antibody or other molecule binds with an affinity of at least about 1x10 7 M 1 , and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
• a non-specific molecule e.g., BSA, casein
• Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
• the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell.
• a Cell Binding Agent can be a protein, polypeptide, peptide or a non- peptidic agent.
• the Cell Binding Agent may be a protein, polypeptide or peptide.
• 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.
• the present invention provides an antibody-drug conjugate (ADC).
• ADC antibody-drug conjugate
• a cell binding agent may be of any kind, and include peptides and non-peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance.
• the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that one cell binding agent is linked to one monomer or dimer pyrrolobenzodiazepine compound. In one embodiment the cell binding agent comprises a peptide that binds integrin a n b 6 . The peptide may be selective for a n b q over XYS.
• the cell binding agent comprises the A20FMDV-Cys polypeptide.
• the A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC.
• a variant of the A20FMDV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted with another amino acid residue.
• polypeptide may have the sequence NAVXXXXXXXXXXXXXXXXXRTC.
• antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour of Immunology 170:4854-4861 ).
• Antibodies may be murine, human, humanized, chimeric, or derived from other species.
• An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001 ) Immuno Biology, 5th Ed., Garland Publishing, New York).
• a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
• An antibody includes a full- length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease.
• the immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass of immunoglobulin molecule.
• the immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
• Antibody fragments comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof.
• antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-ld) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• the term“monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be
• the modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567).
• the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991 ) Nature, 352:624-628; Marks et al (1991 ) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).
• the monoclonal antibodies herein specifically include chimeric antibodies, humanized antibodies and human antibodies.
• cell binding agents include those agents described for use in WO 2007/085930, which is incorporated herein.
• Tumour-associate antigens and cognate antibodies for use in embodiments of the present invention are listed below, and are described in more detail on pages 14 to 86 of WO
• BMPR1 B bone morphogenetic protein receptor-type IB
• Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b)
• PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene)
• STEAP2 (HGNC_8639, IPCA-1 , PCANAP1 , STAMP1 , STEAP2, STMP, prostate cancer associated gene 1 , prostate cancer associated protein 1 , six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein)
• TrpM4 (BR22450, FLJ20041 , TRPM4, TRPM4B, transient receptor potential cation 5 channel, subfamily M, member 4)
• CRIPTO (CR, CR1 , CRGF, CRIPTO, TDGF1 , teratocarcinoma-derived growth factor)
• CD21 CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792)
• CD79b (CD79B, CD793, IGb (immunoglobulin-associated beta), B29)
• FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1 a), SPAP1 B, SPAP1 C)
• EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5)
• PSCA Prostate stem cell antigen precursor
• BAFF-R B cell -activating factor receptor, BLyS receptor 3, BR3
• CD22 B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814)
• CD22 CD22 molecule
• CD79a (CD79A, CD79alpha), immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pi: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q 13.2).
• CXCR5 Bokitt's lymphoma receptor 1 , a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a 10 role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pi: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11 q23.3,
• HLA-DOB Beta subunit of MHC class II molecule (la antigen) that binds peptides and 20 presents them to CD4+ T lymphocytes); 273 aa, pi: 6.56, MW: 30820.
• TM 1 [P] Gene Chromosome: 6p21.3)
• P2X5 Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability
• 422 aa pi: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).
• CD72 B-cell differentiation antigen CD72, Lyb-2
• LY64 Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); 661 aa, pi: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).
• FcRH1 Fc receptor-like protein 1 , a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte
• IRTA2 Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis
• TENB2 (TMEFF2, tomoregulin, TPEF, HPP1 , TR, putative transmembrane
• 35 proteoglycan related to the EGF/heregulin family of growth factors and follistatin); 374 aa)
• PSMA - FOLH1 Fralate hydrolase (prostate-specific membrane antigen) 1
• CEACAM5 Carcinoembryonic antigen-related cell adhesion molecule 5
• EGFRvlll Epidermal growth factor receptor (EGFR), transcript variant 3,
• CD33 (CD33 molecule)
• IL2RA Interleukin 2 receptor, alpha
• NCBI Reference Sequence NM_000417.2
• AXL AXL receptor tyrosine kinase
• CD30 - TNFRSF8 Tumor necrosis factor receptor superfamily, member 8
• BCMA B-cell maturation antigen
• TNFRSF17 Tumor necrosis factor receptor superfamily, member 17
• CT Ags - CTA Cancer Testis Antigens
• CD174 (Lewis Y) - FUT3 (fucosyltransferase 3 (galactoside 3(4)-L-fucosyltransferase, Lewis blood group)
• CLEC14A C-type lectin domain family 14, member A; Genbank accession no. NM175060
• GRP78 - HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)
• GCC - GUCY2C guanylate cyclase 2C (heat stable enterotoxin receptor)
• CD56 - NCMA1 Neuronal cell adhesion molecule 1
• CanAg Tumor associated antigen CA242
• GPNMB Glycoprotein (transmembrane) nmb
• TIM-1 - HAVCR1 Hepatitis A virus cellular receptor 1
• PTK7 protein tyrosine kinase
• CD37 CD37 molecule
• CD138 - SDC1 (syndecan 1 )
• CD74 CD74 molecule, major histocompatibility complex, class II invariant chain
• CD20 - MS4A1 membrane-spanning 4-domains, subfamily A, member 1
• FAP Fibroblast activation protein, alpha
• DKK-1 Dickkopf 1 homolog (Xenopus laevis)
• CD52 CD52 molecule
• V-CAM CD106
• VCAM1 Vascular cell adhesion molecule 1
• the cell binding agent may be labelled, for example to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate.
