WO2022218973A2 - Pyrrolobenzodiazepine conjugates - Google Patents

Pyrrolobenzodiazepine conjugates Download PDF

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Publication number
WO2022218973A2
WO2022218973A2 PCT/EP2022/059733 EP2022059733W WO2022218973A2 WO 2022218973 A2 WO2022218973 A2 WO 2022218973A2 EP 2022059733 W EP2022059733 W EP 2022059733W WO 2022218973 A2 WO2022218973 A2 WO 2022218973A2
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group
methyl
alkyl
phenyl
conjugate according
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PCT/EP2022/059733
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French (fr)
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WO2022218973A3 (en
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Philip Wilson Howard
Luke Masterson
Arnaud Charles Tiberghien
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Medimmune Limited
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    • 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
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present disclosure relates to conjugates comprising pyrrolobenzodiazepine (PBD) or related moity dimers where at least one PBD-related moiety is non-alkylating, and the precursor drug linkers used to make such conjugates.
  • PBD pyrrolobenzodiazepine
  • related moity dimers where at least one PBD-related moiety is non-alkylating
  • PBDs pyrrolobenzodiazepines
  • Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145- 148 (1987)), chicamycin (Konishi, et a/., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J.
  • the PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5'-Pu-GATC-Py-3' interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity.
  • PBD dimer is SG2000 (SJG-136):
  • Dimeric PBD compounds bearing C2 aryl substituents such as SG2202 (ZC-207), are disclosed in WO 2005/085251 : and in W02006/111759, bisulphites of such PBD compounds, for example SG2285 (ZC- 423):
  • 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 2011/130598.
  • 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. If the non-bound N10 position is protected with a capping group, the capping groups exemplified have the same cleavage trigger as the linker to the antibody.
  • WO 2014/057074 describes two specific PBD dimer conjugates bound via the N10 position on one monomer, the other PBD monomer being in imine form.
  • One of the drug-linkers disclosed is SG3249, Tesirine: which, when conjugated to anti-DLL3 rovalpituzumab, is know as rovalpituzumab-tesirine (Rova-T), currently under evaluation for the treatment of small cell lung cancer (Tiberghien, A.C., et al., ACS Med. Chem. Lett., 2016, 7 (11), 983-987; DOI: 10.1021 /acsmedchemlett.6b00062).
  • WO 2015/052322 describes a specific PBD dimer conjugate bound via the N10 position on one monomer, the other PBD monomer being in imine form. It also describes a specific PBD dimer conjugate bound via the N10 position on one monomer, the other PBD monomer having a capping group with the same cleavage trigger as the linker to the antibody:
  • WO201 9/034764 discloses PBD dimer conjugates wherein the PBDs are conjugated to antibodies that are modified so as to have at least one free conjugation site on each heavy chain, and where the conjugation is via each N10 group of the PBD via a linker.
  • WO201 4/096368 discloses PBD dimer conjugates where the PBD moieity which is not linked to the antibody is non-alkylating.
  • conjugates comprising PBD dimers where at least one PBD moiety is non-alkylating (i.e. the released moiety has a secondary amine at the N10 position, rather than an imine or equivalent group), which PBD dimers are conjugated to antibodies that are modified so as to have at least one free conjugation site on each heavy chain, and where the conjugation is via each N10 group of the PBD moiety via a linker.
  • the present disclosure also provides PBD and related dimer drug linkers, where at least one moiety is non-alkylating suitable for conjugating to a modified antibody, where both N10 groups bear linking groups.
  • a first aspect of the present disclosure provides a conjugate of formula I: wherein
  • Ab is a modified antibody having at least one free conjugation site on each heavy chain
  • D represents either group D1 or D2: the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R 2 is selected from the group consisting 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;
  • 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; 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; when there is a single bond present between C2 and C3,
  • R 2 is selected from H, OH, F, diF and , where R 16a and R 16b are independently selected from H, F, C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 16a and R 16b is H, the other is selected from nitrile and a C 1-4 alkyl ester;
  • D' represents either group D'1 or D'2: wherein the dotted line indicates the optional presence of a double bond between C2' and C3'; when there is a double bond present between C2' and C3', R 22 is selected from the group consisting of: (iia) C 5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C 1-7 alkyl, C 3-7 heterocyclyl and bis-oxy-C 1-3 alkylene;
  • each of R 31 , R 32 and R 33 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 12 group is no more than 5;
  • 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; 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; when there is a single bond present between C2' and C3',
  • R 22 is selected from H, OH, F, diF and , where R 26a and R 26b are independently selected from H, F, C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a C 1-4 alkyl ester; R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR', nitro, Me 3 Sn and halo; where R and R' are independently selected from optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups;
  • R 7 is selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR', nitro, Me 3 Sn and halo;
  • R" is a C 3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR N2 (where R N2 is H or C 1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
  • Y and Y' are selected from O, S, and NH;
  • R 11a is: (i) H; or
  • R 6' , R 7' and R 9' are selected from the same groups as R 6 , R 7 and R 9 respectively;
  • R LL1 and R LL2 are linkers connected to the antibody at different sites which are independently: wherein
  • Q x is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;
  • At least one of the N10 positions bearing a linking group releases a non-alkylating PBD (i.e. the released moiety has a secondary amine at the N10 position, rather than an imine or equivalent group).
  • Dimers containing at least one secondary amine moiety are unable to covalently cross-link DNA, and may be less toxic than dimers with two imine moieties (which can covalently cross-link DNA).
  • ADCs which effectively have a drug antibody ratio (DAR) of 1 could offer significant advantages including reduced off-target toxicity and an enhanced therapeutic window by reducing the minimal effective dose requirement over ADCs consisting of heterogeneous mixtures with higher DARs.
  • DAR drug antibody ratio
  • avoiding the presence of a C11 hydroxy group adjacent the carbamate on the N10 nitrogen may increase the stability of the carbamate.
  • the proximity of the hydroxy to the carbonyl of the carbamate allows for the formation of an internal hydrogen bond which could catalyse a more facile nucleophilic attack on the carbonyl.
  • the carbon labelled C3 in D1 is adjacent the ternary N at the ring junction.
  • the carbon labelled C3' in D'1 is adjacent the ternary N at the ring junction.
  • a second aspect of the present disclosure comprises a compound with the formula II: and salts and solvates thereof, wherein D, R 2 , R 6 , R 7 , R 9 , R 11a , Y, R", Y', D', R 6'' , R 7'' , R 9'' and R 22 (including the presence or absence of double bonds between C2 and C3 and C2' and C3' respectively) are as defined in the first aspect of the disclosure;
  • R L1 and R L2 are linkers for connecting to a cell binding agent, which are independently: where Q and X are as defined in the first aspect and G L is a linker for connecting to an antibody.
  • a third aspect of the present disclosure provides the use of a conjugate of the first aspect of the disclosure in the manufacture of a medicament for treating a proliferative disease.
  • the third aspect also provides a conjugate of the first aspect of the disclosure for use in the treatment of a proliferative disease.
  • the third aspect also provides a method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate of the first aspect of the disclosure to a patient in need thereof.
  • a fourth aspect of the present disclosure provides the synthesis of a conjugate of the first aspect of the disclosure comprising conjugating a compound (drug linker) of the second aspect of the disclosure with an antibody as defined in the first aspect of the disclosure.
  • a fifth aspect of the present disclosure provides a method of making a compound of formula IV: from a compound of formula V: using the Mitsunobu reaction; where R 8 ' is selected from:
  • R 6 , R 7 , R 9 , R 11a R 6' , R 7' , R 9' , Y, R", Y', D and D' are as defined in the first aspect of the disclosure;
  • Hal is a halogen, such as Br
  • R L2pre is a precursor to R L2 ;
  • R L1pre is a precursor to R L1 ; and Prot O is a hydroxyl protecting group.
  • 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 1-12 alkyl refers 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).
  • C 1-4 alkyl as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
  • 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 (C 3 ), butyl (C 4 ), pentyl (C 5 ), hexyl (C 6 ) and heptyl (C 7 ).
  • saturated linear alkyl groups include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), n-butyl (C 4 ), n-pentyl (amyl) (C 5 ), n-hexyl (C 6 ) and n-heptyl (C 7 ).
  • 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.
  • C 3-12 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: saturated monocyclic hydrocarbon compounds: cyclopropane (C 3 ), cyclobutane (C 4 ), cyclopentane (C 5 ), cyclohexane (C 6 ), cycloheptane (C 7 ), methylcyclopropane (C 4 ), dimethylcyclopropane (C 5 ), methylcyclobutane (C 5 ), dimethylcyclobutane (C 6 ), methylcyclopentane (C 6 ), dimethylcyclopentane (C 7 ) and methylcyclohexane (C 7 ); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C 3 ), cyclobutene (C 4 ), cyclopentene (C 5 ), cyclohexene (C 6 ), methylcyclopropene (C 4 ), dimethylcyclopropene (C 5 ), methylcycloprop
  • C 3-20 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. C 3-20 , C 3-7 , C 5-6 , etc.
  • the term " C 5-6 heterocyclyl”, 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 1 aziridine (C 3 ), azetidine (C 4 ), pyrrolidine (tetrahydropyrrole) (C 5 ), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C 5 ), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C 5 ), piperidine (C 6 ), dihydropyridine (C 6 ), tetrahydropyridine (C 6 ), azepine (C 7 );
  • O 1 oxirane (C 3 ), oxetane (C 4 ), oxolane (tetrahydrofuran) (C 5 ), oxole (dihydrofuran) (C 5 ), oxane (tetrahydropyran) (C 6 ), dihydropyran (C 6 ), pyran (C 6 ), oxepin (C 7 );
  • N 2 imidazolidine (C 5 ), pyrazolidine (diazolidine) (C 5 ), imidazoline (C 5 ), pyrazoline (dihydropyrazole) (C 5 ), piperazine (C 6 );
  • N 1 O 1 tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ), dihydroisoxazole (C 5 ), morpholine (C 6 ), tetrahydrooxazine (C 6 ), dihydrooxazine (C 6 ), oxazine (C 6 );
  • N 1 S 1 thiazoline (C 5 ), thiazolidine (C 5 ), thiomorpholine (C 6 );
  • O 1 S 1 oxathiole (C 5 ) and oxathiane (thioxane) (C 6 ); and,
  • N 1 O 1 S 1 oxathiazine (C 6 ).
  • substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C 5 ), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C 6 ), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
  • furanoses C 5
  • arabinofuranose such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse
  • pyranoses C 6
  • allopyranose altropyranose
  • glucopyranose glucopyranose
  • mannopyranose gulopyranose
  • idopyranose galactopyranose
  • C 5-20 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 5 to 20 ring atoms.
  • C 5-7 aryl pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 7 ring atoms and the term “C 5-10 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms.
  • each ring has from 5 to 7 ring atoms.
  • the prefixes e.g. C 3-20 , C 5-7 , C 5-6 , C 5-10 , etc.
  • the term " C 5-6 aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.
  • the ring atoms may be all carbon atoms, as in "carboaryl groups".
  • carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C 6 ), naphthalene (C 10 ), azulene (C 10 ), anthracene (C 14 ), phenanthrene (C 14 ), naphthacene (C 18 ), and pyrene (C 16 ).
  • benzene i.e. phenyl
  • C 10 naphthalene
  • azulene C 10
  • anthracene C 14
  • phenanthrene C 14
  • naphthacene C 18
  • pyrene C 16
  • aryl groups which comprise fused rings include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1 H- indene) (C 9 ), indene (C 9 ), isoindene (C 9 ), tetraline (1 ,2,3,4-tetrahydronaphthalene (C 10 ), acenaphthene (C 12 ), fluorene (C 13 ), phenalene (C 13 ), acephenanthrene (C 15 ), and aceanthrene (C 16 ).
  • indane e.g. 2,3-dihydro-1 H- indene
  • indene C 9
  • isoindene C 9
  • acenaphthene C 12
  • fluorene C 13
  • phenalene C 13
  • acephenanthrene C 15
  • the ring atoms may include one or more heteroatoms, as in "heteroaryl groups".
  • heteroaryl groups include, but are not limited to, those derived from:
  • N 1 pyrrole (azole) (C 5 ), pyridine (azine) (C 6 );
  • N 1 O 1 oxazole (C 5 ), isoxazole (C 5 ), isoxazine (C 6 );
  • N 2 O 1 oxadiazole (furazan) (C 5 );
  • N 3 O 1 oxatriazole (C 5 );
  • N 1 S 1 thiazole (C 5 ), isothiazole (C 5 );
  • N 2 imidazole (1 ,3-diazole) (C 5 ), pyrazole (1 ,2-diazole) (C 5 ), pyridazine (1 ,2-diazine) (C 6 ), pyrimidine (1 ,3-diazine) (C 6 ) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (C 6 );
  • N 3 triazole (C 5 ), triazine (C 6 ); and, N 4 : tetrazole (C 5 ).
  • heteroaryl which comprise fused rings include, but are not limited to: C 9 (with 2 fused rings) derived from benzofuran (O 1 ), isobenzofuran (O 1 ), indole (N 1 ), isoindole (N 1 ), indolizine (N 1 ), indoline (N 1 ), isoindoline (N 1 ), purine ( N 4 ) (e.g., adenine, guanine), benzimidazole (N 2 ), indazole (N 2 ), benzoxazole (N 1 O 1 ), benzisoxazole (N 1 O 1 ), benzodioxole (O 2 ), benzofurazan (N 2 O 1 ), benzotriazole ( N 3 ), benzothiofuran (S 1 ), benzothiazole (N 1 S 1 ), benzothiadiazole (N 2 S); C 10 (with 2 fused rings) derived from chromene (O 1
  • C 13 (with 3 fused rings) derived from carbazole (N 1 ), dibenzofuran (O 1 ), dibenzothiophene (S 1 ), carboline (N 2 ), perimidine (N 2 ), pyridoindole (N 2 ); and, C 14 (with 3 fused rings) derived from acridine (N 1 ), xanthene (O 1 ), thioxanthene (S 1 ), oxanthrene (O 2 ), phenoxathiin (O 1 S 1 ), phenazine (N 2 ), phenoxazine (N 1 O 1 ), phenothiazine (N 1 S 1 ), thianthrene (82), 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 C 1-7 alkoxy group, discussed below), a C 3-20 heterocyclyl group (also referred to as a C 3-20 heterocyclyloxy group), or a C 5-20 aryl group (also referred to as a C 5-20 aryloxy group), preferably a C 1-7 alkyl group.
  • Alkoxy -OR, wherein R is an alkyl group, for example, a C 1-7 alkyl group.
  • C 1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n- propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).
  • Oxo (keto, -one): O.
  • R is an acyl substituent, for example, a C 1-7 alkyl group (also referred to as C 1-7 alkylacyl or C 1-7 alkanoyl), a C 3-20 heterocyclyl group (also referred to as C 3-20 heterocyclylacyl), or a C 5-20 aryl group (also referred to as C 5-20 arylacyl), preferably a C 1-7 alkyl group.
  • Carboxy (carboxylic acid): -C( O)OH.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C 1-7 alkyl group (also referred to as C 1-7 alkylamino or di- C 1-7 alkylamino), a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably H or a C 1-7 alkyl group, or, in the case of a "cyclic" amino group, R 1 and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C 1-7 alkyl group (also referred to as C 1-7 alkylamino or di- C 1-7 alkylamino), a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably H or a C 1-7 alkyl group, or, in the case of a "cyclic" amino group, R 1 and R 2 ,
  • Amino groups may be primary (-NH 2 ), secondary (-NHR 1 ), or tertiary (-NHR 1 R 2 ), and in cationic form, may be quaternary (- + NR 1 R 2 R 3 ).
  • Examples of amino groups include, but are not limited to, -NH 2 , -NHCH 3 , -NHC(CH 3 ) 2 , -N(CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , and -NHPh.
  • Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
  • Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C( O)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • Hydroxyl protecting group are well known in the art, for example, in Wuts & Greene 2007. Those groups suitable for use in the present disclosure include substituted methyl ethers, substituted ethyl ethers, methoxy substituted benzyl ethers, silyl ethers and acetates. Of particular relevance are tert-butyldimethylsilyl (TBS) and triisopropylsilyl (TIPS).
  • Amine protecting group Hydroxyl protecting groups are well known in the art, for example, in Wuts & Greene 2007. Those groups suitable for use in the present disclosure include carbamate. Of particular relevance is allyl carbamate (Alloc).
  • Alkylene 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), -CH 2 CH 2 CH 2 CH 2 - (butylene), -CH 2 CH 2 CH 2 CH 2 CH 2 - (pentylene) and -CH 2 CH 2 CH 2 CH-2CH 2 CH 2 CH 2 - (heptylene).
  • Examples of branched saturated C 3-12 alkylene groups include, but are not limited to, -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, -CH(CH 2 CH 3 )-, -CH(CH 2 CH 3 )CH 2 -, and -CH 2 CH(CH 2 CH 3 )CH 2 -.
  • C 3-12 cycloalkylenes examples include, but are not limited to, cyclopentylene (e.g. cyclopent- 1 ,3-ylene), and cyclohexylene (e.g. cyclohex-1 ,4-ylene).
  • C 3-12 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).
  • 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.
  • the Ligand Units for use in the present disclosure are Cell Binding Agents, more specifically modified antibodies, or antigen binding fragments thereof, having at least one conjugation site on each heavy chain.
  • Examples of particular modified antibodies suitable for use according to the present disclosure are disclosed in WO 2012/064733 (filed as PCT/US2011/059775), which is incorporated herein by reference.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific 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.
  • 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.
  • Examples of antibody fragments include F(ab') 2 , and scFv fragments, and dimeric 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.
  • Modified antibodies suitable for use in the present disclosure include those wherein the native interchain cysteine residues have been substituted by amino acid residues lacking thiol groups.
  • the antibodies may comprise at least one additional substitutions in each heavy chain of an amino acid residue comprising a reactive group suitable for conjugation to a linker.
  • the additionally substituted amino acid may be a cysteine or a non-natural amino acid.
  • the position that is substituted may be selected from those set forth below:
  • modified antibodies suitable for use in the present disclosure include the Flexmab structeries disclosed in WO 2012/064733, which is incorporated herein.