• the label may be a biotin label.
• the cell binding agent may be labelled with a radioisotope.
• the Ligand unit may be connected to the Linker unit through a disulfide bond.
• the connection between the Ligand unit and the Drug Linker is formed between a thiol group of a cysteine residue of the Ligand unit and a maleimide group of the Drug 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.
• 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.
• 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 W02008070593, which are incorporated herein.
• the compounds of the present invention may be used in a method of therapy.
• a method of treatment comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate of formula II.
• 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 conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy.
• compositions according to the present invention may comprise, in addition to the active ingredient, i.e. a conjugate 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.
• a pharmaceutically acceptable excipient e.g. cutaneous, subcutaneous, or intravenous.
• 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.
• 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.
• a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
• 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 Conjugates can be used to treat proliferative disease and autoimmune disease.
• 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.
• 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,
• any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.
• autoimmune disease examples 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, Sjogren’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 purpur
• 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, Sjogren’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).
• disorders involving dendritic cells involve disorders of Th1 -lymphocytes.
• the conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate compound of the invention.
• a 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 is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
• a compound of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs, such as chemotherapeutics); surgery; and radiation therapy.
• A“chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action.
• Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids,
• Chemotherapeutic agents include compounds used in“targeted therapy” and conventional chemotherapy.
• chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(ll), CAS No. 15663-27-1 ), carboplatin (CAS No.
• paclitaxel TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.
• trastuzumab HERCEPTIN®, Genentech
• temozolomide 4-methyl-5-oxo- 2, 3, 4,6,8- pentazabicyclo [4.3.0] nona-2,7,9-triene- 9-carboxamide, CAS No. 85622-93-1 ,
• TEMODAR® TEMODAL®, Schering Plough
• tamoxifen (Z)-2-[4-(1 ,2-diphenylbut-1- enyl)phenoxy]-/V,/V-dimethylethanamine
• NOLVADEX® NOLVADEX®
• ISTUBAL® ISTUBAL®
• VALODEX® doxorubicin
• ADRIAMYCIN® doxorubicin
• chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU1 1248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirol
• alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolast
• calicheamicin calicheamicin gammal l, calicheamicin ornegaH (Angew Chem. Inti. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates, such as clodronate; an
• esperamicin as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,
• cyanomorpholino-doxorubicin 2-pyrrolino-doxorubicin and deoxydoxorubicin
• epirubicin esorubicin, idarubicin, nemorubicin, marcellomycin
• mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin
• anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-aza
• etoglucid gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
• pentostatin phenamet
• pirarubicin losoxantrone
• podophyllinic acid 2-ethylhydrazide
• PSK® polysaccharide complex JHS Natural Products, Eugene, OR
• razoxane rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2’, 2”- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
• pipobroman gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin;
• vinblastine etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine
• NAVELBINE® novantrone
• teniposide edatrexate
• daunomycin aminopterin
• capecitabine XELODA®, Roche
• ibandronate CPT-11 ; topoisomerase inhibitor RFS 2000;
• DMFO difluoromethylornithine
• retinoids such as retinoic acid
• pharmaceutically acceptable salts, acids and derivatives of any of the above DMFO
• DMFO difluoromethylornithine
• chemotherapeutic agent also included in the definition of“chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including
• aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca);
• anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog);
• protein kinase inhibitors such as MEK inhibitors (WO 2007/044515);
• lipid kinase inhibitors such as oblimersen (GENASENSE®, Genta Inc.)
• ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;
• vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®
• chemotherapeutic agent are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab
• Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab,
• compositions according to the present invention may comprise, in addition to the active ingredient, i.e. a conjugate compound, 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.
• a pharmaceutically acceptable excipient e.g. 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.
• 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.
• 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.
• a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
• 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.
• conjugate compound While it is possible for the conjugate compound to be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation.
• the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a conjugate compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
• a pharmaceutical composition e.g., formulation, preparation, medicament
• a pharmaceutically acceptable carrier e.g., diluent, or excipient.
• the composition is a pharmaceutical composition comprising at least one conjugate compound, as described herein, together with one or more other
• pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
• pharmaceutically acceptable carriers including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
• the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.
• Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and
• Another aspect of the present invention pertains to methods of making a pharmaceutical composition
• a pharmaceutical composition comprising admixing at least one [ 11 C]-radiolabelled conjugate or conjugate-like compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
• ingredients, materials, compositions, dosage forms, etc. which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• Each carrier, diluent, excipient, etc. must also be“acceptable” in the sense of being compatible with the other ingredients of the formulation.
• the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
• carriers e.g., liquid carriers, finely divided solid carrier, etc.
• the formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
• Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate).
• Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers,
• bacteriostats suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
• excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
• suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
• concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 pg/ml.
• the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• sterile liquid carrier for example water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
• appropriate dosages of the conjugate compound, and compositions comprising the conjugate compound can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
• the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
• the amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
• Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
• a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically about 1 pg to about 10 mg) per kilogram body weight of the subject per day.
• the active compound is a salt, an ester, an amide, a prodrug, or the like
• the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
• the active compound is administered to a human patient according to the following dosage regime: about 100 mg, 3 times daily.
• the active compound is administered to a human patient according to the following dosage regime: about 150 mg, 2 times daily.
• the active compound is administered to a human patient according to the following dosage regime: about 200 mg, 2 times daily.
• the conjugate compound is administered to a human patient according to the following dosage regime: about 50 or about 75 mg, 3 or 4 times daily.
• the conjugate compound is administered to a human patient according to the following dosage regime: about 100 or about 125 mg, 2 times daily.
• the dosage amounts described above may apply to the conjugate (including the PBD moiety and the linker to the antibody) or to the effective amount of PBD compound provided, for example the amount of compound that is releasable after cleavage of the linker.
• an ADC of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
• the molecule is suitably
• mV ⁇ V to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
• a typical daily dosage might range from about 1 mV ⁇ V to 100 mg/kg or more, depending on the factors mentioned above.
• An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
• An exemplary dosing regimen comprises a course of administering an initial loading dose of about 4 mg/kg, followed by additional doses every week, two weeks, or three weeks of an ADC. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
• the drug loading (p) is the average number of PBD drugs per cell binding agent, e.g.
• drug loading may range from 1 to 8 drugs (D) per cell binding agent, i.e. where 1 , 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the cell binding agent.
• Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 8.
• drug loading may range from 1 to 80 drugs (D) per cell binding agent, although an upper limit of 40, 20, 10 or 8 may be preferred.
• Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.
• the average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis.
• the quantitative distribution of ADC in terms of p may also be determined.
• ELISA the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res.
• p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA.
• ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues.
• separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.
• p may be limited by the number of attachment sites on the antibody.
• an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
• Higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.
• an antibody may contain, for example, many lysine residues that do not react with the Drug Linker. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety.
• cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions.
• DTT dithiothreitol
• TCEP TCEP
• the loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of Drug Linker relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.
• Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
• Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol).
• a reducing agent such as DTT (dithiothreitol).
• DTT dithiothreitol
• Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles.
• Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut’s reagent) resulting in conversion of an amine into a thiol.
• Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).
• US 7521541 teaches engineering antibodies by introduction of reactive cysteine amino acids.
• Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721 -2729; US 7521541 ; US 7723485; W02009/052249).
• the engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties.
• the location of the drug moiety can thus be designed, controlled, and known.
• the drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield.
• Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody.
• a drug loading near 2 can be achieved with near homogeneity of the conjugation product ADC.
• the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1 , 2, 3, etc.
• Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value.
• Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.
• antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.
• the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.
• R L pre represents a precursor to the group R L .
• R° is H
• the carboxy group may be protected during synthesis (for example a tert-butyl ester), which can be removed in a final step.
• R L is a group:
• R30 and R31 may also be a nitrogen protecting group and OProt° respectively, when Prot° represents an oxygen protecting group for synthesis, which are removed as appropriate.
• compounds of formula IP may be synthesised by coupling compounds of formulae 2 and 3:
• Hal is selected from I, Cl, and Br
• Prot° represents an oxygen protecting group for synthesis, following by removal of the Prot° group under standard conditions.
• the coupling can be achieved, for example, in refluxing acetone with a base, such as K2CO3.
• Hal-R”-Q Formula 5 where Q is selected from I, Cl, and Br, the reaction can be achieved, for example, in refluxing acetone with a base, such as K2CO 3 . An excess of the compound of Formula 5 is required to achieve the desired product.
• the compound of formula 4 may be synthesised from a compound of Formula 6:
• Prot r is a protecting group for Y’ that is orthogonal to the other protecting groups in the compound.
• the synthesis is achieved by deprotection of Y’, under standard conditions.
• Compounds of formula 3 can be synthesised from compounds of formula 7:
• Prot Y is a protecting group for Y that is orthogonal to the other protecting groups in the compound.
• the synthesis is achieved by deprotection of Y, under standard conditions.
• Compounds of formula 7 can be synthesised from compounds of formula 8:
• the oxidation may be carried out, for example, with Dess-Martin periodinane (or alternatively TPAP/NMO, TFAA/DMSO, SO3. Pyridine complex/DMSO, PDC, PCC, BAIB/TEMPO or under Swern conditions).