  • Flexmabs have cysteines with free thiol groups in the hinge region of the antibody that may be used as conjugation sites for linking through the N1 0 groups of the PBDs of the present disclosure.
  • modified antibodies suitable for use in the present disclosure include those where at least one insertion in each heavy chain of an amino acid residue comprising a reactive group suitable for conjugation to a linker have been made.
  • the inserted amino acid may be a cysteine or a non-natural amino acid.
  • antibodies which have been modified by insertion of a non-natural amino acid at F241 may be used.
  • a non-natural amino acid as employed herein refers to an amino acid which is other than one of the twenty-one naturally occurring amino acids.
  • the non-natural amino acids are generally derived from natural amino acids. Derived from a natural amino acid refers to the fact that the non- natural amino acid is based on (or incorporates) or is similar to the structure of natural amino acid, for example the alkylene chain in lysine may be shortened to provide a 3- carbon chain as opposed to the natural 4 carbon chain but the structural relationship or similarity to lysine still exists.
  • derivatives of natural amino acids include modifications such as incorporating a functional group, lengthening or shortening an alkylene chain, adding one or more substituents to a nitrogen, oxygen, sulfur in a side chain or converting a nitrogen, oxygen or sulfur into a different functional group or a combination of any of the same.
  • modifications such as incorporating a functional group, lengthening or shortening an alkylene chain, adding one or more substituents to a nitrogen, oxygen, sulfur in a side chain or converting a nitrogen, oxygen or sulfur into a different functional group or a combination of any of the same.
  • modification may include removed or replacing an atom naturally found in an amino acid.
  • non-natural amino has a formula (AAII):
  • R x represents an unsaturated group selected from a: i) C 4-9 linear conjugated diene, ii) C 5-14 carbocyclyl comprising a conjugated diene, and iii) a 5 to 14 membered heterocyclyl comprising 1 , 2 or 3 heteroatoms selected O, N and S, and a conjugated diene, wherein i), ii) and iii) may bear up to five substituents, (such as one, two or three substituents) for example, the substituents are independently selected from C 1-3 alkyl, oxo, halogen, sulfo, sulfhydryl, amino, -C 1-3 alkyleneN 3 , or -C 2 - 5alkynyl; and X AA1 represents i) a saturated or unsaturated branched or unbranched C 1-8 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a
  • N, S(O) 0-3 wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino, -C 1-3 alkylene N 3 , or -C 2-5 alkynyl; or ii) together with a carbon from the carbocylcyl or heterocyclyl represents a cyclopropane ring linked to a saturated or unsaturated (in particular saturated) branched or unbranched C 1-6 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a heteroatom selected from
  • amino acid residue referred to in AAII is as defined for AAI above.
  • the amino acid residue refers to an amino acid comprising the -NH 2 and -COOH groups.
  • the amino acid residue in formula AAII may additionally comprise an R group of a natural amino acid.
  • the amino acid residue in formula AAII may be derived from a natural amino acid but have its natural R group replaced with R X -X AA1 -O 0-1 C(O).
  • non-natural amino acid is a residue of the structure of formula (AAIII): wherein
  • X 2 represents -C-, -C(R')-, -CH 2 or O;
  • R' represents H or C 1-3 alkyl
  • R a represents i) a saturated or unsaturated branched or unbranched C 1-8 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O) 0-3 , wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino; or ii) together with a carbon from the 5 membered ring represents a cyclopropane ring linked to a saturated or unsaturated (in particular saturated) branched or unbranched C 1-6 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O) 0-3 , wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino;
  • R b represents H, -OC 1-3 alkyl, C 1-6 alkyl optionally bearing a hydroxyl substituent, -C 1-3 alkyleneN 3 , or -C 2-5 alkynyl;
  • R c represents H, -OC 1-3 alkyl, C 1-6 alkyl optionally bearing a hydroxyl substituent, -C 1-3 alkyleneN 3 , or -C 2-5 alkynyl;
  • R d represents H, -OC 1-3 alkyl, C 1-6 alkyl optionally bearing a hydroxyl substituent, -C 1-3 alkyleneN 3 , or -C 2-5 alkynyl;
  • R e represents H, saturated or unsaturated (in particular saturated) branched or unbranched C 1-8 alkylene chain, wherein one or more carbons are optionally replaced by -O- and the chain is optionally substituted by one or more halogen atoms (such as iodo), N 3 or -C 2 . 5alkynyl.
  • R a is -(CH 2 )mC(O)-, -CH 2 (CH 3 )C(O)-, -(CH 2 )mCH 2 OC(O)-, -CHCHCH 2 OC(O)-, or -OCH 2 CH 2 COC(O)- and m represents 0 or 1.
  • R b is H, -OC 1-3 alkyl, -CH 3 , -CH(CH 3 ) 2 , CH 2 OH, -CH 2 N 3 , or -CCH.
  • R c is H, -OC 1-3 alkyl, -CH 3 , -CH(CH 3 ) 2 , CH 2 OH, -CH 2 N 3 , or -CCH.
  • R d is H, -OC 1-3 alkyl, -CH 3 , -CH(CH 3 ) 2 , CH 2 OH, -CH 2 N 3 , or -CCH.
  • R e represents H or -CH 2 OCH 2 CH 2 N 3 .
  • the non-natural amino acid has the structure of formula (AAlllb): wherein R a , R b , R c , R d , R e and X 2 are defined above.
  • the non-natural amino acid has the structure of formula (AAlllc): (AAlllc) wherein R a , R b , R c , R d , R e are defined above and X 2 ' is -C- or -CR' as defined above.
  • the non-natural amino acid is selected from:
  • Antibodies which have been modified by the insertion of CP2-NNAA are of particular use in the present invention. These are described in as described in WO2019/224340, and Roy et al., MAbs 12 (1): 1684749 (doi:10.1080/19420862.2019.1684749), which are both incorporated herein by reference.
  • antibodies with this insertion at F241 may be used, e.g. modified Herceptin.
  • PBD-analogue dimer is/are attached to said non-natual amino acid(s).
  • the antibody may be to a tumour-associated antigen, for example: HER2 (ErbB2); EPHA2 (EPH receptor A2); CD19; IL2RA (Interleukin 2 receptor, alpha).
  • HER2 ErbB2
  • EPHA2 EPH receptor A2
  • CD19 CD19
  • IL2RA Interleukin 2 receptor, alpha
  • Tumour-associate antigens and cognate antibodies for use in embodiments of the present disclosure are listed below, and are described in more detail on pages 14 to 86 of WO 2017/186894, which is incorporated herein.
  • BMPR1B bone morphogenetic protein receptor-type IB
  • MPF MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin
  • Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b)
  • Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B)
  • PSCA hlg (2700050C12Rik, C530008O16Rik, 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
  • 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, CD79 ⁇ , IGb (immunoglobulin-associated beta), B29
  • FcRH2 IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1 B, SPAP1C
  • 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), pl: 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 role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3,
  • HLA-DOB Beta subunit of MHC class II molecule (la antigen) that binds peptides and 20 presents them to CD4+ T lymphocytes); 273 aa, pl: 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 pl: 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);
  • 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 differentiation
  • IRTA2 Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies; 977 aa, pl: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1 q21 )
  • TENB2 (TMEFF2, tomoregulin, TPEF, HPP1 , TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin); 374 aa)
  • PSMA - FOLH1 Falate 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 (Neural cell adhesion molecule 1 )
  • GPNMB Glycoprotein (transmembrane) nmb
  • TIM-1 - HAVCR1 Hepatitis A virus cellular receptor 1
  • PTK7 protein tyrosine kinase
  • CD37 CD37 molecule
  • 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
  • CS 1 - SLAMF7 SLAMF7
  • V-CAM CD106
  • VCAM1 Vascular cell adhesion molecule 1
  • ASCT2 ASC transporter 2, also known as SLC1 A5
  • ASCT2 antibodies are described in WO 2018/089393, which is incorporated herein by reference.
  • the Ligand unit may be connected to the Linker unit through a disulfide bond.
  • 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.
  • Other possible groups for linking, and the resulting linking groups, are shown below.
  • 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. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit.
  • 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 W02008/070593, which are incorporated herein.
  • the compounds of the present disclosure 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 I.
  • 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 disclosure 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.
  • cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
  • malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma
  • subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
  • 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,
  • the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th 1 -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 amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose.
  • the drug loading (p) is the average number of PBD drugs per cell binding agent, e.g. antibody. In the present disclosure, this is always 1. However, any composition may comprise antibodies where a PBD is conjugated and antibodies where a PBD is not conjugated. Thus for a composition, the drug loading (or DAR) may be less than 1 , for example 0.75 and higher, 0.80 and higher, 0.85 and higher, 0.90 and higher or 0.95 or higher.
  • the Therapeutic Index of a particular drug-linker/conjugate can be calculated by dividing the maximum tolerated single dose (MTD) of a non-targeted ADC in rat, by the minimal effective single dose (MED) of a comparable targeted ADC in mouse.
  • the MED may be the single dose necessary to achieve tumour stasis in an in vivo model at 28 days.
  • a key step in the synthesis of compounds (drug-linkers) of formula II (and similar compounds) is the synthesis of compounds of formula IV: from compounds of formula V: by using a Mitsunobu reaction to close the ring.
  • R 8 ' should be selected from:
  • R 8 When R 8 is (Vc), compounds of formula II may be synthesised from compounds of formula IV by converting R L1-pre into R L1 and R L2-pre into R L2 .
  • the precursors of R L1 and R L2 will comprise:
  • Prot N is an amine protecting group, such as Alloc.
  • R 8 ' is (Vb)
  • compounds of formula IV where R 8 is (Vc) may be synthesised by coupling a compound of formula Via: to the compound of formula IV when R 8 is -Y'-R"-HaL
  • compounds of formula IV where R 8 is (Vc) may be synthesised by coupling a compound of formula Vlb: to the compound of formula IV when R 8 is -OH.
  • the compound of formula VII may be symmetrical, i.e. both moieties linked by -Y'-R"-Y- may be identical.
  • the removal of only one Prot o group may be achieved by a statistical approach, as illustrated in the examples.
  • Antibodies can be conjugated to the Drug Linker compound generally as described in Doronina et al., Nature Biotechnology, 2003, 21 , 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37 °C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5'-dithiobis(2- nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved. The reduced antibody is then cooled to 0°C and alkylated with 3 eguivalents of drug-linker per antibodyl.
  • TCEP tris(carboxyethyl)phosphine hydrochloride
  • the reaction is guenched by the addition of 5 eguivalents 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 ⁇ m 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-guenched drug-linker.
  • the following preferences may apply to all aspects of the disclosure as described above, or may relate to a single aspect.
  • the preferences may be combined together in any combination.
  • 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 22 is the same as R 2 .
  • Y and Y' are both O.
  • R" is a C 3-7 alkylene group with no substituents. In some of these embodiments, R" is a C 3 , C 5 or C 7 alkylene. In particular, R" may be a C 3 or C 5 alkylene.
  • R" is a group of formula: where r is 1 or 2.
  • R" is a group of formula: where r is 1 or 2.
  • R and R' are independently selected from optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups, wherein the optional substituents are selected from C 1-12 alkyl, C 3-20 heterocyclyl, C 5-20 aryl, halo, hydroxy, ether, alkoxy, oxo, acyl, carboxy, ester, amino, amido, nitro, and cyano.
  • R 9 is H.
  • R 6 is selected from H, OH, OR, SH, NH 2 , 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, NH 2 , NHR, NRR', and halo.
  • R 7 is selected from H, OH and OR, where R is selected from optionally substituted C 1-7 alkyl, C 3-10 heterocyclyl and C 5-10 aryl groups.
  • R may be more preferably a C 1-4 alkyl group, which may or may not be substituted.
  • a substituent of interest is a C 5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7- positions are OMe and OCH 2 Ph.
  • Other substituents of particular interest are dimethylamino (i.e.
  • D and D' are D1 and D'1 respectively.
  • D and D' are D2 and D'2 respectively.
  • R 2 is selected from:
  • 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;
  • 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; 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.
  • 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 C 8-10 aryl, for example a quinolinyl or isoquinolinyl group.
  • the quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position.
  • 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 C 5-10 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 C 5-7 aryl group
  • a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably ⁇ or ⁇ to the bond to the remainder of the compound. Therefore, where the C 5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para- position.
  • R 2 is a C 8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. 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 C 5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
  • R 2 when R 2 is a C 5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C 1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C 5-7 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 C 5-10 aryl group is C 1-7 alkyl, it may preferably be a C 1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).
  • a substituent on R 2 when R 2 is a C 5-10 aryl group is C 3-7 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 C 1-4 alkyl groups. If the C 6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
  • 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.
  • Particularly preferred substituted 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 2 group is 4-nitrophenyL R 2 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.
  • 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 11 is H.
  • R 12 is H.
  • R 13 is H.
  • R 11 and R 12 are H.
  • R 11 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 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, C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 16a and R 16b is H, the other is selected from nitrile and a C 1-4 alkyl ester.
  • R 2 is H.
  • R 2 is
  • 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 C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
  • the group which is not H is selected from methyl and ethyl.
  • R 22 is selected from: (a) C 5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C 1-7 alkyl, C 3-7 heterocyclyl and bis-oxy- C 1-3 alkylene;
  • each of R 31 , R 32 and R 33 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 22 group is no more than 5;
  • 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; 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.
  • R 22 When R 22 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 22 is preferably phenyl.
  • R 22 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
  • R 22 When R 22 is a C 5-10 aryl group, it may be a C 8-10 aryl, for example a quinolinyl or isoquinolinyl group.
  • the quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position.
  • 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 22 is a C 5-10 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 22 is C 5-7 aryl group
  • a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably ⁇ or y to the bond to the remainder of the compound. Therefore, where the C 5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para- position.
  • R 22 is a C 8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. 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 22 substituents, when R 22 is a C 5-10 aryl group
  • R 22 when R 22 is a C 5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
  • R 22 when R 22 is a C 5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C 1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C 5-7 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 22 when R 22 is a C 5-10 aryl group is C 1-7 alkyl, it may preferably be a C 1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).
  • a substituent on R 22 when R 22 is a C 5-10 aryl group is C 3-7 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 C 1-4 alkyl groups. If the C 6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom. If a substituent on R 22 when R 22 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 22 when R 22 is a C 5-10 aryl group is ester, this is preferably methyl ester or ethyl ester.
  • R 22 is a C 5-10 aryl group
  • substituents when R 22 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 22 are dimethylaminopropyloxy and carboxy.
  • Particularly preferred substituted R 22 groups when R 22 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 22 group is 4-nitrophenyL R 22 groups of particular interest include 4-(4- methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
  • R 22 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 22 When R 22 is C 3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
  • each of R 31 , R 32 and R 33 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 22 group is no more than 5. In some embodiments, the total number of carbon atoms in the R 22 group is no more than 4 or no more than 3. In some embodiments, one of R 31 , R 32 and R 33 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 31 , R 32 and R 33 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 are not H are methyl.
  • R 31 is H.
  • R 32 is H.
  • R 33 is H.
  • R 31 and R 32 are H.
  • R 31 and R 33 are H.
  • R 32 and R 33 are H.
  • R 22 group of particular interest is:
  • R 22 is , one of R 25a and R 25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl.
  • 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 24 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 24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R 24 is selected from H and methyl.
  • R 22 is H or , where R 26a and R 26b are independently selected from H, F, C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a C 1-4 alkyl ester.
  • R 22 is H.
  • R 22 is
  • R 26a and R 26b are both H.
  • R 26a and R 26b are both methyl.
  • R 26a and R 26b are H, and the other is selected from C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
  • the group which is not H is selected from methyl and ethyl.
  • R 11a is H.
  • R 11a is OH
  • R 11a is OR A , where R A is C 1-4 alkyl. In some of these embodiments, R A is methyl.
  • R A is C 1-4 alkyl. In some of these embodiments, R A is methyl.
  • R 2a and R 22a are the same and are selected from:
  • R 1a is selected from methyl and benzyl
  • R LL1 , R LL2 and R 11a are as defined above.
  • both R 2 and R 22 comprise no more than 3 carbon atoms.
  • R 2 may be selected from:
  • R 2 may be selected from:
  • R 22 may be selected from:
  • R 22 may be selected from:
  • both R 2 and R 22 comprise no more than 2 carbon atoms.
  • R 2 may be selected from:
  • R 2 may be selected from:
  • R 22 may be selected from:
  • R 22 may be selected from:
  • both R 2 and R 22 comprise no more than 1 carbon atom.
  • R 2 may be methyl.
  • R 2 may be selected from: (i) H;
  • R 22 may be methyl.
  • R 22 may be selected from: (i) H;
  • the use of the glucuronide capping unit in these drug linkers is believed to be particularly advantageous, as it significantly increases the hydrophilicity of the drug linker, making the drug linkers easier to conjugate to a ligand unit.
  • G L may be selected from
  • Ar represents a C 5-6 arylene group, e.g. phenylene, and X 1 represents C 1-4 alkyl.
  • 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 may be selected from: where CBA represents the point of connection to the modified antibody, Ar represents a C 5- 6 arylene group, e.g. phenylene and X 1 represents C 1-4 alkyl.
  • G LL is selected from G LL1-1 and G LL1-2 . In some of these embodiments, G LL is G LL1-1 . In other embodiments, G LL is G LL1-1A . G LL1-1A may be formed by a Diels-Alder reaction between G L1-1 and a spirocyclopropyl- cyclopentadiene of formula: . Such a group can be incorporated into the antibody via the addition of a linker or by incorporating a non-natural amino acid into the polypeptide sequence, as described in WO2019/224340, which is incorporated herein by reference).
  • a may be 0, 1 , 2, 3, 4 or 5.
  • a 0 to 3.
  • a 0 or 1 .
  • a 0.
  • b may be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16.
  • b is 0 to 12.
  • 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.
  • d is 0 to 3. In some of these embodiments, 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, Vai, 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:
  • 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:
  • dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
  • Q 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.
  • Tripeptide linkers of particular interest are:
  • Q is a tetrapeptide residue.
  • the amino acids in the tetrapeptide may be any combination of natural amino acids and non-natural amino acids.
  • the tetrapeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide then is a recognition site for cathepsin.