• Dess-Martin periodinane or alternatively TPAP/NMO, TFAA/DMSO, SO3. Pyridine complex/DMSO, PDC, PCC, BAIB/TEMPO or under Swern conditions).
• Conjugates can be prepared as previously described. Antibodies can be conjugated to the Drug Linker compound 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.
• TCEP tris(carboxyethyl)phosphine hydrochloride
• 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 pm 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.
• R 6’ , R 7’ , R 9’ , and Y’ are selected from the same groups as R 6 , R 7 , R 9 and Y respectively. In some embodiments, R 6’ , R 7’ , R 9’ , and Y’ are the same as R 6 , R 7 , R 9 and Y respectively.
• R 2 is the same as R 2 .
• Y and Y’ are preferably O.
• n is 1. In other embodiments, n is 2. In other embodiments, n is 3.
• n is 1. In other embodiments, m is 2. In other embodiments, m is 3. In certain embodiments, n and m are the same. In some of these embodiments, n and m are 1. In other of these embodiments, n and m are 2. In other of these embodiments, n and m are 3.
• Q is N. In other embodiments, Q is CH.
• R° is H. In other embodiments, R° is methyl. In other
• R° is ethyl. In other embodiments, R° is iso-propyl. In other embodiments, R° is benzyl.
• R" is of formula I la:
• R° 1 is selected from the group consisting of H and methyl.
• R 9 is H.
• R 6 is selected from H, OH, OR, SH, NH2, nitro and halo, and may be selected from H or halo. In some of these embodiments R 6 is H.
• R 7 is selected from H, OH, OR, SH, SR, NH2, NHR, NRR’, and halo.
• R 7 is selected from H, OH and OR, where R is selected from optionally substituted C 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 OCH2PI7.
• Other substituents of particular interest are dimethylamino (i.e.
• R 2 is selected from:
• each of R 11 , R 12 and R 13 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 R 2 group is no more than 5;
• R 15a and R 15b are 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
• R 14 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.
• R 2 When R 2 is a C 5-10 aryl group, it may be a C 5-7 aryl group.
• a C 5-7 aryl group may be a phenyl group or a C 5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl.
• R 2 is preferably phenyl.
• R 2 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
• R 2 When R 2 is a C 5-10 aryl group, it may be a Cs-io 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.
• 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.
• R 2 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.
• R 2 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 b or y 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.
• R 2 is a Ce-io 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).
• R 2 substituents, when R 2 is a C 5-10 aryl group
• R 2 when R 2 is a C5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
• R 2 when R 2 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).
• R 2 when R 2 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).
• R 2 when R 2 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be C 6 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 Ci -4 alkyl groups.
• the said further substituent may be on the second nitrogen ring atom.
• a substituent on R 2 when R 2 is a C 5-10 aryl group is bis-oxy-C- 1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
• R 2 when R 2 is a C 5-10 aryl group is ester, this is preferably methyl ester or ethyl ester.
• R 2 is a C 5-10 aryl group
• substituents when R 2 is a C 5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl.
• Other particularly preferred substituents for R 2 are dimethylaminopropyloxy and carboxy.
• R 2 groups when R 2 is a C 5-10 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 R 12 group is 4- nitrophenyl.
• R 12 groups of particular interest include 4-(4-methylpiperazin-1 -yl)phenyl and 3,4-bisoxymethylene-phenyl.
• R 2 is C 1-5 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.
• R 2 When R 2 is C 3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
• each of R 11 , R 12 and R 13 are independently selected from H, C 1-3 saturated alkyl, C 2-3 alkenyl, C 2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 2 group is no more than 5. In some embodiments, the total number of carbon atoms in the R 2 group is no more than 4 or no more than 3. In some embodiments, one of R 11 , R 12 and R 13 is H, with the other two groups being selected from H, C 1-3 saturated alkyl, C 2-3 alkenyl, C 2-3 alkynyl and cyclopropyl.
• two of R 11 , R 12 and R 13 are H, with the other group being selected from H, C 1-3 saturated alkyl, C 2-3 alkenyl, C 2-3 alkynyl and cyclopropyl.
• 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.
• R 1 1 is H.
• R 12 is H.
• R 13 is H.
• R 1 1 and R 12 are H.
• R 1 1 and R 13 are H.
• R 12 and R 13 are H.
• R 2 group of particular interest is:
• R 15a and R 15b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl.
• the group which is not H is optionally substituted phenyl.
• the phenyl optional substituent is halo, it is preferably fluoro.
• the phenyl group is unsubstituted.
• R 2 is R 14 is selected from: H; C 1-3 saturated alkyl; C 2-3 alkenyl; C 2-3 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.
• R 14 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R 14 is selected from H and methyl.
• R 2 is H or , where R 16a and R 16b are independently selected from H, F, Ci -4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and Ci -4 alkyl ester; or, when one of R 16a and R 16b is H, the other is selected from nitrile and a Ci -4 alkyl ester.
• R 2 is H.
• R 16a and R 16b are both H.
• R 16a and R 16b are both methyl.