  • Tetrapeptide linkers of particular interest are:
  • NH - represents the N-terminus
  • 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.
  • the first aspect of the disclosure comprises a conjugate of formula Id:
  • n is an integer from 2 to 8
  • X is selected from:
  • the first aspect of the disclosure comprises a conjugate of formula le: where m is an integer from 2 to 8, and X is selected from:
  • the Drug linker (D L ) is of formula (lid): where m is an integer from 2 to 8, and X is as defined above.
  • R L1 and R L2 are different.
  • R LL1 and R LL2 are different.
  • differences may only be in the G groups, such that the remainder of the linking groups are the same (so that the cleavage triggers are the same).
  • the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups: In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
  • Compounds of particular interest include those of the examples.
  • Flash chromatography was performed using a Biotage Isolera OneTM using gradient elution on SNAP UltraTM columns starting from either 88% hexane/EtOAc or 99.9% DCM/MeOH until all UV active components (detection at 214 and 254 nm) 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 aluminum plates. Visualization of TLC was achieved with UV light or iodine vapor unless otherwise stated. Extraction and chromatography solvents were bought and used without further purification from VWR UK.
  • the column was a Waters Acquity UPLC® BEH Shield RP18 1 .7 ⁇ m 2.1 mm x 50 mm fitted with a Waters Acquity UPLC® BEH Shield RP18 VanGuard pre-column, 130 A, 1.7 ⁇ m, 2.1 mm x 5 mm at 50 °C.
  • Method 1 Gradient started with initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 min 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 min with a sample injection volume of 2 ⁇ L and detection at 223 nm and 254 nm.
  • Method 2 Gradient started with initial composition 25% B held over 25 seconds, then increased from 25% B to 100% B over a 1 min 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 min with a sample injection volume of 2 pL and detection at 223 nm and 254 nm.
  • Method 3 Gradient started with initial composition 5% B held over 1 min 25 seconds, then increased from 5% B to 100% B over a 9 min 35 seconds period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 10 seconds and held there for 2 min. The total duration of the gradient run was 15.0 min with a sample injection volume of 2 pL and detection at 223 nm and 254 nm.
  • the preparative HPLC conditions were as follows: reverse-phase UPLC was carried out on a Shimazdzu Prominence® machine using a Phenomenex® Gemini NX 5 ⁇ m C18 column 150 mm x 21.2 mm fitted with a Phenomenex® Gemini SecurityGuard PREP Cartridge NX C18 15 mm x 21.2 mm at 50 °C. Eluents used were solvent A (H 2 O with 0.01% formic acid) and solvent B (CH 3 CN with 0.01% formic acid).
  • Tetrakisacetonitrile copper(l) triflate (33.1 mg, 0.0878 mmol) was added to a solution of 5 (620 mg, 0.439 mmol), and Stahl tempo solution in acetonitrile (0.439 mL, 0.0878 mmol, 0.200 mol/b) in dichloromethane (12.0 mL), under an atmosphere of air (air ballon).
  • the solution was stirred at 34°C for 18h, when completion was observed by LCMS.
  • the solution was loaded directly on a biotage samplet, dried, and eluted on a 50g Ultra column with 4/1 DCM/MeOH in DCM, gradient from 15% to 30%. Elution around 20%.
  • the reaction mixture was partitioned between DCM (10 mL), 1 M aqueous ammonium chloride (10 mL). The organic layer was decanted through an isolera cartridge, and the volatiles were removed under vacuum. Half of this material (229 mg, 0.184 mmol) was dissolved in DCM (5.00 mL) and methanol (0.2 mL), followed by mal-amido-peg8-acid (245 mg, 0.405 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (78.0 mg, 0.407 mmol). The reaction was allowed to proceed at room temperature for 45 min when completion was observed by LCMS.
  • the reaction mixture was concentrated (2 mL), loaded on a 3g biotage silica samplet and dried under vacuum.
  • the samplet was loaded on a 25g Ultra Biotage column, and eluted (gradient 15/90 to 60/40 of 20% MeOH in DCM / DCM in 12CV; Elution from around 40/60; gradient allowed to increase to avoid streaking). All fractions were analysed by TLC (15% MeOH in DCM). The pure fractions were pooled. The solvent was removed by evaporation to give 7 (390 mg, 0.163 mmol, 88.5% Yield). The purity was 94.5%.
  • Diisopropyl azodicarboxylate (0.406 mL, 2.02 mmol, 98 mass%) was added to a solution of 9 (1 .45 g, 0.918 mmol) and triphenylphosphine (0.726 g, 2.75 mmol) in tetrahydrofuran (26.0 mL).
  • the reaction was heated at 40°C for 2h, at which point a satisfactory amount of product was formed by LCMS.
  • the volatiles were removed under vacuum and the residue was purified by chromatography (50g Ultra, Biotage, EtOAc/EtOH 4/1 in Hexane, gradient from 20% to 57%. Elution around 45% upwards.
  • the reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL), followed by saturated aqueous hydrogen carbonate (50 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified with a first chromatography (50 g ultra, 4/1 DCM/MeOH in DCM, gradient from 5% to 25%, elution from 24% to 25%); The pure fractions were pooled and the volatiles removed under vacuum to give 11 (544 mg, 0.376 mmol, 81 .5% Yield) as a white solid.
  • Tetrakisacetonitrile copper(l) triflate (27.1 mg, 0.0719 mmol) was added to a solution of 11 (520 mg, 0.359 mmol) and Stahl TEMPO solution in acetonitrile (0.359 mL, 0.0718 mmol, 0.2 mol/L) in dichloromethane (8.00 mL), under an atmosphere of air (air balloon). The solution was stirred at 34°C for 18h, when completion was observed by LCMS. The solution was loaded directly, and eluted, on a 50g Ultra column with 4/1 DCM/MeOH in DCM, gradient from 15% to 30% Elution around 20 to 22%.
  • the reaction mixture was partitioned between DCM (10 mL), 1 M aqueous ammonium chloride (10 mL). The organic layer was decanted through an isolera cartridge, and the volatiles were removed under vacuum to give the crude deprotected amine (177 mg, 0.139 mmol), which was redissolved in dichloromethane (5.00 mL) and methanol (0.2 mL), followed by addition of mal-amido-peg8-acid (245 mg, 0.405 mmol, 98 mass%) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (78.0 mg, 0.407 mmol, 100 mass%). The reaction was allowed to proceed at room temperature for 45 min when completion was observed by LCMS. The reaction mixture was concentrated (2 mL), loaded on a 3g biotage silica samplet and dried under vacuum.
  • the solution was stirred at -35°C for 30 min at which point completion was indicated by LCMS and TLC (EtOAc / hexane 1/3).
  • the solution was diluted with ethyl acetate (100 mL) and water (100 mL).
  • the organic layer was washed further with 0.02 N HCI (100 mL), followed by saturated bicarbonate (100 mL), and brine (50 mL).
  • the organics were dried with magnesium sulfate and concentrated down to around 50 mL under vacuum.
  • the solution was diluted with toluene (100 mL).
  • the solution underwent two vacuum/argon cycles, followed by addition of potassium phosphate dibasic (36.7 g, 206 mmol, 98.0 mass%), 4-methoxyphenylboronic acid (7.01 g, 44.7 mmol), tetrakis(triphenylphosphine)palladium(0) (800 mg, 0.689 mmol), and water (30 mL).
  • the reaction was stirred under argon at room temperature for 1 h, at which point TLC and LCMS indicated reaction completion.
  • the mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL) and brine (50 mL). The organics were dried over magnesium sulfate and concentrated under vacuum.
  • Zinc 39.7 g, 606 mmol was added to mixture of water (5.50 mL), acetic acid (5.50 mL, 95.9 mmol) and ethanol (88.0 mL) at 0°C.
  • a further 20g of zinc was added, together with another 5 mL of acid and water. The reaction was allowed to warm up to room temperature slowly over 3h, at which point completion was observed. The solids were removed by filtration over a bed of celite.
  • Triphosgene (1.46 g, 4.87 mmol, 99 mass%) was added to a stirred solution of 16 (8.70 g, 13.6 mmol) in dry dichloromethane (87.0 mL, 99.5 mass%) at -10°C, followed by dry triethylamine (4.18 mL, 29.8 mmol, 99.5 mass%). The mixture was allowed to warm up to room temperature.
  • the reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL), followed by saturated aqueous hydrogen carbonate (50 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified with a first chromatography (340 g ultra, EtOAc/Hexanes 35/65 up to 100/0 in 6 CV. Elution from 90/10); The pure fractions were pooled and the volatiles removed under vacuum to give 18 (11.8 g, 12.7 mmol, 99.6% Yield) as a yellow solid.
  • Lithium acetate (1.3 g, 20 mmol) was added to a solution of 19 (8.40 g, 9.21 mmol) in DMF (42.0 mL, 543 mmol) and water (1.7 mL, 94 mmol). The solution was stirred at 40°C for 1 h and was then partitioned between 2-MeTHF (250 mL) and water (400 mL). The organics were washed with brine (150 mL) and dried over magnesium sulfate. The solution was concentrated under vacuum. The residue was purified by chromatography (100g Ultra, gradient 50/50 EtOAc / Hexane up 100% EtOAc). The pure fractions were concentrated and dried under vacuum. The solids were taken up in diethylether (100 mL), collected by filtration and dried to give 20 (5.0 g, 6.6 mmol, 72% Yield) as an off-white powder.
  • the mixture was heated at 50°C for 1 .5h, at which point LCMS showed conversion of the phenol to the mono-alkylated and bis-alkylated products.
  • the mixture was decanted, and the supernatant was evaporated to dryness.
  • the residue was loaded on a 10g silica sample!, dried under vacuum, and loaded on top of a 50g Ultra Biotage column.
  • the mixture was purified by chromatography (gradient EtOAc / Hexane 25/75 up to 100% EtOAc, followed by EtOAc / EtOH 90/10). The fractions were pooled and the volatiles were removed under vacuum to give the product 21 (462 mg, 0.492 mmol, 37.2% Yield), and 22 (531 mg, 0.329 mmol, 49.8% Yield), as pale yellow solids.
  • Gradient started with initial composition 10% B which increased to 50% B over a 6 minute period, then further increased to 95% over a 2 minute period before returning to 10% B over 5 seconds and held there for 2 minutes.
  • the total duration of the gradient run was 10 minutes with a sample injection volume of 0.1 ⁇ L and detection at 220 nm.
  • a GreenSep Basic (3 x 15 cm, 5 ⁇ m) column was used with a flowrate of 60 mL/min at a temperature of 35 °C at a pressure of 100 bar.
  • Mobile phases used were A (CO 2 ) and B (MeOH with 0.1 % 2 M NH 3 -MeOH).
  • An isocratic gradient of 35% B was used with a total gradient duration of 9 minutes with detection at 220 nm.
  • a 350 g (3-25 ⁇ m, 100 A) column was used with a flowrate of 100 mL/min.
  • Mobile phases used were solvent A (H 2 O with 0.01 % formic acid) and solvent B (CH 3 CN with 0.01 % formic acid).
  • LiOAc (0.8 eq.) was added to a solution of compound 18 (1.0 eq.) in DMF (5 V) and water (0.1 V) under a nitrogen atmosphere at between 20 °C and 30 °C. The reaction was stirred at this temperature for 8 hours at which point LCMS showed reaction completion.
  • the solution was diluted with EtOAc (20 V) and cooled to between 0 °C and 10 °C. Aq. citric acid (0.2 M, 10 V) was slowly added and the resultant layers were separated.
  • the aqueous phase was further extracted with EtOAc (10 V x 2).
  • the combined organic layers were washed with brine (5 V) before the brine phase was extracted with EtOAc (5 V). The organic layers were combined and concentrated to dryness.
  • LiOAc (0.8 eq.) was added to a solution of compound 16 (1 .0 eq.) in DMF (5 V) and water (0.1 V) under a nitrogen atmosphere at between 20 °C and 30 °C. The reaction was stirred at this temperature for 8 hours at which point LCMS showed reaction completion.
  • the solution was diluted with MTBE (5 V) and washed with water (5 V x 2).
  • the aqueous phase was extracted with MTBE (5 V) and the combined organic layers were filtered through a silica gel pad and concentrated.
  • Heptane (10 V) was added to the crude product and after 30 minutes at 25 °C the slurry was filtered and the cake was washed with heptane (1.5 V) to give 21. 90% yield on 35 g scale. Retention time: 8.26 min (HPLC Method 1 ).
  • reaction was stirred at 34 °C for 48 hours at which point LCMS showed reaction completion.
  • the reaction mixture was concentrated and the resultant material was purified by column chromatography (0-10% MeOH in DCM). The pure fractions were pooled and the volatiles removed under vacuum to give 12.
  • MAL-dPEG® 8 -acid (2.93 eq.) and EDCI (2.95 eq.) were added to a solution of compound 12A (1 .0 eq.) in DCM (25 V) and MeOH (1 V) at between 20 °C and 25 °C under an atmosphere of argon in the dark.
  • the reaction was stirred at 50 °C for 2 hours at which point LCMS showed reaction completion.
  • the reaction was concentrated and the resultant material was purified by column chromatography (0-20% MeOH in DCM). The pure fractions were pooled and the volatiles removed under vacuum.
  • Herceptin, R347 and 1 c1 antibodies engineered to have cysteine inserted between the 239 and 240 positions were produced following the methods described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI:
  • DTT DL-dithiothreitol
  • PBS phosphate-buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • the reduced antibody was buffer exchanged, via spin filter centrifugation, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
  • Compound 7 was added as a DMSO solution (2.5 molar equivalent/antibody, 333 nanomoles, in 2.0 mL DMSO) to 18 mL of this reoxidised antibody solution (20 mg, 133 nanomoles) for a 10% (v/v) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (1.67 micromoles, 16.7 ⁇ L at 100 mM) for 30 min at room temperature, then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
  • a 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 20 micromoles, 400 ⁇ L) to a 14.6 mL solution of antibody (30 mg, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL.
  • 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, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
  • Compound 13 was added as a DMSO solution (2.5 molar equivalent/antibody, 0.5 micromoles, in 3.0 mL DMSO) to 27 mL of this reoxidised antibody solution (30 mg, 200 nanomoles) for a 10% (v/v) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.5 micromoles, 25 ⁇ L at 100 mM) for 30 min at room temperature.
  • the conjugation mixture was diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->25% then 25->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min.
  • Drug-to-antibody ratio 1 (DAR 1 ) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • 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 and 1 mM EDTA to remove all the excess reducing agent.
  • TMF Tangential Flow Filtration unit
  • Compound 7 was added as a DMSO solution (2.5 molar equivalent/antibody, 3.33 micromoles, in 10 mL DMSO) to 90 mL of this reoxidised antibody solution (200 mg, 1.33 micromoles) for a 10% (v/v ) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of N- acetyl cysteine (10 micromoles, 100 ⁇ L at 100 mM) for 30 min at room temperature.
  • the conjugation mixture was sterile filtered, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto 2 x 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP columns, eluting with 0->20% then 20->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 120 mL (12 CV) at 5 mL/min.
  • HIC Hydrophobic Interaction Chromatography
  • Drug-to-antibody ratio 1 (DAR 1) fractions were pooled and buffer exchanged into 25 mM Histidine, 205 mM Sucrose pH 6.0 via TFF using mPES, MidiKros® 30 kDa fiber filter with 115 cm 2 surface area, sterile-filtered and analysed.
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • 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 and 1 mM EDTA to remove all the excess reducing agent.
  • TMF Tangential Flow Filtration unit
  • Compound 13 was added as a DMSO solution (2.0 molar equivalent/antibody, 2.67 micromoles, in 10 mL DMSO) to 90 mL of this reoxidised antibody solution (200 mg, 1.33 micromoles) for a 10% (v/v ) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of N- acetyl cysteine (8 micromoles, 80 ⁇ L at 100 mM) for 30 min at room temperature.
  • the conjugation mixture was sterile filtered, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto 2 x 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP columns, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 150 mL (15 CV) at 5 mL/min.
  • HIC Hydrophobic Interaction Chromatography
  • Drug-to- antibody ratio 1 (DAR 1 ) fractions were pooled and buffer exchanged into 25 mM Histidine, 205 mM Sucrose pH 6.0 via TFF using mPES, MidiKros® 30 kDa fiber filter with 115 cm 2 surface area, sterile-filtered and analysed.
  • a 1 M solution of DL-d ith ioth reitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 33.3 micromoles, 33.3 ⁇ L) to a 25 mL solution of antibody (50 mg, 333 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL.
  • 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, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
  • Compound 7 was added as a DMSO solution (2.5 molar equivalent/antibody, 417 nanomoles, in 2.5 mL DMSO) to 22.5 mL of this reoxidised antibody solution (25 mg, 167 nanomoles) for a 10% (v/v) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.08 micromoles, 20.8 ⁇ L at 100 mM) for 30 min at room temperature.
  • the conjugation mixture was diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 4 mL/min.
  • Drug-to-antibody ratio 1 (DAR 1 ) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
  • a 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 20 micromoles, 400 ⁇ L) to a 14.6 mL solution of antibody (30 mg, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL.
  • 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, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
  • Compound 13 was added as a DMSO solution (2.5 molar equivalent/antibody, 0.5 micromoles, in 3.0 mL DMSO) to 27 mL of this reoxidised antibody solution (30 mg, 200 nanomoles) for a 10% (v/v) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of N-acetyl cysteine (2.5 micromoles, 25 ⁇ L at 100 mM) for 30 min at room temperature.
  • the conjugation mixture was diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min.
  • Drug-to-antibody ratio 1 (DAR 1 ) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • the reduced antibody was buffer exchanged, via spin filter centrifugation using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing 30 mM Histidine, 30 mM Arginine pH 6.8 and 1 mM EDTA to remove all the excess reducing agent.
  • Compound 24 was added as a DMSO solution (3.0 molar equivalent/antibody, 0.8 micromoles, in 8 mL DMSO) to 72 mL of this reoxidised antibody solution (40 mg, 267 nanomoles) for a 10% (v/v ) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at +37 °C with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.4 micromoles, 24 ⁇ L at 100 mM) for 30 min at room temperature.