• R 16a and R 16b are H, and the other is selected from Ci -4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
• the group which is not H is selected from methyl and ethyl.
• R 11a is OH
• R 11a is OR A . In some of these embodiments, R A is methyl. In some embodiments, R 11a is selected from OH, OR A , where R A is Ci -4 alkyl, and SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation.
• R 30 is H, and R 31 is OH. In other embodiments, R 30 is H, and R 31 is OR A . In some of these embodiments, R A is methyl.
• R 30 and R 31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
• R 30 is H and R 31 is SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation.
• M is a monovalent pharmaceutically acceptable cation, and is more preferably Na + .
• z is preferably 3.
• R L is of formula Ilia.
• R LL is of formula Ilia’.
• G L may be selected from
• G L is selected from G L1 1 and G L1 2 . In some of these embodiments, G L is G L1 - 1 .
• G LL G LL may be selected from:
• G LL is selected from G LL1 1 and G LL1 2 . In some of these
• G LL is G LL1 1 .
• a 0 to 5
• b 0 to 16
• c 0 or 1
• d 0 to 5.
• a may be 0, 1 , 2, 3, 4 or 5.
• a is 0 to 3. In some of these
• a is 0 or 1. In further embodiments, a is 0. b may be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16. In some embodiments, b is 0 to 12. In some of these embodiments, b is 0 to 8, and may be 0, 2, 4 or 8. c may be 0 or 1 . d may be 0, 1 , 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these
• d is 1 or 2. In further embodiments, d is 2.
• a is 0, c is 1 and d is 2, and b may be from 0 to 8. In some of these embodiments, b is 0, 4 or 8.
• Q is an amino acid residue.
• the amino acid may a natural amino acids or a non-natural amino acid.
• Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, lie, Arg, and Trp, where Cit is citrulline.
• Q comprises a dipeptide residue.
• the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids.
• the dipeptide comprises natural amino acids.
• 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.
• Q is selected from:
• Cit is citrulline
• Q is selected from:
• Q is selected from co -Phe-Lys- NH , co -Val-Cit- NH and co -Val-Ala- NH .
• dipeptide combinations of interest include:
• Q x is a tripeptide residue.
• the amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids.
• the tripeptide comprises natural amino acids.
• the linker is a cathepsin labile linker
• the tripeptide is the site of action for cathepsin-mediated cleavage.
• the tripeptide then is a recognition site for cathepsin.
• 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.
• R L is of formula II lb.
• R LL is is formula 11 lb’.
• R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group.
• both R L1 and R L2 are H.
• R L1 is H and R L2 is methyl.
• both R L1 and R L2 are methyl.
• R L1 and R L2 together with the carbon atom to which they are bound form a cyclopropylene group.
• R L1 and R L2 together with the carbon atom to which they are bound form a cyclobutylene group.
• e is 0.
• e is 1 and the nitro group may be in any available position of the ring. In some of these embdoiments, it is in the ortho position. In others of these embodiments, it is in the para position.
• the first aspect of the invention comprises a compound of
• R 2a and R 2a are the same and are selected from:
• R 7a and R 7a are the same and are selected from methoxy and benzyloxy;
• R°, R L1 and R 11a are as defined above.
• the drug linkers (D L ) are of formula Ilia, Nib or I lie:
• R 2a and R 2a are the same and are selected from: R 7a and R 7a are the same and are selected from methoxy and benzyloxy;
• R°, R L1a and R 11a are as defined above.
• Flash chromatography was performed using a Biotage Isolera 1TM using gradient elution with hexane/EtOAc or CFhCh/MeOH mixtures as indicated until all UV active components eluted from the column. The gradient was manually held whenever substantial elution of UV active material was observed. Fractions were checked for purity using thin-layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Extraction and chromatography solvents were bought and used without further purification from VWR U.K. All fine chemicals were purchased from Sigma-Aldrich or VWR.
• TLC thin-layer chromatography
• the analytical LC/MS conditions were as follows: Positive mode electrospray mass spectrometry was performed using a Shimadzu Nexera®/Prominence® LCMS-2020. Mobile phases used were solvent A (FhO with 0.1 % formic acid) and solvent B (CH3CN with 0.1 % formic acid).
• LCMS-A Gradient for 3-minute run: Initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 seconds’ period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes at a flow rate of 0.8 mL/min.