  • the conjugation mixture was sterile filtered, concentration to ⁇ 30 mL, diluted ⁇ 4x with 10 mM sodium phosphate pH 6.0 CHT Buffer A and loaded onto a 5 mL Bio-Scale Mini CHT ceramic hydroxyapatite 40 ⁇ m Type II cartridge, eluting with 0->100% 10 mM sodium phosphate, 1 M sodium chloride pH 6.0 CHT Buffer B over 100 mL (20 CV) at 3.5 mL/min.
  • High monomeric purity fractions were pooled, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min.
  • Drug-to-antibody ratio 1 (DAR 1) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile- filtered and analysed.
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • the reduced antibody was buffer exchanged, via spin filter centrifugation using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing 30 mM Histidine, 30 mM Arginine pH 6.8 and 1 mM EDTA to remove all the excess reducing agent.
  • Compound 24 was added as a DMSO solution (3.0 molar equivalent/antibody, 0.8 micromoles, in 8 mL DMSO) to 72 mL of this reoxidised antibody solution (40 mg, 267 nanomoles) for a 10% (v/v ) final DMSO concentration.
  • the solution left to react at room temperature for 20 hours at +37 °C with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.4 micromoles, 24 ⁇ L at 100 mM) for 30 min at room temperature.
  • the conjugation mixture was sterile filtered, concentration to ⁇ 30 mL, diluted ⁇ 4x with 10 mM sodium phosphate pH 6.0 CHT Buffer A and loaded onto a 5 mL Bio-Scale Mini CHT ceramic hydroxyapatite 40 ⁇ m Type II cartridge, eluting with 0->100% 10 mM sodium phosphate, 1 M sodium chloride pH 6.0 CHT Buffer B over 100 mL (20 CV) at 3.5 mL/min.
  • High monomeric purity fractions were pooled, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min.
  • Drug-to-antibody ratio 1 (DAR 1) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile- filtered and analysed.
  • Anti-HER2 trastuzumab-derived antibodies were expressed containing a lysine analogue bearing a cyclopentadiene as the reactive group (SCpHK/CP2) at F241 (EU numbering according to Kabat) according to the method described in Roy et al., MAbs 12 (1): 1684749 (doi:10.1080/19420862.2019.1684749).
  • Fractions were individually analysed by RP-HPLC and fractions containing > 90% bridged heavy chain with 1 molecule of 13, ⁇ 5% of unconjugated heavy chain, and ⁇ 5% of heavy chain conjugated to 1 molecule of 13 were pooled and then concentrated and formulated in 20 mM His/His HCI, 240 mM sucrose pH 6.0 by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile- filtered and analysed.
  • the in vitro activity of ADCs was measured in the Her2-expressing cell line NCI-N87 and the Her2 negative cell line MDA-MB-468.
  • the concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask are measured by trypan blue staining, and counted using the LUNA-IITM Automated Cell Counter. Cells were diluted to 2x10 5 /ml, dispensed (50 ⁇ L per well) into 96-well flat-bottom plates.
  • a stock solution (1 ml) of antibody drug conjugate (ADC) (20 ⁇ g/m I) 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 ⁇ L into 900 ⁇ L of cell culture medium.
  • ADC dilution was dispensed (50 ⁇ L per well) into 4 replicate wells of the 96-well plate, containing 50 ⁇ L cell suspension seeded the previously. Control wells received 50 ⁇ L cell culture medium.
  • the 96-well plate containing cells and ADCs was incubated at 37°C in a CO 2 -gassed incubator for the exposure time.
  • MTS MTS (Promega) was dispensed (20 ⁇ L per well) into each well and incubated for 4 hours at 37°C in the CO 2 -gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC 50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope.
  • ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87.
  • MDA-MB- 468 and NCI-N87 were cultured in RPMI 1640 with Glutamax + 10% (v/v) HyCloneTM Fetal Bovine Serum.
  • the in vitro activity of ADCs was measured in the Her2-expressing cell line NCI-N87.
  • the concentration and viability of cells from a sub-confluent (80-90% confluency) T175 flask are measured and counted using the Vi-Cell BLU automated cell viability analyzer. Cells were diluted to 2.5x10 5 /ml, dispensed (80 ⁇ L per well) into 96-well, white wall, flat- bottom plates.
  • a 5x stock solution of antibody drug conjugate (ADC) (200 ⁇ g/ml ) was made by dilution of
  • ADC into cell culture medium A set of 4-fold dilutions of stock ADC were made in a 96-well
  • U-bottomed plate by serial transfer of 75 ⁇ L into 225 ⁇ L of cell culture medium.
  • ADC dilution was dispensed (20 ⁇ L per well) into 2 replicate wells of the 96-well plate, containing 80 ⁇ L cell suspension seeded the previously. Control wells received 20 ⁇ L cell culture medium.
  • the 96-well plate containing cells and ADCs was incubated at 37°C in a CO 2 -gassed incubator for the exposure time.
  • cell viability was measured by CellTiterGlo® assay.
  • CellTiterGlo® Promega was prepared according to manufacturers instructions (100 ⁇ L per well) into each well and incubated for 15 minutes at 4°C on a plate shaker.
  • Well luminescence was measured using an Envision plate reader.
  • Percentage cell survival was calculated from the mean luminescence in the 2 ADC-treated wells compared to the mean absorbance in the 6 control untreated wells (100%).
  • IC 50 was determined from the dose- response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope.
  • ADC incubation times were 6 days for NCI-N87.
  • NCI-N87 were cultured in RPMI 1640 with D-Glucose, HEPES, L-Glutamine, Sodium Bicarbonate, Sodium Pyruvate + 10% (v/v) GibcoTM HI Fetal Bovine Serum.
  • mice Female severe combined immune-deficient mice (Fox Chase SCID®, C.B-17 lIcr-Prkdcscid, Charles River) were:
  • mice 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'cobs TM 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 G sodium, 100 ⁇ g/mL streptomycin sulfate and 25 ⁇ g/mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO 2 and 95% air.
  • mice were sorted according to calculated tumour size into groups each consisting of ten animals with individual tumour volumes ranging from:
  • A. 108 to 172 mm 3 and group mean tumour volumes of 131 mm 3 ;
  • Tumours were measured in two dimensions using calipers, and volume was calculated using the formula:
  • A.ConjA (2 mg/kg) and ConjG (5 mg/kg) was administered intravenously once on Day 1 (qd x 1).
  • a vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 51.
  • ConjB (2 and 6 mg/kg) was administered intravenously once on Day 1 (qd x 1 ).
  • a vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 93.
  • TTE time to endpoint
  • Tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 800 mm 3 or at the end of the study, whichever came first. Animals that exited the study for tumor volume endpoint were documented as euthanized for tumor progression (TP), with the date of euthanasia.
  • TTE time to endpoint
  • TTE time to endpoint
  • the calculated TTE is usually less than the TP date, the day on which the animal was euthanized for tumor size. Animals with tumors that did not reach the endpoint volume were assigned a TTE value equal to the last day of the study. In instances in which the log-transformed calculated TTE preceded the day prior to reaching endpoint or exceeded the day of reaching tumor volume endpoint, a linear interpolation was performed to approximate the TTE. Any animal classified as having died from NTR (non-treatment-related) causes due to accident (NTRa) or due to unknown etiology (NTRu) were excluded from TTE calculations (and all further analyses).
  • TTE tumor growth delay
  • TGD T - C, expressed in days, or as a percentage of the median TTE of the control group:
  • T median TTE for a treatment group
  • TGI Tumor growth inhibition
  • the data set for TGI analysis included all animals in a group, except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.
  • Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day.
  • the MTV (n) was defined as the median tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume.
  • Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study.
  • 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.
  • mice were weighed daily on Days 1-5, then twice per week until the completion of the study. The mice were observed frequently for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs were recorded when observed. Individual body weight was monitored as per protocol, and any animal with weight loss exceeding 30% for one measurement or exceeding 25% for three consecutive measurements was euthanized as a TR death. Group mean body weight loss was also monitored according to CR Discovery Services protocol. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed.
  • BW body weight
  • NTR deaths are further categorized as follows: NTRa describes deaths due to accidents or human error; NTRm is assigned to deaths thought to result from tumor dissemination by invasion and/or metastasis based on necropsy results; NTRu describes deaths of unknown causes that lack available evidence of death related to metastasis, tumor progression, accident or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as NTRu.
  • GraphPad Prism 8.0 for Windows was used for all statistical analysis and graphical presentations. Study groups experiencing toxicity beyond acceptable limits (>20% group mean body weight loss or greater than 10% treatment-related deaths) or having fewer than five evaluable observations, were not included in the statistical analysis.
  • the MTV for the groups dosed with ConjA and ConjG were 288 and 379 mm 3 which corresponded to significant TGIs of 50 and 34%, respectively, (P ⁇ 0.001 , Mann- Whitney (U-test).
  • the MTV for Groups dosed at 2mg/kg and 6 mg/kg were 126 and 32 mm 3 which corresponded to significant TGIs of 72 and 93%, respectively, (P ⁇ 0.001 , Mann- Whitney U-test) both of which attained the 60% threshold for potential therapeutic activity.
  • mice Female severe combined immunodeficient mice (Fox Chase SCID®, CB17/lcr- Prkdcscidl ⁇ co ⁇ crCrl, Charles River) were nine weeks old with a body weight (BW) range of 17.2 to 24.3 g 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 fiber.
  • the mice were housed on irradiated Enrich-o'cobsTM Laboratory Animal Bedding in static microisolators 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
  • JIMT-1 human breast carcinoma cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 100 units/mL penicillin G sodium, 100 ⁇ g/mL streptomycin sulfate, 25 ⁇ g/mL gentamicin, and 2 mM glutamine. Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO 2 and 95% air.
  • DMEM Dulbecco's Modified Eagle's Medium
  • mice were sorted according to calculated tumor size into groups each consisting of ten animals with individual tumor volumes ranging from 108 to 144 mm 3 and group mean tumor volume of 122 mm 3 . Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
  • the agents were administered i.v. via tail vein injection.
  • the dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal.
  • ConjA 4 mg/kg
  • ConjB (1 and 2 mg/kg
  • ConjG 10 mg/kg
  • a vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 81 .
  • TTE time to endpoint
  • TGD Tumor Growth Delay
  • TGI Tumor Growth Inhibition
  • MTV Regression Responses
  • Toxicity Statistical and Graphical Analyses
  • the group treated with ConjA at 4 mg/kg had a median TTE of 65.4 days, which corresponded to TGD of 22.5 days (52%). All the animals reached the tumor volume endpoint by Day 81.
  • the ConjA regimen resulted in a significant overall survival difference versus controls (P ⁇ 0.001 , logrank).
  • the MTV for the group was 327 mm 3 which corresponded to significant TGI of 55% (P ⁇ 0.001 , Mann-Whitney U test test) but did not attain the 60% threshold for potential therapeutic activity.
  • the MTV for the 1 mg/kg and 2 mg/kg groups were 188 and 135 mm 3 which corresponded to significant TGIs of 74 and 81% (P ⁇ 0.001 , Mann-Whitney U test) and attained the 60% threshold for potential therapeutic activity.
  • the group treated with ConjG at 10 mg/kg had a median TTE of 59.9 days, which corresponded to TGD of 17.0 days (40%).
  • TGD 17.0 days
  • the ConjG regimen resulted in a significant overall survival difference versus controls (P ⁇ 0.001 , logrank).
  • the MTV for the group was 405 mm 3 which corresponded to significant TGI of 44% (P ⁇ 0.001 , Mann-Whitney U test).
  • mice Female athymic nude mice (Crl:NU(NCr)-Foxn7nu, Charles River) were eight weeks old on Day 1 of the study and had a BW range of 18.5 to 25.8 g. 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 fiber. The mice were housed on irradiated Enrich-o'cobsTM bedding in static microisolators on a 12-hour light cycle at 20-22 °C (68-72 °F) and 40-60% humidity.
  • ad libitum water reverse osmosis, 1 ppm Cl
  • NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber.
  • the mice were housed on irradiated Enrich-o'cobsTM
  • 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
  • the androgen-independent PC-3 tumor line was derived from a human prostatic cancer metastatic to bone, and displayed the morphology of a poorly-differentiated adenocarcinoma (Kaighn, ME, et al., Invest Urol, 1979; 17(1 ): 16-23).
  • the PC-3 cells were cultured in RPMI-1640 Medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 0.075% sodium bicarbonate, 2 mM glutamine, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin sulfate and 25 ⁇ g/mL gentamicin.
  • the cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO 2 and 95% air.
  • mice were sorted according to calculated tumor size into ten groups each consisting of ten animals with individual tumor volumes ranging from 100 to 162 mm 3 and group mean tumor volumes of 126 to 128 mm 3 . Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
  • T umour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm 3 of tumour volume.
  • the agents were administered i.v. via tail vein injection.
  • the dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal.
  • ConjE (6 mg/kg) and ConjF (6 mg/kg) were administered intravenously once on Day 1 (qd x 1).
  • a vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 80.
  • TTE time to endpoint
  • TGD Tumor Growth Delay
  • TGI Tumor Growth Inhibition
  • MTV Regression Responses
  • Toxicity Statistical and Graphical Analyses
  • the group treated with ConjE at 6 mg/kg had a median TTE of 74.4 days, corresponding to TGD of 44.0 days (145%).
  • the group had 100% regression responses consisting of three PRs and seven CRs, two of which remained as TFSs on Day 80.
  • Six animals reached the 800 mm 3 tumor volume endpoint leaving four end of study survivors with an MTV of 123 mm 3 .
  • the treatment produced significant survival benefit compared to vehicle-treated controls (P ⁇ 0.001).
  • the group treated with ConjF at 6 mg/kg had a median TTE of 80.0 days, corresponding to TGD of 49.6 days (163%).
  • the group had 100% regression responses consisting of ten CRs, all of which were TFSs on Day 80.
  • the end of study survivors had a Day 80 MTV of 1 mm 3 .
  • the treatment produced significant survival benefit compared to vehicle-treated controls (P ⁇ 0.001).
  • mice Female athymic nude mice (Hsd:Athymic Nude-Foxn1 nu , Envigo) were 5-7 weeks old with a body weight (BW) range of 20.1 to 26.6 grams at the start of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and Envigo Teklad 2918 Global Protein Rodent Diet consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on Envigo Teklad 7097 1 ⁇ 4 inch corncob bedding and in Techniplast Greenline GM500 cages on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% humidity. All animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.
  • AALAC Association for Assessment of Laboratory Animal Care
  • IACUC Institutional Animal Care and Use Committee
  • Human NCI-N87 gastric carcinoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 0.3g/L L-Glutamine. The cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO 2 and 95% air.
  • mice were sorted according to calculated tumor size into groups consisting of 6 animals (Untreated group) or 3 animals (treated groups) when tumors were approximately 155mm 3 in size. Randomization was performed using the deterministic randomization method built into the Study Log software package (Studylog Systems, Inc., South San Francisco, CA).
  • Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
  • T umor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm 3 of tumor volume.
  • Treatment began on Day 10 with established subcutaneous NCI-N87 tumors.
  • a single intravenous dose of ConjJ at 0.25, 0.5, 0.75, 1 , 2, 3, or 4 mg/kg was administered in ADC buffer (20mM Histidine, 240mM Sucrose pH6).
  • ADC buffer (20mM Histidine, 240mM Sucrose pH6).
  • the Untreated group served as the control group for efficacy analysis.
  • the dosing volume was 0.2 mL per 20 grams of body weight (10 mb/kg), and was adjusted to the body weight of each individual animal. Tumors were measured twice per week until the study was ended on Day 55.
  • Tumor growth inhibition (TGI) analysis evaluates the difference in mean tumor volumes (MTVs) of treated and control mice. For this study, the endpoint for determining TGI was Day 55. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:
  • the data set for TGI analysis included all animals in a group, except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.
  • Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day.
  • the MTV (n) was defined as the mean tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume.
  • Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study.
  • 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 initial 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.
  • TR treatment-related
  • Both group and individual body weight were monitored as per protocol, and any animal with weight loss exceeding 20% for one measurement was euthanized as a TR death.
  • Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths.
  • BW body weight
  • Dosing was suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed. Deaths were classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy.
  • NTR non- treatment-related
  • TGI tumour growth inhibition
  • mice Female athymic nude mice (Hsd:Athymic Nude-Foxn1 nu , Envigo) were 5-7 weeks old with a body weight (BW) range of 20.8 to 26.9 grams at the start of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and Envigo Teklad 2918 Global Protein Rodent Diet consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on Envigo Teklad 7097 1 ⁇ 4 inch corncob bedding and in Techniplast Greenline GM500 cages on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% humidity. All animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.
  • AALAC Association for Assessment of Laboratory Animal Care
  • IACUC Institutional Animal Care and Use Committee
  • JIMT-1 human breast carcinoma cells were grown in Dulbecco's Modified Eagle's
  • DMEM fetal bovine serum
  • D-Glucose 1 g/L D-Glucose
  • L-Glutamine 1 g/L L-Glutamine
  • 25mM HEPES 1 g/L Glutamine
  • 110mg/L Sodium Pyruvate a fetal bovine serum
  • Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO 2 and 95% air.
  • the JIMT-1 cells used for implantation were harvested during log phase growth and resuspended in cold phosphate buffered saline (PBS) containing 50% Cultrex Basement Membrane Extract BME3 (Bio-Techne).
  • PBS cold phosphate buffered saline
  • BME3 Cultrex Basement Membrane Extract BME3
  • mice were sorted according to calculated tumor size into groups consisting of 6 animals (Untreated group) or 3 animals (treated groups) when tumors were approximately 190mm 3 in size. Randomization was performed using the deterministic randomization method built into the Study Log software package (Studylog Systems, Inc., South San Francisco, CA). Tumors were measured twice per week.
  • Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
  • Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm 3 of tumor volume.
  • Treatment began on Day 7 with established subcutaneous JIMT-1 tumors.
  • Untreated group served as the control group for efficacy analysis.
  • the dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was adjusted to the body weight of each individual animal.
  • TGI Tumor Growth Inhibition
  • MTV Regression Responses
  • Toxicity and Statistical and Graphical Analyses were all calculated as for Example 5.
  • the endpoint for determining TGI was Day 42 since some untreated tumors were removed due to ulcerations or excessive volume as the study progressed.
  • Ab is a modified antibody having at least one free conjugation site on each heavy chain
  • D represents either group D1 or D2: the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R 2 is selected from the group consisting of:
  • 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;
  • one of R 15a and R 15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
  • 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; when there is a single bond present between C2 and C3,
  • R 2 is selected from H, OH, F, diF and , where R 16a and R 16b are independently selected from H, F, C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 16a and R 16b is H, the other is selected from nitrile and a C 1-4 alkyl ester;
  • D' represents either group D'1 or D'2: wherein the dotted line indicates the optional presence of a double bond between C2' and C3'; when there is a double bond present between C2' and C3', R 22 is selected from the group consisting of:
  • R 31 , R 32 and R 33 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 22 group is no more than 5;
  • 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; 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; when there is a single bond present between C2' and C3',
  • R 22 is selected from H, OH, F, diF and where R 26a and R 26b are independently selected from H, F, C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a C 1-4 alkyl ester; R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR', nitro, Me 3 Sn and halo; where R and R' are independently selected from optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups;
  • R 7 is selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR', nitro, Me 3 Sn and halo;
  • R" is a C 3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR N2 (where R N2 is H or C 1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
  • Y and Y' are selected from O, S, and NH;
  • R 11a is:
  • R 6' ', R 7' and R 9' are selected from the same groups as R 6 , R 7 and R 9 respectively; and R LL1 and R LL2 are linkers connected to the antibody at different sites which are independently wherein
  • Q x is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;
  • R 14 is selected from H, methyl, ethyl, ethenyl and ethynyl.
  • R 24 is selected from H, methyl, ethyl, ethenyl and ethynyl.
  • a conjugate according to statement 1 which is of formula la-1 , la-2 or la-3:
  • R 2a and R 22a are the same and are selected from:
  • R 1a is selected from methyl and benzyl
  • R LL1 , R LL2 and R 11a are as defined in statement 1 .
  • G LL is selected from: where CBA represents the point of connection to the modified antibody Ar represents a C 5-6 arylene group, e.g. phenylene and X 1 represents C 1-4 alkyl.
  • conjugate according to statement 89 wherein the antibody or antibody fragment is an antibody which binds to one or more tumor-associated antigens or cell- surface receptors selected from (1 )-(89):

Abstract

A conjugate of formula (I) wherein Ab is a modified antibody having at least one free conjugation site on each heavy chain; D represents either group D1 or D2. D' represents either group D'1 or D'2.

Description

PYRROLOBENZODIAZEPINE CONJUGATES
The present disclosure relates to conjugates comprising pyrrolobenzodiazepine (PBD) or related moity dimers where at least one PBD-related moiety is non-alkylating, and the precursor drug linkers used to make such conjugates.
Background
Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793- 5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145- 148 (1987)), chicamycin (Konishi, et a/., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41 , 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41 , 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:
Figure imgf000003_0001
They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham- VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents.
It has been previously disclosed that the biological activity of this molecules can be potentiated by joining two PBD units together through their C8/C'-hydroxyl functionalities via a flexible alkylene linker (Bose, D.S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, D.E., et al., J. Org. Chem., 61 , 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5'-Pu-GATC-Py-3' interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity.
One example of a PBD dimer is SG2000 (SJG-136):
Figure imgf000004_0001
(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001 ); Alley, M.C., et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J.A., et al., Cancer Research, 64, 6693-6699 (2004)) which has been involved in clinical trials as a standalone agent, for example, NCT02034227 investigating its use in treating Acute Myeloid Leukemia and Chronic Lymphocytic Leukemia (see: https://www.clinicaltrials.gov/ct2/show/NCT02034227).
Dimeric PBD compounds bearing C2 aryl substituents, such as SG2202 (ZC-207), are disclosed in WO 2005/085251 :
Figure imgf000004_0002
and in W02006/111759, bisulphites of such PBD compounds, for example SG2285 (ZC- 423):
Figure imgf000005_0001
These compounds have been shown to be highly useful cytotoxic agents (Howard, P.W., et al., Bioorg. Med. Chem. (2009), doi: 10.1016/j.bmcl.2009.09.012).
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 2011/130598. 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. If the non-bound N10 position is protected with a capping group, the capping groups exemplified have the same cleavage trigger as the linker to the antibody.
WO 2014/057074 describes two specific PBD dimer conjugates bound via the N10 position on one monomer, the other PBD monomer being in imine form. One of the drug-linkers disclosed is SG3249, Tesirine:
Figure imgf000005_0002
which, when conjugated to anti-DLL3 rovalpituzumab, is know as rovalpituzumab-tesirine (Rova-T), currently under evaluation for the treatment of small cell lung cancer (Tiberghien, A.C., et al., ACS Med. Chem. Lett., 2016, 7 (11), 983-987; DOI: 10.1021 /acsmedchemlett.6b00062). Further conjugates of this drug-linker with an engineered version of tratuzumab and a humanized antibody against human CD19 also began trials in early 2017 by ADC Therapeutics SA (Abstracts #51 and #52 in Proceedings of the American Association for Cancer Research, Volume 58, April 2017).
WO 2015/052322 describes a specific PBD dimer conjugate bound via the N10 position on one monomer, the other PBD monomer being in imine form. It also describes a specific PBD dimer conjugate bound via the N10 position on one monomer, the other PBD monomer having a capping group with the same cleavage trigger as the linker to the antibody:
Figure imgf000006_0001
WO201 9/034764 discloses PBD dimer conjugates wherein the PBDs are conjugated to antibodies that are modified so as to have at least one free conjugation site on each heavy chain, and where the conjugation is via each N10 group of the PBD via a linker.
WO201 4/096368 discloses PBD dimer conjugates where the PBD moieity which is not linked to the antibody is non-alkylating.
Disclosure
The present disclosure provides conjugates comprising PBD dimers where at least one PBD moiety is non-alkylating (i.e. the released moiety has a secondary amine at the N10 position, rather than an imine or equivalent group), which PBD dimers are conjugated to antibodies that are modified so as to have at least one free conjugation site on each heavy chain, and where the conjugation is via each N10 group of the PBD moiety via a linker.
The present disclosure also provides PBD and related dimer drug linkers, where at least one moiety is non-alkylating suitable for conjugating to a modified antibody, where both N10 groups bear linking groups.
A first aspect of the present disclosure provides a conjugate of formula I:
Figure imgf000007_0001
wherein
Ab is a modified antibody having at least one free conjugation site on each heavy chain;
D represents either group D1 or D2:
Figure imgf000007_0002
the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R2 is selected from the group consisting of:
(ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
(ib) C1-5 saturated aliphatic alkyl;
(ic) C3-6 saturated cycloalkyl;
Figure imgf000008_0001
, wherein each of R11, R12 and R13 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 R2 group is no more than 5;
(ie)
Figure imgf000008_0002
, wherein one of R15a and R15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(if)
Figure imgf000008_0003
, where R14 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,
R2 is selected from H, OH, F, diF and , where R16a and R16b are
Figure imgf000008_0004
independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl ester;
D' represents either group D'1 or D'2:
Figure imgf000008_0005
wherein the dotted line indicates the optional presence of a double bond between C2' and C3'; when there is a double bond present between C2' and C3', R22 is selected from the group consisting of: (iia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
(iib) C1-5 saturated aliphatic alkyl;
(iic) C3-6 saturated cycloalkyl;
(iid)
Figure imgf000009_0001
, wherein each of R31, R32 and R33 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;
(iie) , wherein one of R25a and R25b is H and the other is selected from:
Figure imgf000009_0002
phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(iif)
Figure imgf000009_0003
, where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2' and C3',
R22 is selected from H, OH, F, diF and
Figure imgf000009_0004
, where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo; where R and R' are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo;
R" is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y' are selected from O, S, and NH;
R11a is: (i) H; or
(ii) OH or ORA, where RA is C1-4 alkyl;
R6' , R7' and R9' are selected from the same groups as R6, R7 and R9 respectively; and
RLL1 and RLL2 are linkers connected to the antibody at different sites which are independently:
Figure imgf000010_0003
wherein
Q is:
Figure imgf000010_0001
, where Qx is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;
X is:
Figure imgf000010_0002
where a = 0 to 5, b = 0 to 16, c = 0 or 1 , d = 0 to 5; GLL is a linker connected to the antibody.
At least one of the N10 positions bearing a linking group releases a non-alkylating PBD (i.e. the released moiety has a secondary amine at the N10 position, rather than an imine or equivalent group). Dimers containing at least one secondary amine moiety are unable to covalently cross-link DNA, and may be less toxic than dimers with two imine moieties (which can covalently cross-link DNA).
It is thought that such ADCs which effectively have a drug antibody ratio (DAR) of 1 could offer significant advantages including reduced off-target toxicity and an enhanced therapeutic window by reducing the minimal effective dose requirement over ADCs consisting of heterogeneous mixtures with higher DARs.
Without wishing to be bound by theory, avoiding the presence of a C11 hydroxy group adjacent the carbamate on the N10 nitrogen may increase the stability of the carbamate. The proximity of the hydroxy to the carbonyl of the carbamate allows for the formation of an internal hydrogen bond which could catalyse a more facile nucleophilic attack on the carbonyl.
For the avoidance of doubt, the carbon labelled C3 in D1 is adjacent the ternary N at the ring junction. The carbon labelled C3' in D'1 is adjacent the ternary N at the ring junction.
A second aspect of the present disclosure comprises a compound with the formula II:
Figure imgf000011_0002
and salts and solvates thereof, wherein D, R2, R6, R7, R9, R11a, Y, R", Y', D', R6'' , R7'' , R9'' and R22 (including the presence or absence of double bonds between C2 and C3 and C2' and C3' respectively) are as defined in the first aspect of the disclosure;
RL1 and RL2 are linkers for connecting to a cell binding agent, which are independently:
Figure imgf000011_0001
where Q and X are as defined in the first aspect and GL is a linker for connecting to an antibody. A third aspect of the present disclosure provides the use of a conjugate of the first aspect of the disclosure in the manufacture of a medicament for treating a proliferative disease. The third aspect also provides a conjugate of the first aspect of the disclosure for use in the treatment of a proliferative disease. The third aspect also provides a method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate of the first aspect of the disclosure to a patient in need thereof.
One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.
A fourth aspect of the present disclosure provides the synthesis of a conjugate of the first aspect of the disclosure comprising conjugating a compound (drug linker) of the second aspect of the disclosure with an antibody as defined in the first aspect of the disclosure.
A fifth aspect of the present disclosure provides a method of making a compound of formula IV:
Figure imgf000012_0001
from a compound of formula V:
Figure imgf000013_0001
using the Mitsunobu reaction; where R8' is selected from:
(a) OMe, OCH2Ph, OH and -Y'-R"-Hal;
(b)
(
Figure imgf000013_0002
R6, R7, R9, R11a R6' , R7' , R9' , Y, R", Y', D and D' are as defined in the first aspect of the disclosure;
Hal is a halogen, such as Br;
RL2pre is a precursor to RL2;
RL1pre is a precursor to RL1; and ProtO is a hydroxyl protecting group.
The presence of the carbamate on the pre-N10 prevents the Mitsunobu reaction from proceeding beyond the formation of the secondary amine.
Definitions
Substituents
The phrase "optionally substituted" as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.
Unless otherwise specified, the term "substituted" as used herein, pertains to a parent group which bears one or more substituents. The term "substituent" is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
Examples of substituents are described in more detail below. C1-12 alkyl: The term " C1-12 alkyl " as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). The term "C1-4 alkyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C7).
Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7). Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).
C2-12 Alkenyl: The term " C2-12 alkenyl" as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, - CH=CH2), 1 -propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1- methylvinyl, -C(CH3)=CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).
C2-12 alkynyl: The term "C2-12 alkynyl" as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and 2-propynyl (propargyl, -CH2-C≡CH). C3-12 cycloalkyl: The term " C3-12 cycloalkyl" as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and saturated polycyclic hydrocarbon compounds: norcarane (C7), norpinane (C7), norbornane (C7). C3-20 heterocyclyl: The term "C3-20 heterocyclyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
In this context, the prefixes (e.g. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term " C5-6heterocyclyl", as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:
N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);
S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3: trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1 O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
N1 S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);
N2O1: oxadiazine (C6);
O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1O1S1: oxathiazine (C6).
Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
C5-20 aryl: The term " C5-20 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 20 ring atoms. The term "C5-7 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 7 ring atoms and the term "C5-10 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.
In this context, the prefixes (e.g. C3-20, C5-7, C5-6, C5-10, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term " C5-6 aryl" as used herein, pertains to an aryl group having 5 or 6 ring atoms.
The ring atoms may be all carbon atoms, as in "carboaryl groups".
Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).
Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1 H- indene) (C9), indene (C9), isoindene (C9), tetraline (1 ,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (C16).
Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroaryl groups". Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:
N1: pyrrole (azole) (C5), pyridine (azine) (C6);
O1: furan (oxole) (C5);
S1: thiophene (thiole) (C5);
N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);
N2O1: oxadiazole (furazan) (C5); N3O1: oxatriazole (C5);
N1S1: thiazole (C5), isothiazole (C5);
N2: imidazole (1 ,3-diazole) (C5), pyrazole (1 ,2-diazole) (C5), pyridazine (1 ,2-diazine) (C6), pyrimidine (1 ,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (C6);
N3: triazole (C5), triazine (C6); and, N4 : tetrazole (C5).
Examples of heteroaryl which comprise fused rings, include, but are not limited to: C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine ( N4 ) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole ( N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S); C10(with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4 );
C11 (with 2 fused rings) derived from benzodiazepine (N2);
C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and, C14(with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxathiin (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (82), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.
Halo: -F, -Cl, -Br, and -I.
Hydroxy: -OH.
Ether: -OR, wherein R is an ether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7alkyl group. Alkoxy: -OR, wherein R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n- propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).
Oxo (keto, -one): =O.
Acyl (keto): -C(=O)R, wherein R is an acyl substituent, for example, a C1-7 alkyl group (also referred to as C1-7 alkylacyl or C1-7alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, -C(=O)CH3 (acetyl), -C(=O)CH2CH3 (propionyl), -C(=O)C(CH3)3 (t-butyryl), and -C(=O)Ph (benzoyl, phenone).
Carboxy (carboxylic acid): -C(=O)OH.
Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C(=O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH3, -C(=O)OCH2CH3, -C(=O)OC(CH3)3, and -C(=O)OPh.
Amino: -NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di- C1-7 alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a "cyclic" amino group, R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (-NH2), secondary (-NHR1), or tertiary (-NHR1R2), and in cationic form, may be quaternary (-+NR1R2R3). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, -NHC(CH3)2, -N(CH3)2, -N(CH2CH3)2, and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3, and -C(=O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
Nitro: -NO2.
Cyano (nitrile, carbonitrile): -CN.
Hydroxyl protecting group: Hydroxyl protecting groups are well known in the art, for example, in Wuts & Greene 2007. Those groups suitable for use in the present disclosure include substituted methyl ethers, substituted ethyl ethers, methoxy substituted benzyl ethers, silyl ethers and acetates. Of particular relevance are tert-butyldimethylsilyl (TBS) and triisopropylsilyl (TIPS).
Amine protecting group: Hydroxyl protecting groups are well known in the art, for example, in Wuts & Greene 2007. Those groups suitable for use in the present disclosure include carbamate. Of particular relevance is allyl carbamate (Alloc).
Alkylene C3-12 alkylene: The term "C3-12 alkylene", as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
Examples of linear saturated C3-12 alkylene groups include, but are not limited to, -(CH2)n- where n is an integer from 3 to 12, for example, -CH2CH2CH2- (propylene), -CH2CH2CH2CH2- (butylene), -CH2CH2CH2CH2CH2- (pentylene) and -CH2CH2CH2CH-2CH2CH2CH2- (heptylene).
Examples of branched saturated C3-12 alkylene groups include, but are not limited to, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-, and -CH2CH(CH2CH3)CH2-. Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene, and alkynylene groups) include, but are not limited to, -CH=CH-CH2-, -CH2-CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -
CH=CH-CH=CH-CH2-CH2-, -CH=CH-CH2-CH=CH-, -CH=CH-CH2-CH2-CH=CH-, and -CH2-
C≡C-CH2-.
Examples of branched partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, -CH=CH-CH(CH3)- and - C≡C-CH(CH3)-.
Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent- 1 ,3-ylene), and cyclohexylene (e.g. cyclohex-1 ,4-ylene).
Examples of alicyclic partially unsaturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1 ,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1 ,4-ylene; 3-cyclohexen-1 ,2-ylene; 2,5-cyclohexadien- 1 ,4-ylene).
Ligand Unit
The Ligand Units for use in the present disclosure are Cell Binding Agents, more specifically modified antibodies, or antigen binding fragments thereof, having at least one conjugation site on each heavy chain. Examples of particular modified antibodies suitable for use according to the present disclosure are disclosed in WO 2012/064733 (filed as PCT/US2011/059775), which is incorporated herein by reference.
Antibodies
The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific 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. Examples of antibody fragments include F(ab')2, and scFv fragments, and dimeric 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.
Modified antibodies suitable for use in the present disclosure include those wherein the native interchain cysteine residues have been substituted by amino acid residues lacking thiol groups. The antibodies may comprise at least one additional substitutions in each heavy chain of an amino acid residue comprising a reactive group suitable for conjugation to a linker. The additionally substituted amino acid may be a cysteine or a non-natural amino acid. The position that is substituted may be selected from those set forth below:
Figure imgf000022_0001
Figure imgf000023_0001
Examples of modified antibodies suitable for use in the present disclosure include the Flexmab structuires disclosed in WO 2012/064733, which is incorporated herein. Such Flexmabs have cysteines with free thiol groups in the hinge region of the antibody that may be used as conjugation sites for linking through the N1 0 groups of the PBDs of the present disclosure.
Other examples of modified antibodies suitable for use in the present disclosure include those where at least one insertion in each heavy chain of an amino acid residue comprising a reactive group suitable for conjugation to a linker have been made. The inserted amino acid may be a cysteine or a non-natural amino acid. Some of these are described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 10.1021/acs.molpharmaceut.6b00995) and WO2015/157595. In particular, antibodies which have been modified by insertion of a cysteine after the S239 position (ie. between positions 239 and 240) are of use.