• LCMS-B Gradient for 15-minute run: Initial composition 5% B held over 1 minute, then increased from 5% B to 100% B over a 9 minute period. The composition was held for 2 minutes at 100% B, then returned to 5% B in 10 seconds and held there for 2 minutes 50 seconds. The total duration of the gradient run was 15.0 minutes at a flow rate of 0.6 mL/minute. Detection was monitored at 254 nm, 223 nm and 214 nm. ACE Excel 2 C18-AR, 2 m, 3.0 x 100mm fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre- column, 130A, 1 7pm, 2.1 mm x 5 mm. Intermediates
• I6 is Compound 152 of WO 2018/182341
• Example 1 3-((((11 S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11 -hydroxy- 7-methoxy-2-methylene-5-oxo-2,3,5,10,11 ,11a-hexahydro-1 H-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepin-8-yl)oxy)methyl)-5-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a- tetrahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8-yl)oxy)methyl)benzoic acid (19)
• Example 2 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)-3- methylbutanamido)propanamido)benzyl (11 S,11aS)-11 -hydroxy-7-methoxy-8-((3-((((S)- 7-methoxy-2-methylene-5-oxo-2,3,5,11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-2-methylene-5-oxo- 2,3,11 ,11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine-10(5H)-carboxylate (27)
• a microwave vial was charged with a magnetic stirrer, CHCI3 (1 mL) 29 (100 mg, 0.06999 mmol, 1.0 eq.) SnMesOH (127 mg, 0.6999 mmol, 10 eq.) and irradiated at 100 °C for 10 h.
• the reaction mixture was filtered through Celite® and the filtrate concentrated in vacuo.
• the resulting residue was dissolved in THF (1 mL) and H2O (1 mL), K2CO3 (20 mg, 0.1400 mmol, 2.0 eq.) and allyl chloroformate (14 pL, 0.1400 mmol, 2.0 eq.) were added.
• a microwave vial was charged with a magnetic stirrer bar, (CH2CI)2 (0.5 mL) 40 (100 mg, 64.62 pmol, 1.0 eq.), SnMesOH (35.1 mg, 193.9 pmol, 3.0 eq.) and the vial sealed.
• the mixture was heated to 80 °C for 4 h, cooled to rt, poured into H2O, extracted with 10%
• DTT DL-dithiothreitol
• PBS phosphate-buffered saline pH 7.4
• EDTA ethylenediaminetetraacetic acid
• the reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.
• the reduced antibody was buffer exchanged, via spin filter centrifugation, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
• a 50 mM solution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 4.5 micromoles, 90 pl_) in DMSO was added and the reoxidation mixture was allowed to react for 2-3 hours at room temperature with gentle (50 rpm) shaking at an antibody concentration of 1.5 mg/ml_
• DTT DL-dithiothreitol
• PBS phosphate-buffered saline pH 7.4
• EDTA ethylenediaminetetraacetic acid
• the reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, the reduced antibody was buffer exchanged, via
• Compound 27 was added as a DMSO solution (12 molar equivalent/antibody, 1.2 micromole, in 1.5 mL DMSO) to 15.0 mL of this reoxidised antibody solution (15.3 mg, 0.1 pmoles) for a 10% (v/v) final DMSO concentration.
• the solution left to react at room temperature for 2 hours at room temperature with gentle shaking.
• the conjugation was quenched by addition of N- acetyl cysteine (6.0 micromoles, 60 pL at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
• DTT DL-dithiothreitol
• PBS phosphate-buffered saline pH 7.4
• EDTA ethylenediaminetetraacetic acid
• the reduction mixture was allowed to react at room temperature for 3-4 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.
• the reduced antibody was buffer exchanged, via spin filter centrifugation, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
• Compound 33 was added as a DMSO solution (10 molar equivalent/antibody, 1 micromole, in 0.75 mL DMSO) to 6.75 mL of this reoxidised antibody solution (15 mg, 100 nanomoles) for a 10% (v/v) final DMSO
• the solution was mixed for 4 hours at room temperature (0.5-1.0 mL propylene glycol added after 2 hours to aid solubility), then the conjugation was quenched by addition of N- acetyl cysteine (5 micromoles, 50 pL at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
• DTT DL-dithiothreitol
• PBS phosphate-buffered saline pH 7.4
• EDTA ethylenediaminetetraacetic acid
• the reduction mixture was allowed to react at room temperature for 20 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.
• the reduced antibody was buffer exchanged, via tangential flow filtration (TFF) using a Spectrum Labs KrosFlo Research Hi system with a 30 kDa MWCO, 115 cm 2 surface area hollow fibre filter module at 50 mL/min in PBS with a transmembrane pressure (TMP) of 0.5-1.0 bar, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent, recovery 97 mg (667 nanomoles).
• TFP transmembrane pressure
• Compound 34 was added as a DMSO solution (15 molar equivalent/antibody, 1.4 micromoles, in 1.4 mL DMSO) to 12.6 mL of this reoxidised antibody solution (14 mg, 93 nanomoles) for a 10% (v/v) final DMSO concentration.
• the solution was mixed for 2 hours at room temperature, stored at +4 °C for 16 hours then the conjugation was quenched by addition of N- acetyl cysteine (3.7 micromoles, 37 mI_ at 100 mM).
• Reaction mixture was purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, further purified by TFF (800 mL permeated), sterile-filtered and analysed.
• UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris 3.6u XB- C18 150 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjD at 280 nm and 330 nm shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 34, consistent with a drug-per-antibody ratio (DAR) of 1.81 molecules of Compound 34 per antibody.