In some embodiments, antibodies which have been modified by insertion of a non-natural amino acid at F241 (EU numbering according to Kabat) may be used. A non-natural amino acid as employed herein refers to an amino acid which is other than one of the twenty-one naturally occurring amino acids. The non-natural amino acids are generally derived from natural amino acids. Derived from a natural amino acid refers to the fact that the non- natural amino acid is based on (or incorporates) or is similar to the structure of natural amino acid, for example the alkylene chain in lysine may be shortened to provide a 3- carbon chain as opposed to the natural 4 carbon chain but the structural relationship or similarity to lysine still exists. Thus, derivatives of natural amino acids include modifications such as incorporating a functional group, lengthening or shortening an alkylene chain, adding one or more substituents to a nitrogen, oxygen, sulfur in a side chain or converting a nitrogen, oxygen or sulfur into a different functional group or a combination of any of the same. Usually the majority of modifications will be the addition of structure in the non-natural amino acid. However, modification may include removed or replacing an atom naturally found in an amino acid.
In some embodiments the non-natural amino has a formula (AAII):
Rx-XAA1-O0-1C(O)-amino-acid-residue (AAII) wherein:
Rx represents an unsaturated group selected from a: i) C4-9 linear conjugated diene, ii) C5-14 carbocyclyl comprising a conjugated diene, and iii) a 5 to 14 membered heterocyclyl comprising 1 , 2 or 3 heteroatoms selected O, N and S, and a conjugated diene, wherein i), ii) and iii) may bear up to five substituents, (such as one, two or three substituents) for example, the substituents are independently selected from C1-3 alkyl, oxo, halogen, sulfo, sulfhydryl, amino, -C1-3 alkyleneN3, or -C2- 5alkynyl; and XAA1 represents i) a saturated or unsaturated branched or unbranched C1-8 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a heteroatom selected from O,
N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino, -C1-3 alkylene N3, or -C2-5alkynyl; or ii) together with a carbon from the carbocylcyl or heterocyclyl represents a cyclopropane ring linked to a saturated or unsaturated (in particular saturated) branched or unbranched C1-6 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a heteroatom selected from
O, N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino, -C1-3 alkylene N3, or -C2-5alkynyl and
-O0-1C(O)- is linked through a side chain of an amino acid.
The amino acid residue referred to in AAII is as defined for AAI above.
In the context of formula AAII, the amino acid residue refers to an amino acid comprising the -NH2 and -COOH groups. The amino acid residue in formula AAII may additionally comprise an R group of a natural amino acid. Alternatively, the amino acid residue in formula AAII may be derived from a natural amino acid but have its natural R group replaced with RX-XAA1-O0-1C(O).
In one embodiment the non-natural amino acid is a residue of the structure of formula (AAIII):
Figure imgf000025_0001
wherein
X2 represents -C-, -C(R')-, -CH2 or O;
R' represents H or C1-3 alkyl,
Ra represents i) a saturated or unsaturated branched or unbranched C1-8 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino; or ii) together with a carbon from the 5 membered ring represents a cyclopropane ring linked to a saturated or unsaturated (in particular saturated) branched or unbranched C1-6 alkylene chain, wherein at least one carbon (for example 1 , 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino;
Rb represents H, -OC1-3 alkyl, C1-6alkyl optionally bearing a hydroxyl substituent, -C1-3 alkyleneN3, or -C2-5 alkynyl;
Rc represents H, -OC1-3 alkyl, C1-6alkyl optionally bearing a hydroxyl substituent, -C1-3 alkyleneN3, or -C2-5 alkynyl;
Rd represents H, -OC1-3 alkyl, C1-6alkyl optionally bearing a hydroxyl substituent, -C1-3 alkyleneN3, or -C2-5 alkynyl;
Re represents H, saturated or unsaturated (in particular saturated) branched or unbranched C1-8 alkylene chain, wherein one or more carbons are optionally replaced by -O- and the chain is optionally substituted by one or more halogen atoms (such as iodo), N3 or -C2. 5alkynyl.
In one embodiment Ra is -(CH2)mC(O)-, -CH2(CH3)C(O)-, -(CH2)mCH2OC(O)-, -CHCHCH2OC(O)-, or -OCH2CH2COC(O)- and m represents 0 or 1.
In one embodiment Rb is H, -OC1-3 alkyl, -CH3, -CH(CH3)2, CH2OH, -CH2N3, or -CCH.
In one embodiment Rc is H, -OC1-3 alkyl, -CH3, -CH(CH3)2, CH2OH, -CH2N3, or -CCH.
In one embodiment Rd is H, -OC1-3 alkyl, -CH3, -CH(CH3)2, CH2OH, -CH2N3, or -CCH.
In one embodiment Re represents H or -CH2OCH2CH2N3.
In one embodiment the non-natural amino acid is a residue of the structure of formula
(AAllla):
Figure imgf000026_0001
wherein Ra, Rb, Rc, Rd, Re and X2 are defined above.
In one embodiment the non-natural amino acid has the structure of formula (AAlllb):
Figure imgf000026_0002
wherein Ra, Rb, Rc, Rd, Re and X2 are defined above.
In one embodiment the non-natural amino acid has the structure of formula (AAlllc):
Figure imgf000027_0001
(AAlllc) wherein Ra, Rb, Rc, Rd, Re are defined above and X2' is -C- or -CR' as defined above.
Generally compounds, for example formula (AAIII), (AAllla), (AAlllb) and (AAlllc) will at most contain only one azide group.
In one embodiment the non-natural amino acid is selected from the group comprising:
Figure imgf000027_0002
Figure imgf000028_0001
In some embodiments, the non-natural amino acid is selected from:
Figure imgf000028_0002
Antibodies which have been modified by the insertion of CP2-NNAA are of particular use in the present invention. These are described in as described in WO2019/224340, and Roy et al., MAbs 12 (1): 1684749 (doi:10.1080/19420862.2019.1684749), which are both incorporated herein by reference. In particular, antibodies with this insertion at F241 (EU numbering according to Kabat) may be used, e.g. modified Herceptin. In some embodiments, there is provided a conjugate of a PBD-dimer or PBD-analogue dimer conjugated to an antibody which is modified at one or both F241 positions with a non-natural amino acid as described above. The or both linkers between the PBD-dimer or
PBD-analogue dimer is/are attached to said non-natual amino acid(s).
Reference is made to the targets listed on pages 60 to 62 of WO 2012/064733, which is incorporated herein. In some embodiments, the antibody may be to a tumour-associated antigen, for example: HER2 (ErbB2); EPHA2 (EPH receptor A2); CD19; IL2RA (Interleukin 2 receptor, alpha).
Tumour-associate antigens and cognate antibodies for use in embodiments of the present disclosure are listed below, and are described in more detail on pages 14 to 86 of WO 2017/186894, which is incorporated herein.
(1) BMPR1B (bone morphogenetic protein receptor-type IB)
(2) E16 (LAT1 , SLC7A5)
(3) STEAP1 (six transmembrane epithelial antigen of prostate)
(4) 0772P (CA125, MUC16)
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin)
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b)
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B)
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene)
(9) ETBR (Endothelin type B receptor)
(10) MSG783 (RNF124, hypothetical protein FLJ20315)
(11) STEAP2 (HGNC_8639, IPCA-1 , PCANAP1 , STAMP1 , STEAP2, STMP, prostate cancer associated gene 1 , prostate cancer associated protein 1 , six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein)
(12) TrpM4 (BR22450, FLJ20041 , TRPM4, TRPM4B, transient receptor potential cation
5 channel, subfamily M, member 4)
(13) CRIPTO (CR, CR1 , CRGF, CRIPTO, TDGF1 , teratocarcinoma-derived growth factor)
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792)
(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29) (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1 B, SPAP1C)
(17) HER2 (ErbB2)
(18) NCA (CEACAM6)
(19) MDP (DPEP1 )
(20) IL20R-alpha (IL20Ra, ZCYTOR7)
(21) Brevican (BCAN, BEHAB)
(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5)
(23) ASLG659 (B7h)
(24) PSCA (Prostate stem cell antigen precursor)
(25) GEDA
(26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3)
(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) (27a) CD22 (CD22 molecule)
(28) CD79a (CD79A, 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), pl: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q 13.2).
(29) CXCR5 (Burkitt'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 role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3,
(30) HLA-DOB (Beta subunit of MHC class II molecule (la antigen) that binds peptides and 20 presents them to CD4+ T lymphocytes); 273 aa, pl: 6.56, MW: 30820. TM: 1 [P] Gene Chromosome: 6p21.3)
(31) 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), pl: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa, pl: 8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3).
(33) 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, pl: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12). (34) 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 differentiation); 429 aa, pl: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1 q21 -1 q22)
(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies); 977 aa, pl: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1 q21 )
(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1 , TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin); 374 aa)
(37) PSMA - FOLH1 (Folate hydrolase (prostate-specific membrane antigen) 1)
(38) SST (Somatostatin Receptor; note that there are5 subtypes)
(38.1 ) SSTR2 (Somatostatin receptor 2)
(38.2) SSTR5 (Somatostatin receptor 5)
(38.3) SSTR1
(38.4) SSTR3
(38.5) SSTR4
AvB6 - Both subunits (39+40)
(39) ITGAV (Integrin, alpha V)
(40) ITGB6 (Integrin, beta 6)
(41) CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5)
(42) MET (met proto-oncogene; hepatocyte growth factor receptor)
(43) MUC1 (Mucin 1 , cell surface associated)
(44) CA9 (Carbonic anhydrase IX)
(45) EGFRvlll ( Epidermal growth factor receptor (EGFR), transcript variant 3,
(46) CD33 (CD33 molecule)
(47) CD19 (CD19 molecule)
(48) IL2RA (Interleukin 2 receptor, alpha); NCBI Reference Sequence: NM_000417.2);
(49) AXL (AXL receptor tyrosine kinase)
(50) CD30 - TNFRSF8 (Tumor necrosis factor receptor superfamily, member 8)
(51) BCMA (B-cell maturation antigen) - TNFRSF17 (Tumor necrosis factor receptor superfamily, member 17)
(52) CT Ags - CTA (Cancer Testis Antigens)
(53) CD174 (Lewis Y) - FUT3 (fucosyltransferase 3 (galactoside 3(4)-L-fucosyltransferase, Lewis blood group)
(54) CLEC14A (C-type lectin domain family 14, member A; Genbank accession no. NM175060) (55) GRP78 - HSPA5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)
(56) CD70 (CD70 molecule) L08096
(57) Stem Cell specific antigens. For example:
• 5T4 (see entry (63) below)
• CD25 (see entry (48) above)
• CD32
• LGR5/GPR49
• Prominin/CD133
(58) ASG-5
(59) ENPP3 (Ectonucleotide pyrophosphatase/phosphodiesterase 3)
(60) PRR4 (Proline rich 4 (lacrimal))
(61) GCC - GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor)
(62) Liv-1 - SLC39A6 (Solute carrier family 39 (zinc transporter), member 6)
(63) 5T4, Trophoblast glycoprotein, TPBG - TPBG (trophoblast glycoprotein)
(64) CD56 - NCMA1 (Neural cell adhesion molecule 1 )
(65) CanAg (Tumor associated antigen CA242)
(66) FOLR1 (Folate Receptor 1)
(67) GPNMB (Glycoprotein (transmembrane) nmb)
(68) TIM-1 - HAVCR1 (Hepatitis A virus cellular receptor 1)
(69) RG-1/Prostate tumor target Mindin - Mindin/RG-1
(70) B7-H4 - VTCN1 (V-set domain containing T cell activation inhibitor 1
(71) PTK7 (PTK7 protein tyrosine kinase 7)
(72) CD37 (CD37 molecule)
(73) CD138 - SDC1 (syndecan 1)
(74) CD74 (CD74 molecule, major histocompatibility complex, class II invariant chain)
(75) Claudins - CLs (Claudins)
(76) EGFR (Epidermal growth factor receptor)
(77) Her3 (ErbB3) - ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian))
(78) RON - MST1 R (macrophage stimulating 1 receptor (c-met-related tyrosine kinase))
(79) EPHA2 (EPH receptor A2)
(80) CD20 - MS4A1 (membrane-spanning 4-domains, subfamily A, member 1 )
(81) Tenascin C - TNG (Tenascin C)
(82) FAP (Fibroblast activation protein, alpha)
(83) DKK-1 (Dickkopf 1 homolog (Xenopus laevis)
(84) CD52 (CD52 molecule) (85) CS1 - SLAMF7 (SLAM family member 7)
(86) Endoglin - ENG (Endoglin)
(87) Annexin A1 - ANXA1 (Annexin A1 )
(88) V-CAM (CD106) - VCAM1 (Vascular cell adhesion molecule 1 )
An additional tumour-associate antigen and cognate antibodies of interest are: (89) ASCT2 (ASC transporter 2, also known as SLC1 A5).
ASCT2 antibodies are described in WO 2018/089393, which is incorporated herein by reference.
Connection of Linker unit to Ligand unit
The Ligand unit may be connected to the Linker unit through a disulfide bond.
In one embodiment, 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. Other possible groups for linking, and the resulting linking groups, are shown below.
The cysteine residues of the Ligand unit may be available for reaction with the functional group of the Linker unit to form a connection. In other embodiments, for example where the Ligand unit is an antibody, the thiol groups of the antibody may participate in interchain disulfide bonds. These interchain bonds may be converted to free thiol groups by e.g. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit.
In some embodiments, the cysteine residue is an introduced into the heavy or light chain of an antibody. Positions for cysteine insertion by substitution in antibody heavy or light chains include those described in Published U.S. Application No. 2007-0092940 and International Patent Publication W02008/070593, which are incorporated herein.
Methods of Treatment
The compounds of the present disclosure 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 of formula I. The term "therapeutically effective amount" is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
A conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy.
Pharmaceutical compositions according to the present disclosure, and for use in accordance with the present disclosure, 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. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
The Conjugates can be used to treat proliferative disease and autoimmune disease. The term "proliferative disease" pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo. Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, 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, Dressier's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti- phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampler's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.
In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th 1 -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). Generally, disorders involving dendritic cells involve disorders of Th1- lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmunie disorder is a T cell-mediated immunological disorder.
In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose.
Drug loading
The drug loading (p) is the average number of PBD drugs per cell binding agent, e.g. antibody. In the present disclosure, this is always 1. However, any composition may comprise antibodies where a PBD is conjugated and antibodies where a PBD is not conjugated. Thus for a composition, the drug loading (or DAR) may be less than 1 , for example 0.75 and higher, 0.80 and higher, 0.85 and higher, 0.90 and higher or 0.95 or higher.
Therapeutic Index
The Therapeutic Index of a particular drug-linker/conjugate can be calculated by dividing the maximum tolerated single dose (MTD) of a non-targeted ADC in rat, by the minimal effective single dose (MED) of a comparable targeted ADC in mouse. The MED may be the single dose necessary to achieve tumour stasis in an in vivo model at 28 days.
General synthetic routes
Mitsunobu
A key step in the synthesis of compounds (drug-linkers) of formula II (and similar compounds) is the synthesis of compounds of formula IV:
Figure imgf000037_0001
from compounds of formula V: by using a Mitsunobu reaction to close the ring.
Figure imgf000037_0002
The reaction may be carried out under conventional conditions, using triphenylphosphine and an azodicarboxylate such as diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD). In particular, for the synthesis of compounds of formula II, R8' should be selected from:
(a) OH and -Y'-R"-Hal;
(b)
Figure imgf000038_0001
Further derivation
When R8 is (Vc), compounds of formula II may be synthesised from compounds of formula IV by converting RL1-pre into RL1 and RL2-pre into RL2. Typically the precursors of RL1 and RL2 will comprise:
Figure imgf000038_0002
Where ProtN is an amine protecting group, such as Alloc. The conversion may be carried out by removal of the amine protecting group and reaction with the remainder of the RL1/RL2 group (i.e. HO-C(=O)-X-GL). When R8' is (Vb), compounds of formula IV where R8' is (Vc) may be synthesised by removal of the hydroxyl protecting group, such as TBS, followed by oxidative ring closure, for example using a Cu/TEMPO catalyst system (TEMPO = 2,2,6,6-tetramethylpiperidine- N-oxyl).
When R8 is -Y'-R"-Hal, compounds of formula IV where R8 is (Vc) may be synthesised by coupling a compound of formula Via:
Figure imgf000039_0001
to the compound of formula IV when R8 is -Y'-R"-HaL
When R8 is -OH, and Y' is O, compounds of formula IV where R8 is (Vc) may be synthesised by coupling a compound of formula Vlb:
Figure imgf000039_0002
to the compound of formula IV when R8 is -OH.
Compounds of formula V may be synthesised from compounds of formula VII:
Figure imgf000040_0001
by removal of the hydroxyl protecting group (Prot°) where R8' is selected from:
(a) OMe, OCH2Ph, OH and -Y'-R"-Hal;
(b)
Figure imgf000040_0002
and R6, R7, R9, R11a , R6'', R7'', R9'', Y, R", Y', D and D' are as defined in the first aspect of the disclosure.
Where R8' is (Vb), the compound of formula VII may be symmetrical, i.e. both moieties linked by -Y'-R"-Y- may be identical. The removal of only one Proto group may be achieved by a statistical approach, as illustrated in the examples. Synthesis of Drug Conjugates
Antibodies can be conjugated to the Drug Linker compound generally as described in Doronina et al., Nature Biotechnology, 2003, 21 , 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37 °C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5'-dithiobis(2- nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved. The reduced antibody is then cooled to 0°C and alkylated with 3 eguivalents of drug-linker per antibodyl. After 1 hour, the reaction is guenched by the addition of 5 eguivalents of N- acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the levels of remaining NAC-guenched drug-linker.
Further Preferences
The following preferences may apply to all aspects of the disclosure as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
R6' , R7'' , R9' and Y' are selected from the same groups as R6, R7, R9 and Y respectively. In some embodiments, R6' , R7'' , R9' and Y' are the same as R6, R7, R9 and Y respectively.
In some embodiments, R22 is the same as R2.
Dimer link
In some embodiments, Y and Y' are both O.
In some embodiments, R" is a C3-7 alkylene group with no substituents. In some of these embodiments, R" is a C3, C5 or C7 alkylene. In particular, R" may be a C3 or C5 alkylene.