• DAR drug-per-antibody ratio
• a 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added Trastuzumab (40 molar equivalent/antibody, 27 pmol, 536 pL) to a 25 mL solution of antibody (100 mg, 0.67 micromoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.0 mg/mL.
• DTT DL-dithiothreitol
• PBS phosphate-buffered saline pH 7.4
• EDTA ethylenediaminetetraacetic acid
• the reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, the reduced antibody was buffer exchanged, via
• the reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of 1.7 mg/mL
• Compound 44 was added as a DMSO solution (10 molar equivalent/antibody, 1.0 micromole, in 1.5 mL DMSO) to 15.0 ml. of this reoxidised antibody solution (15.3 mg, 0.1 pmoles) for a 10% ( v/v ) final DMSO concentration.
• the solution left to react at room temperature for 2 hours at room temperature with gentle shaking.
• conjugation was quenched by addition of N- acetyl cysteine (5.0 micromoles, 50 mI_ at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
• N- acetyl cysteine 5.0 micromoles, 50 mI_ at 100 mM
• DTT DL-dithiothreitol
• PBS phosphate-buffered saline pH 7.4
• EDTA ethylenediaminetetraacetic acid
• the reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.
• the reduced antibody was buffer exchanged, via Tangential Flow Filtration unit (TFF) using mPES, MidiKros ® 30 kDa fiber filter with 115 cm 2 surface area, into a reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove all the excess reducing agent.
• the reduced antibody was centrifuged for 3 min at 4000 rpm and then filtered using 0.22 mM membrane filter.
• Compound 45 was added as a DMSO solution (10 molar equivalent/antibody, 1.0 micromole, in 1.5 mL DMSO) to 15.0 ml. of this reoxidised antibody solution (15.3 mg, 0.1 pmoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 2 hours at room temperature with gentle shaking. Then the conjugation was quenched by addition of N- acetyl cysteine (5.0 micromoles, 50 pl_ at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
• N- acetyl cysteine 5.0 micromoles, 50 pl_ at 100 mM
• the cell concentration and viability are measured by trypan blue staining, and counting using the LUNA-IITM Automated Cell Counter. Cells were diluted to 2x10 5 /ml, dispensed (50 pi /well) into 96-well flat-bottom plates and incubated overnight before use.
• a stock solution (1 ml) of antibody drug conjugate (ADC) (20 pg/ml) was made by dilution of filter-sterilised ADC into cell culture medium.
• a set of 8x 10-fold dilutions of stock ADC were made in a 24-well plate by serial transfer of 100 mI into 900 mI of cell culture medium.
• ADC dilution was dispensed (50 mI/ well) into 4 replicate wells of the 96-well plate, containing 50 mI cell suspension seeded the previous day. Control wells received 50 mI cell culture medium.
• the 96-well plate containing cells and ADCs was incubated at 37C in a C02-gassed incubator for the exposure time. At the end of the incubation period, cell viability was measured by MTS assay.
• X is log(concentration).
• ADC incubation times were 4 and 7 days for MDA MB 468 and NCI N87 respectively.
• Cell growth medium for MDA MB 468 was RPMI 1640 with glutamax, 10% (v/v) HyCloneTM Fetal Bovine Serum and for NCI N87 was RPMI 1640 with glutamax, 10% (v/v) HyCloneTM Fetal Bovine Serum.
• mice Female severe combined immune-deficient mice (Fox Chase SCID®, C.B-17 l ⁇ cr-Prkdcscid, Charles River) were eight to twelve weeks old on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on irradiated Enricho’cobsTM Laboratory Animal Bedding in static micro-isolators on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% humidity.
• CR Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care.
• the animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.
• AALAC Laboratory Animal Care International
• Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, 100 pg/mL streptomycin sulfate and 25 pg/mL gentamicin.
• the cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air.
• the NCI-N87 cells used for implantation were harvested during log phase growth and resuspended in phosphate buffered saline (PBS) containing 50% MatrigelTM (BD).
• PBS phosphate buffered saline
• BD MatrigelTM
• mice On the day of tumor implant, each test mouse was injected subcutaneously in the right flank with 1 x 10 7 cells (0.1 ml. cell suspension), and tumor growth was monitored as the average size approached the target range of 100 to 150 mm 3 . Eleven days later, designated as Day 1 of the study, mice were sorted according to calculated tumor size into groups each consisting of ten animals and equivalent group mean tumor volumes. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
• Tumour Volume (mm 3 ) 0.5 (w 2 x I)
• Tumour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm 3 of tumour volume
• Treatment outcome was determined from percent tumor growth delay (%TGD), defined as the percent increase in median TTE for treated versus control mice, with differences between groups deemed statistically significant at P ⁇ 0.05 using logrank survival analysis.
• Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal.
• PR partial regression
• CR complete regression
• the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm 3 for one or more of these three measurements.
• the tumor volume was less than 13.5 mm 3 for three consecutive measurements during the course of the study.
• Treatment tolerability was assessed by body weight measurements and frequent observation for signs of treatment-related side effects. Treatment tolerability was assessed by body weight measurements and frequent observation for signs of treatment-related side effects.
• Phenol I3 (240 mg, 0.5 mmol, 2 eq.) and dibromo-m-xylene 21 (81 mg, 0.253 mmol, 1 eq.) were solubilised in dry DMF (20 ml.) before TBAI (1 1 mg, 0.03 mmol, 0.1 eq.) and K 2 CO 3 (70 mg, 0.5 mmol, 2 eq) were added.
• the mixture was heated to 60°C for an hour at which point the reaction was completed.
• DMF was removed under vacuum and the crude product was purified by Biotage chromatography to give pure product 46 (273 mg, 97% yield).
• ester 46 60mg, 0.054 mmol, 1 eq. was solubilised in CH2CI2 (5 ml.) before PdP(Ph3)4 (3.5 mg, 0.003 mmol, 0.05 eq.) and pyrrolidine (1 1 mI_, 0.135 mmol, 2.5 eq.) were added and the mixture left to stir until complete.
• the organics were diluted with more CH2CI2 and washed with sat NFUCIf aq) , water and brine before being dried with MgS0 4 , filtered and the volatiles removed in vacuo.
• the crude material was purified by Biotage chromatography to give pure product 47 (17.8 mg, 48% yield).
• Ester 46 (185 mg, 0.17 mmol, 1 eq.) was solubilised in a 1 :1 :1 mixture of THF/H 2 0/Me0H (10 ml.) and the mixture was stirred for 15h and after work up, product 48 (59 mg, 32% yield) was isolated.
• ester 48 (59mg, 0.054 mmol, 1 eq.) was solubilised in CH2CI2 (5 mL) before PdP(Ph3)4 (3.5 mg, 0.003 mmol, 0.05 eq.) and pyrrolidine (1 1 pL, 0.135 mmol, 2.5 eq.) were added and the mixture left to stir until complete.
• the organics were diluted with more CH2CI2 and washed with sat NhUCI ⁇ q ) , water and brine before being dried with MgS0 4 , filtered and the volatiles removed in vacuo.
• the crude material was purified by Biotage chromatography to give pure product 49 (17.08 mg, 48% yield).
• Phenol I5 400 mg, 0.8152 mmol, 2.1 eq.
• bromide 56 103 mg, 0.3902 mmol, 1.0 eq.
• K 2 CO 3 1 15 mg, 0.8321 mmol, 2.1 eq.
• the potency of each of the PBD molecules were measured via in vitro cytotox assays in the carcinoma cell line K562.
• Solid PBD material was dissolved in DMSO to a 2 mM stock solution, from which eight serial dilutions were made at a 1 :10 ratio in DMSO and stored at -20°C until use.
• Adherent K562 cells were washed with D-PBS and detached with Trypsin-EDTA, cell density and viability were then determined in duplicate by Trypan blue exclusion assay using an automated cell counter (LUNA-IITM).
• Cell suspension was diluted to 1 x 10 5 cells/ml in growth media (RPMI 1640 with Glutamax + 10% (v/v) HyCloneTM Fetal Bovine Serum) and vortexed before dispensing 2mL per well into sterile 3 mL polypropylene plates. Warhead dilutions were then dispensed into the appropriate wells at 10 mI/well and mixed by repeat pipetting.
• the potency of each of the PBD molecules were measured via in vitro cytotoxicity assays in the carcinoma cell line NCI-N87.
• Solid PBD material was dissolved in DMSO to a 2 mM stock solution, from which eight serial dilutions were made at a 1 :10 ratio in DMSO and stored at -20°C until use.
• Adherent NCI-N87 cells were washed with D-PBS and detached with Trypsin-EDTA, cell density and viability were then determined in duplicate by Trypan blue exclusion assay using an automated cell counter (LUNA-IITM).
• Cell suspension was diluted to 1 x 10 5 cells/ml in growth media (RPMI 1640 with Glutamax + 10% (v/v) HyCloneTM Fetal Bovine Serum) and vortexed before dispensing 2ml_ per well into sterile 3 ml. polypropylene plates. Warhead dilutions were then dispensed into the appropriate wells at 10 mI/well and mixed by repeat pipetting. For control wells 10 m I of DMSO was dispensed onto 2 ml.
• mice Male Sprague Dawley rats (Envigo, Inc) were dosed once by slow bolus intravenous injection via the tail vein with ADC.
• the vehicle for dilution used was 25 mM Histidine-HCI, 7% sucrose, 0.02% Polysorbate 80, pH 6.0.
• Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, clinical pathology (clinical chemistry, hematology, and coagulation), and gross pathology findings. All animals were terminated on Study Day (SD) 29.
• Test article- associated changes in the hematology data were indicative of myelotoxicity and included decreases in all cell lineages (red blood cells, reticulocytes, platelets, neutrophils, lymphocytes, monocytes and eosinophils in most dose groups. The values generally increased at subsequent intervals, indicating reversibility.
• Bzl benzyl where Bzl-OMe is methoxybenzyl and Bzl-Me is methylbenzene

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