In other embodiments, R" is a group of formula:
Figure imgf000042_0001
where r is 1 or 2.
In other embodiments, R" is a group of formula:
Figure imgf000042_0002
where r is 1 or 2.
R6 to R9
In some embodiments, R and R' are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups, wherein the optional substituents are selected from C1-12 alkyl, C3-20 heterocyclyl, C5-20 aryl, halo, hydroxy, ether, alkoxy, oxo, acyl, carboxy, ester, amino, amido, nitro, and cyano.
In some embodiments, R9 is H.
In some embodiments, R6 is selected from H, OH, OR, SH, NH2, nitro and halo, and may be selected from H or halo. In some of these embodiments R6 is H.
In some embodiments, R7 is selected from H, OH, OR, SH, SR, NH2, NHR, NRR', and halo. In some of these embodiments R7 is selected from H, OH and OR, where R is selected from optionally substituted C1-7 alkyl, C3-10 heterocyclyl and C5-10 aryl groups. R may be more preferably a C1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7- positions are OMe and OCH2Ph. Other substituents of particular interest are dimethylamino (i.e. -NMe2); -(OC2H4)qOMe, where q is from 0 to 2; nitrogen-containing C6 heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl.
These embodiments and preferences apply to R9'', R6' and R7' respectively. D and D’
In some embodiments, D and D' are D1 and D'1 respectively.
In some embodiments, D and D' are D2 and D'2 respectively.
R2
When there is a double bond present between C2 and C3, R2 is selected from:
(a) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1 -3 alkylene;
(b) C1-5 saturated aliphatic alkyl;
(c) C3-6 saturated cycloalkyl;
(d)
Figure imgf000043_0001
, wherein each of R11, R12 and R13 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 R2 group is no more than 5;
(e)
Figure imgf000043_0002
, wherein one of R15a and R15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and
(f) , where R14 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3
Figure imgf000043_0003
alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
When R2 is a C5-10 aryl group, it may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R2 is preferably phenyl. In other embodiments, R2 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
When R2 is a C5-10 aryl group, it may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3- yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1 -yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
When R2 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
Where R2 is C5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para- position.
Where R2 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R2 substituents, when R2 is a C5-10 aryl group
If a substituent on R2 when R2 is a C5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
If a substituent on R2 when R2 is a C5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
If a substituent on R2 when R2 is a C5-10 aryl group is C1-7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).
If a substituent on R2 when R2 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
If a substituent on R2 when R2 is a C5-10 aryl group is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
If a substituent on R2 when R2 is a C5-10 aryl group is ester, this is preferably methyl ester or ethyl ester.
Particularly preferred substituents when R2 is a C5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl. Other particularly preferred substituents for R2 are dimethylaminopropyloxy and carboxy.
Particularly preferred substituted R2 groups when R2 is a C5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R2 group is 4-nitrophenyL R2 groups of particular interest include 4-(4- methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
Other R2 groups
When R2 is C1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
When R2 is C3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
When R2 is
Figure imgf000045_0001
, each of R11, R12 and R13 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 R2 group is no more than 5. In some embodiments, the total number of carbon atoms in the R2 group is no more than 4 or no more than 3.
In some embodiments, one of R11, R12 and R13 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
In other embodiments, two of R11, R12 and R13 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that re not H are methyl.
In some embodiments, R11 is H.
In some embodiments, R12 is H.
In some embodiments, R13 is H.
In some embodiments, R11 and R12 are H.
In some embodiments, R11 and R13 are H.
In some embodiments, R12 and R13 are H.
An R2 group of particular interest is:
Figure imgf000046_0001
When R2 is
Figure imgf000046_0002
, one of R15a and R15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. When R2 is
Figure imgf000047_0001
, R14 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
In some embodiments, R14 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R14 is selected from H and methyl.
When there is a single bond present between C2 and C3,
R2 is H or
Figure imgf000047_0002
, where R16a and R16b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl ester.
In some embodiments, R2 is H.
In some embodiments, R2 is
Figure imgf000047_0003
In some embodiments, it is preferred that R16a and R16b are both H.
In other embodiments, it is preferred that R16a and R16b are both methyl.
In further embodiments, it is preferred that one of R16a and R16b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl.
R22
When there is a double bond present between C2' and C3', R22 is selected from: (a) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy- C1-3 alkylene;
(b) C1-5 saturated aliphatic alkyl;
(c) C3-6 saturated cycloalkyl;
(d)
Figure imgf000048_0001
, wherein each of R31, R32 and R33 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 R22 group is no more than 5;
(e) , wherein one of R25a and R25b is H and the other is selected from: phenyl,
Figure imgf000048_0003
which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and
(f)
Figure imgf000048_0002
, where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
When R22 is a C5-10 aryl group, it may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R22 is preferably phenyl. In other embodiments, R22 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
When R22 is a C5-10 aryl group, it may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1 -yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred. When R22 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
Where R22 is C5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or 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.
Where R22 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R22 substituents, when R22 is a C5-10 aryl group
If a substituent on R22 when R22 is a C5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
If a substituent on R22 when R22 is a C5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
If a substituent on R22 when R22 is a C5-10 aryl group is C1-7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).
If a substituent on R22 when R22 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom. If a substituent on R22 when R22 is a C5-10 aryl group is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
If a substituent on R22 when R22 is a C5-10 aryl group is ester, this is preferably methyl ester or ethyl ester.
Particularly preferred substituents when R22 is a C5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl. Other particularly preferred substituents for R22 are dimethylaminopropyloxy and carboxy.
Particularly preferred substituted R22 groups when R22 is a C5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R22 group is 4-nitrophenyL R22 groups of particular interest include 4-(4- methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
Other R22 groups
When R22 is C1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
When R22 is C3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
When R22 is
Figure imgf000050_0001
, each of R31, R32 and R33 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 R22 group is no more than 5. In some embodiments, the total number of carbon atoms in the R22 group is no more than 4 or no more than 3. In some embodiments, one of R31, R32 and R33 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
In other embodiments, two of R31, R32 and R33 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl.
In some embodiments, R31 is H.
In some embodiments, R32 is H.
In some embodiments, R33 is H.
In some embodiments, R31 and R32 are H.
In some embodiments, R31 and R33 are H.
In some embodiments, R32 and R33 are H.
An R22 group of particular interest is:
Figure imgf000051_0001
When R22 is , one of R25a and R25b is H and the other is selected from:
Figure imgf000051_0002
phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
When R22 is
Figure imgf000051_0003
, R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
In some embodiments, R24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R24 is selected from H and methyl.
When there is a single bond present between C2' and C3',
R22 is H or
Figure imgf000052_0001
, where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester.
In some embodiments, R22 is H.
In some embodiments, R22 is
Figure imgf000052_0002
In some embodiments, it is preferred that R26a and R26b are both H.
In other embodiments, it is preferred that R26a and R26b are both methyl.
In further embodiments, it is preferred that one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl.
R11a
In some embodiments, R11a is H.
In some other embodiments, R11a is OH.
In some other embodiments, R11a is ORA, where RA is C1-4 alkyl. In some of these embodiments, RA is methyl. In some embodiments of the first aspect of the present disclosure are of formula la-1 , la-2 or la-3:
Figure imgf000053_0001
where the dotted line represents the possible presence of a double bond between C2 and C3 and C2' and C3'; where there is no double bond between C2 and C3 and C2' and C3', R2a and R22a are the same and are selected from:
(a) H; and
(b)
Figure imgf000053_0002
where there is a double bond between C2 and C3 and C2' and C3' R2a and R22a are the same and are selected from:
Figure imgf000054_0001
R1a is selected from methyl and benzyl;
RLL1, RLL2 and R11a are as defined above.
In some embodiments of the present disclosure both R2 and R22 comprise no more than 3 carbon atoms.
Thus in these embodiments where there is a double bond present between C2 and C3, R2 may be selected from:
(i) Methyl;
(ii) Ethyl; and
(iii) Propyl;
Figure imgf000054_0002
(iv) Cyclopropyl; Thus in these embodiments where there is no double bond present between C2 and C3, R2 may be selected from:
(i) H;
Figure imgf000055_0001
; and
(ii)
Figure imgf000055_0002
Thus in these embodiments where there is a double bond present between C2' and C3', R22 may be selected from:
(i) Methyl;
(ii) Ethyl; and
(iii) Propyl;
Figure imgf000055_0003
(iv) Cyclopropyl;
Thus in these embodiments where there is no double bond present between C2' and C3', R22 may be selected from:
(i) H;
Figure imgf000055_0004
; and
Figure imgf000055_0005
In some of these embodiments both R2 and R22 comprise no more than 2 carbon atoms.
Thus in these embodiments where there is a double bond present between C2 and C3, R2 may be selected from:
(i) Methyl;
Figure imgf000055_0006
(ii) Ethyl; and Thus in these embodiments where there is no double bond present between C2 and C3, R2 may be selected from:
(i) H;
Figure imgf000056_0002
(ii)
Figure imgf000056_0001
and
Thus in these embodiments where there is a double bond present between C2' and C3', R22 may be selected from:
(i) Methyl;
Figure imgf000056_0003
(ii) Ethyl; and
Thus in these embodiments where there is no double bond present between C2' and C3', R22 may be selected from:
(i) H;
Figure imgf000056_0004
(ii)
Figure imgf000056_0005
; and
In further of these embodiments both R2 and R22 comprise no more than 1 carbon atom.
Thus in these embodiments where there is a double bond present between C2 and C3, R2 may be methyl. Thus in these embodiments where there is no double bond present between C2 and C3, R2 may be selected from: (i) H; and
Figure imgf000056_0006
Thus in these embodiments where there is a double bond present between C2' and C3', R22 may be methyl. Thus in these embodiments where there is no double bond present between C2' and C3', R22 may be selected from: (i) H; and
Figure imgf000057_0001
Without wishing to be bound by theory, where the substituent at the C2 position of the PBD dimers are small, the use of the glucuronide capping unit in these drug linkers is believed to be particularly advantageous, as it significantly increases the hydrophilicity of the drug linker, making the drug linkers easier to conjugate to a ligand unit.
These embodiments and preferences also apply to the second aspect of the disclosure.
GL
GL may be selected from
Figure imgf000057_0002
Figure imgf000058_0001
where Ar represents a C5-6 arylene group, e.g. phenylene, and X1 represents C1-4 alkyl.
In some embodiments, GL is selected from GL1-1 and GL1-2. In some of these embodiments, GL is GL1-1.
GLL
GLL may be selected from:
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000060_0001
where CBA represents the point of connection to the modified antibody, Ar represents a C5- 6 arylene group, e.g. phenylene and X1 represents C1-4 alkyl.
In some embodiments, GLL is selected from GLL1-1 and GLL1-2. In some of these embodiments, GLL is GLL1-1. In other embodiments, GLL is GLL1-1A. GLL1-1A may be formed by a Diels-Alder reaction between GL1-1 and a spirocyclopropyl- cyclopentadiene of formula:
Figure imgf000060_0002
. Such a group can be incorporated into the antibody via the addition of a linker or by incorporating a non-natural amino acid into the polypeptide sequence, as described in WO2019/224340, which is incorporated herein by reference).
X
X is:
Figure imgf000060_0003
where 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. In some embodiments, a is 0 to 3. In some of these embodiments, 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, 11 , 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 embodiments, d is 1 or 2. In further embodiments, d is 2.
In some embodiments of X, 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
In one embodiment, Q is an amino acid residue. The amino acid may a natural amino acids or a non-natural amino acid.
In one embodiment, Q is selected from: Phe, Lys, Vai, Ala, Cit, Leu, lie, Arg, and Trp, where Cit is citrulline.
In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, Q is selected from:
CO-Phe-Lys-NH,
CO-Val-Ala-NH,
CO-Val-Lys-NH,
CO-Ala-Lys-NH,
CO-Val-Cit-NH,
CO-Phe-Cit-NH,
CO-Leu-Cit-NH,
CO-lle-Cit-NH,
CO-Phe-Arg-NH, and
CO-Trp-Cit-NH; where Cit is citrulline.
Preferably, Q is selected from:
CO-Phe-Lys-NH,
CO-Val-Ala-NH, CO-Val-Lys-NH,
CO-Ala-Lys-NH,
CO-Val-Cit-NH.
Most preferably, Q is selected from CO-Phe-Lys-NH, CO-Val-Cit-NH and CO-Val-Ala-NH.
Other dipeptide combinations of interest include:
CO-Gly-Gly-NH,
CO-Pro-Pro-NH, and
CO-Val-Glu-NH.
Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where 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. Tripeptide linkers of particular interest are:
NH-Glu-Val-Ala-C=O NH-Glu-Val-Cit-C=O NH-αGlu-Val-Ala-C=O NH-αGlu-Val-Cit-C=O
In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide then is a recognition site for cathepsin. Tetrapeptide linkers of particular interest are:
NH -Gly-Gly-Phe-Gly C=O; and
NH -Gly-Phe-Gly-Gly C=O .
In some embodiments, the tetrapeptide is: NH -Gly-Gly-Phe-GlyC=O. In the above representations of peptide residues, NH- represents the N-terminus, and - C=O represents the C-terminus of the residue.
Glu represents the residue of glutamic acid, i.e.:
Figure imgf000063_0001
αGlu represents the residue of glutamic acid when bound via the α-chain, i.e.:
Figure imgf000063_0002
In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.
In one particular embodiment, the first aspect of the disclosure comprises a conjugate of formula Id:
Figure imgf000064_0001
where m is an integer from 2 to 8, and X is selected from:
Figure imgf000064_0003
In another particular embodiment, the first aspect of the disclosure comprises a conjugate of formula le:
Figure imgf000064_0002
where m is an integer from 2 to 8, and X is selected from:
Figure imgf000065_0002
In one particular embodiment, the second aspect of the disclosure, the Drug linker (DL) is of formula (lid):
Figure imgf000065_0001
where m is an integer from 2 to 8, and X is as defined above.
In some embodiments, RL1 and RL2 are different.
In some embodiments, RLL1 and RLL2 are different.
In particular, in embodiments where the linking groups are different, differences may only be in the G groups, such that the remainder of the linking groups are the same (so that the cleavage triggers are the same).
In some embodiments of the present disclosure, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
Figure imgf000065_0003
In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
Figure imgf000066_0001
Compounds of particular interest include those of the examples.
Examples
Flash chromatography was performed using a Biotage Isolera One™ using gradient elution on SNAP Ultra™ columns starting from either 88% hexane/EtOAc or 99.9% DCM/MeOH until all UV active components (detection at 214 and 254 nm) 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 aluminum plates. Visualization of TLC was achieved with UV light or iodine vapor unless otherwise stated. Extraction and chromatography solvents were bought and used without further purification from VWR UK. All fine chemicals were purchased from Sigma-Aldrich or TCI Europe unless otherwise stated. Pegylated reagents were obtained from Quanta Biodesign US via Stratech UK, or Purepeg. The LC-MS conditions were as follows: positive mode electrospray mass spectrometry was performed using a Waters Acquity UPLC® (ultra-high- performance liquid chromatography) fitted with an SQ2 mass detector. Mobile phases used were solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid) at a flow rate of 0.8 mL/min. The column was a Waters Acquity UPLC® BEH Shield RP18 1 .7 μm 2.1 mm x 50 mm fitted with a Waters Acquity UPLC® BEH Shield RP18 VanGuard pre-column, 130 A, 1.7 μm, 2.1 mm x 5 mm at 50 °C.
Method 1 : Gradient started with initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 min 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 min with a sample injection volume of 2 μL and detection at 223 nm and 254 nm. Method 2: Gradient started with initial composition 25% B held over 25 seconds, then increased from 25% B to 100% B over a 1 min 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 min with a sample injection volume of 2 pL and detection at 223 nm and 254 nm.
Method 3: Gradient started with initial composition 5% B held over 1 min 25 seconds, then increased from 5% B to 100% B over a 9 min 35 seconds period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 10 seconds and held there for 2 min. The total duration of the gradient run was 15.0 min with a sample injection volume of 2 pL and detection at 223 nm and 254 nm.
The preparative HPLC conditions were as follows: reverse-phase UPLC was carried out on a Shimazdzu Prominence® machine using a Phenomenex® Gemini NX 5μm C18 column 150 mm x 21.2 mm fitted with a Phenomenex® Gemini SecurityGuard PREP Cartridge NX C18 15 mm x 21.2 mm at 50 °C. Eluents used were solvent A (H2O with 0.01% formic acid) and solvent B (CH3CN with 0.01% formic acid). All UPLC experiments were performed with gradient conditions: initial composition 15% B increased to 55% B over a 15-min period then increased to 100% B over 2 min, held for 1 min at 100% B, then returned to 13% B in 0.1 min and held there for 1 .9 min. The total duration of the gradient run was 20.0 min. Flow rate was 20.0 mL/min and with detection at 254 and 280 nm.
Example 1
Figure imgf000068_0001
Figure imgf000069_0001
Compound 1 is described in Tiberghien et al., Journal of Organic Chemistry, 2019 DOI: 10.1021 /acs.joc.8b02876
(a) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[5-[5-[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2 -methoxy- phenoxy]pentoxy]-2-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methyl-2,3- dihydropyrrole-1 -carbonyl]-4-methoxy-phenyl]carbamate (2)
1 ,5-dibromopentane (427 pb, 3.14 mmol) was added to a mixture of 1 (5.00 g, 6.28 mmol, 1 eq), potassium carbonate (0.955 g, 6.91 mmol) and tetrabutylammonium iodide (2.32 g, 6.28 mmol) in acetone (30.0 mL, 410 mmol). The mixture was heated at 50°C for 20h at which point LCMS showed a large amount of product in the reaction. The mixture was partitioned between ethyl acetate (150 mL) and water (100 mL). The organic phase was dried over magnesium sulphate and concentrated under vacuum. The residue was purified by chromatography (100g ultra, gradient 10% to 40% EtOAc/EtOH 4/1 in isohexane). The purest fractions were pooled and the volatiles were removed under vacuum to give the product 2 (3.93 g, 2.37 mmol, 75.4%) yield as a pale yellow foam. bC-MS (method 2) 2.39 min, ES+ m/z 1661.3 [M+H ]. (b) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[5-[5-[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2 -methoxy- phenoxy]pentoxy]-2-[(2S)-2-(hydroxymethyl)-4-methyl-2,3-dihydropyrrole-1- carbonyl]-4-methoxy-phenyl]carbamate (3)
Compound 2 (3.80 g, 2.29 mmol) was dissolved in a mixture of tetrahydrofuran (19.0 mL), acetic acid (15.2 mL), water (7.60 mL) and methanol (3.80 mL) and stirred at room temperature. Progression of the reaction was closely followed by LC/MS with a target of completion in 6 hours. When the reaction rate was found too slow, the temperature was raised to 30°C, with the occasional addition of acetic acid.
A correct profile of 1/2/1 bis alcohol/half alcohol/bis tbs was obtained after 6 hours. The reaction mixture was diluted with ethyl acetate (200 mL) and washed with water (200 mL), followed by saturated aqueous hydrogen carbonate (200 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified with a first chromatography (100 g ultra, ethyl acetate / EtOH 4/1 in hexane; elution of desired product around 50%). A monomeric phenol impurity was removed during a second chromatography (100 g ultra, DCM/MeOH, elution after 6 CV at 4.8 % MeOH in DCM). The pure fractions were pooled and the volatiles removed under vacuum to give 3 (1 .328 g, 0.8591 mmol, 37.5% Yield) as a white solid. LC-MS (method 2) 2.10 min, ES+ m/z 1547.1 [M+H].
(c) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[5-[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2 -methoxy- phenoxy]pentoxy]-2-methoxy-8-methyl-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1 - c] [1 ,4]benzodiazepine-5-carboxylate (4)
Diisopropyl azodicarboxylate (0.372 mL, 1.85 mmol) was added to a solution of 3 (A, 1.30 g, 0.841 mmol, 100 mass%) and triphenylphosphine (0.665 g, 2.52 mmol) in tetrahydrofuran (26.0 mL). The reaction was heated at 40°C for 2h, at which point a satisfactory amount of product was formed by LCMS. The volatiles were removed under vacuum and the residue was purified by chromatography (50g Ultra, Biotage, EtOAc/EtOH 4/1 in Hexane, gradient from 20% to 57%. Elution around 45% upwards). The pure fractions were pooled to give 4 (1 .010 g, 0.6611 mmol, 78.6% Yield). LC-MS (method 2)
2.13 min, ES+ m/z 1528.8 [M+H],
(d) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[5-[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2- (hydroxymethyl)-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2 -methoxy- phenoxy]pentoxy]-2-methoxy-8-methyl-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1 - c] [1 ,4]benzodiazepine-5-carboxylate (5) p-Toluenesulfonic acid (60.0 mg, 0.345 mmol) was added to a solution of 4 (980 mg, 0.641 mmol) in tetrahydrofuran (4.0 mL), acetic acid (1.5 mL), water (1.0 mL) and methanol (1.5 mL) and stirred at room temperature. The reaction was found to be progressing rapidly and complete after 15 min by LCMS.
The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL), followed by saturated aqueous hydrogen carbonate (50 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified by chromatography (50 g ultra, 4/1 DCM/MeOH in DCM, gradient from 5% to 32%, elution from 24% to 28%). The pure fractions were pooled and the volatiles removed under vacuum to give 5 (B, 640 mg, 0.453 mmol, 100 mass%, 70.6% Yield) as a white solid. LC-MS (method 2) 1.71 min, ES+ m/z 1414.5 [M+H],
(e) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6S,6aS)-3-[5-[[(6aS)-5-[[4-[[(2S)-2- [[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2-methoxy-8-methyl-11- oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]oxy]pentoxy]-6-hydroxy-2- methoxy-8-methyl-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1 -c][1 ,4]benzodiazepine-5- carboxylate (6)
Tetrakisacetonitrile copper(l) triflate (33.1 mg, 0.0878 mmol) was added to a solution of 5 (620 mg, 0.439 mmol), and Stahl tempo solution in acetonitrile (0.439 mL, 0.0878 mmol, 0.200 mol/b) in dichloromethane (12.0 mL), under an atmosphere of air (air ballon). The solution was stirred at 34°C for 18h, when completion was observed by LCMS. The solution was loaded directly on a biotage samplet, dried, and eluted on a 50g Ultra column with 4/1 DCM/MeOH in DCM, gradient from 15% to 30%. Elution around 20%. The pure fractions were pooled and the volatiles removed under vacuum to give 6 (535 mg, 0.379 mmol, 86.4% Yield) as a white solid. LC-MS (method 2) 1.71 min, ES+ m/z 1412.5 [M+H].
(f) [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 - yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop anoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (6S,6aS)-3-[5- [[(6aS)-5-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 - yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop anoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2- methoxy-8-methyl-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1 -c][1 ,4]benzodiazepin-3- yl]oxy]pentoxy]-6-hydroxy-2-methoxy-8-methyl-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1 - c] [1 ,4]benzodiazepine-5-carboxylate (7)
Compound 6 (520 mg, 0.368 mmol) was dissolved in a mixture of dichloromethane (8.00 mL, 99.5 mass%) and pyrrolidine (77.3 μL, 0.921 mmol). The atmosphere was purged with argon. A catalytic amount of tetrakis(triphenylphosphine)palladium(0) (8.56 mg, 0.00737 mmol) was added and the reaction allowed to proceed for 30 min, when LCMS showed completion.
The reaction mixture was partitioned between DCM (10 mL), 1 M aqueous ammonium chloride (10 mL). The organic layer was decanted through an isolera cartridge, and the volatiles were removed under vacuum. Half of this material (229 mg, 0.184 mmol) was dissolved in DCM (5.00 mL) and methanol (0.2 mL), followed by mal-amido-peg8-acid (245 mg, 0.405 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (78.0 mg, 0.407 mmol). The reaction was allowed to proceed at room temperature for 45 min when completion was observed by LCMS. The reaction mixture was concentrated (2 mL), loaded on a 3g biotage silica samplet and dried under vacuum. The samplet was loaded on a 25g Ultra Biotage column, and eluted (gradient 15/90 to 60/40 of 20% MeOH in DCM / DCM in 12CV; Elution from around 40/60; gradient allowed to increase to avoid streaking). All fractions were analysed by TLC (15% MeOH in DCM). The pure fractions were pooled. The solvent was removed by evaporation to give 7 (390 mg, 0.163 mmol, 88.5% Yield). The purity was 94.5%. LC-MS (method 3) 6.89 min, ES+ m/z 1197.4 [(M+2H)/2 ] (method 1 ) 1.71 min, ES+ m/z 1197.5 [(M+2H)/2]. 1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 2H), 8.16 (d, J = 6.9 Hz, 2H), 7.99 (t, J = 5.5 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.66 - 7.44 (m, 4H), 7.40 - 7.23 (m, 1 H), 7.23 - 7.09 (m, 3H), 7.08 - 7.01 (m, 2H), 6.99 (s, 4H), 6.93 (s, 1 H), 6.75 (s, 1 H), 6.71 - 6.57 (m, 3H), 5.64 - 5.49 (m, 1 H), 5.18 - 4.98 (m, 3H), 4.93 - 4.76 (m, 2H), 4.45 - 4.31 (m, 2H), 4.21 (dd, J = 8.6, 6.7 Hz, 2H), 4.07 - 3.88 (m, 5H), 3.88 - 3.71 (m, 8H), 3.70 - 3.62 (m, 2H), 3.62 - 3.55 (m, 8H), 3.55 - 3.43 (m, 56H), 3.36 (t, J = 5.9 Hz, 4H), 3.20 - 3.10 (m, 4H), 2.98 - 2.76 (m, 2H), 2.45 (t, J = 6.8 Hz, 2H), 2.39 (t, J = 6.5 Hz, 2H), 2.33 (dt, J = 8.1 , 5.4 Hz, 4H), 1.96 (q, J = 6.8 Hz, 2H), 1.83 - 1.66 (m, 10H), 1.61 - 1.48 (m, 2H), 1.29 (d, J = 7.1 Hz, 6H), 0.84 (dd, J = 15.4, 6.7 Hz, 12H).
Example 2
Figure imgf000073_0001
Figure imgf000074_0001
(a) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[5-[[3-[[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2 -methoxy- phenoxy]methyl]phenyl]methoxy]-2-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4- methyl-2,3-dihydropyrrole-1-carbonyl]-4-methoxy-phenyl]carbamate (8)
1 ,3-bis(bromomethyl)benzene (829 mg, 3.14 mmol) was added to a mixture of 1 (5.00 g, 6.28 mmol), potassium carbonate (955 mg, 6.91 mmol) and tetrabutylammonium iodide (2.32 g, 6.28 mmol) in acetone (30.0 mL). The mixture was heated at 40°C for 1 ,5h, followed by stirring at 23°C for 18h at which point LCMS showed reaction completion. The mixture was partitioned between ethyl acetate (150 mL) and water (100 mL). The organic phase was dried over magnesium sulphate and concentrated under vacuum. The residue was dry-loaded and purified by chromatography (100g ultra, gradient 10% to 41% EtOAc/EtOH 4/1 in isohexane, elution at 41%). The fractions were pooled and the volatiles were removed under vacuum to give the product 8 (3.81 g, 2.25 mmol, 71 .6 % yield) as a pale yellow solid. LC-MS (method 2) 2.41 min, ES+ m/z 1695.8 [M+H].1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 2H), 9.07 (s, 2H), 8.14 (d, J = 7.1 Hz, 2H), 7.65 - 7.54 (m, 5H), 7.49 - 7.43 (m, 3H), 7.39 (s, 2H), 7.33 - 7.26 (m, 4H), 7.22 (d, J = 8.7 Hz, 2H), 6.82 (s, 2H), 6.04 (s, 2H), 5.96 - 5.83 (m, 2H), 5.29 (dt, J = 17.2, 1.8 Hz, 2H), 5.20 - 5.13 (m, 2H), 5.10 (s, 4H), 5.02 (d, J = 3.0 Hz, 4H), 4.54 - 4.36 (m, 8H), 3.90 (dd, J = 8.8, 6.8 Hz, 2H), 3.82 (s, 2H), 3.74 (s, 6H), 3.67 (s, 2H), 2.67 (d, J = 1 .8 Hz, 2H), 2.43 - 2.32 (m, 2H), 1 .99 (d, J =
1 .8 Hz, 2H), 1.60 (s, 6H), 1.30 (d, J = 7.1 Hz, 6H), 0.92 - 0.78 (m, 30H), 0.02 (d, J = 11 .6 Hz, 12H).
(b) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[5-[[3-[[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2 -methoxy- phenoxy]methyl]phenyl]methoxy]-2-[(2S)-2-(hydroxymethyl)-4-methyl-2,3- dihydropyrrole-1 -carbonyl]-4-methoxy-phenyl]carbamate (9)
Compound 8 (3.80 g, 2.24 mmol) was dissolved in a mixture of tetra hydrofuran (19.0 mL), acetic acid (7.6 mL, 130 mmol), water (7.60 mL) and methanol (3.80 mL) and stirred at 30°C for 3 to 4 hours. Progression of the reaction was closely followed by LC/MS. A desired profile of 1/2/1 bis alcohol/half alcohol/bis tbs was obtained after 4 hours. The reaction mixture was diluted with ethyl acetate (200 mL) and washed with water (200 mL), followed by saturated aqueous hydrogen carbonate (200 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified by chromatography (100 g ultra, Ethyl acetate / EtOH 4/1 in hexane; gradient from 25% up to 100% in 24 CV; elution of desired product around 50%) to give 9 (1 .51 g, 0.956 mmol, 42.6% Yield) as a white solid. LC-MS (method 2) 2.10 min, ES+ m/z 1581.3 [M+H].1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 2H), 9.06 (s, 2H), 8.14 (d, J = 7.0 Hz, 2H), 7.65 - 7.54 (m, 5H), 7.49 - 7.43 (m, 3H), 7.42 - 7.26 (m, 6H), 7.22 (d, J = 8.8 Hz, 2H), 6.89 (s, 1 H), 6.82 (s, 1 H), 6.05 (s, 1 H), 5.98 (s, 1 H), 5.95 - 5.83 (m, 2H), 5.30 (dq, J = 17.1 , 1.8 Hz, 2H), 5.16 (dt, J = 10.4, 1.6 Hz, 2H), 5.12 - 4.95 (m, 8H), 4.83 (s, 1 H), 4.56 - 4.35 (m, 8H), 3.94 - 3.86 (m, 2H), 3.85 - 3.79 (m, 1 H), 3.78 - 3.71 (m, 6H), 3.66 (s, 2H), 3.52 (s, 1 H), 2.75 - 2.59 (m, 2H), 2.46 - 2.29 (m, 2H), 1 .99 (d, J = 1.8 Hz, 2H), 1.59 (d, J = 5.7 Hz, 6H), 1.30 (d, J = 7.0 Hz, 6H), 0.92 - 0.79 (m, 21 H), 0.02 (d, J = 11.4 Hz, 6H).
(c) [4-[[(2S)-2-[[(2S)-210(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2 -methoxy- phenoxy]methyl]phenyl]methoxy]-2-methoxy-8-methyl-11-oxo-6a,7-dihydro-6H- pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (10)
Diisopropyl azodicarboxylate (0.406 mL, 2.02 mmol, 98 mass%) was added to a solution of 9 (1 .45 g, 0.918 mmol) and triphenylphosphine (0.726 g, 2.75 mmol) in tetrahydrofuran (26.0 mL). The reaction was heated at 40°C for 2h, at which point a satisfactory amount of product was formed by LCMS. The volatiles were removed under vacuum and the residue was purified by chromatography (50g Ultra, Biotage, EtOAc/EtOH 4/1 in Hexane, gradient from 20% to 57%. Elution around 45% upwards. The purest fractions were pooled and concentrated under vacuum to give 10 (739 mg, 0.473 mmol, 51.6% yield) as a white solid. LC-MS (method 2) 2.13 min, ES+ m/z 1563.1 [M+H], 1H NMR (400 MHz, DMSO-d6) 5 10.09 - 9.85 (m, 2H), 9.07 (s, 1 H), 8.23 - 8.02 (m, 2H), 7.67 - 7.50 (m, 5H), 7.49 - 7.34 (m, 4H), 7.30 (d, J = 8.5 Hz, 2H), 7.26 - 7.14 (m, 3H), 7.09 (d, J = 11.0 Hz, 2H), 6.82 (s, 1 H), 6.67 - 6.59 (m, 1 H), 6.05 (s, 1 H), 5.96 - 5.82 (m, 2H), 5.36 - 5.23 (m, 2H), 5.20 - 5.13 (m, 2H), 5.13 - 4.98 (m, 6H), 4.97 - 4.79 (m, 2H), 4.54 - 4.34 (m, 6H), 4.11 - 3.94 (m, 2H), 3.93 - 3.86 (m, 2H), 3.86 - 3.71 (m, 6H), 3.68 (s, 1 H), 3.54 (s, 1 H), 2.91 - 2.75 (m, 1 H), 2.74 - 2.61 (m, 1 H), 2.45 - 2.21 (m, 2H), 2.05 - 1 .90 (m, 2H), 1 .74 (s, 3H), 1 .60 (s, 3H), 1.37 - 1.22 (m, 6H), 0.93 - 0.75 (m, 21 H), 0.11 - -0.09 (m, 6H).
(d) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[5-[[4-[[(2S)-2-[[(2S)-2- (allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]-4-[(2S)-2- (hydroxymethyl)-4-methyl-2,3-dihydropyrrole-1-carbonyl]-2-methoxy- phenoxy]methyl]phenyl]methoxy]-2-methoxy-8-methyl-11-oxo-6a,7-dihydro-6H- pyrrolo[2,1 -c][1 ,4]benzodiazepine-5-carboxylate (11 ) p-Toluenesulfonic acid (60.0 mg, 0.345 mmol) was added to a solution of 10 (720 mg, 0.461 mmol) in tetrahydrofuran (4.0 mL), acetic acid (1 .5 mL), water (1 .0 mL) and methanol (1 .5 mL) and stirred at room temperature. The reaction was found to be progressing rapidly and complete after 20 min by LCMS.
The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL), followed by saturated aqueous hydrogen carbonate (50 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified with a first chromatography (50 g ultra, 4/1 DCM/MeOH in DCM, gradient from 5% to 25%, elution from 24% to 25%); The pure fractions were pooled and the volatiles removed under vacuum to give 11 (544 mg, 0.376 mmol, 81 .5% Yield) as a white solid. LC-MS (method 2) 1.74 min, ES+ m/z 1448.7 [M+H], 1H NMR (400 MHz, DMSO-d6) δ 10.12 - 9.82 (m, 2H), 9.05 (s, 1 H), 8.22 - 8.04 (m, 2H), 7.64 - 7.49 (m, 5H), 7.48 - 7.34 (m, 4H), 7.31 (d, J = 8.3 Hz, 2H), 7.22 (dd, J = 8.9, 3.5 Hz, 3H), 7.09 (d, J = 11.9 Hz, 2H), 6.89 (s, 1 H), 6.67 - 6.59 (m, 1 H), 5.97 (s, 1 H), 5.96 - 5.84 (m, 2H), 5.30 (ddt, J = 16.9, 3.3, 1.7 Hz, 2H), 5.16 (ddd, J = 10.4, 3.5, 1.8 Hz, 2H), 5.12 - 4.97 (m, 6H), 4.96 - 4.78 (m, 2H), 4.53 - 4.35 (m, 7H), 4.09 - 3.95 (m, 2H), 3.89 (t, J = 7.8 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 3.64 (s, 1 H), 3.53 (s, 2H), 2.83 (d, J = 16.2 Hz, 1 H), 2.71 - 2.58 (m, 1 H), 2.40 (d, J = 16.8 Hz, 1 H), 2.28 (d, J = 16.7 Hz, 1 H), 2.06 - 1.89 (m, 2H), 1.74 (s, 3H), 1.59 (s, 3H), 1.35 - 1.21 (m, 6H), 0.95 - 0.75 (m, 12H).
(e) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6S,6aS)-3-[[3-[[(6aS)-5-[[4-[[(2S)-2- [[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2-methoxy-8-methyl-11- oxo-6a,7-dihydro-6H-pyrrolo[2,1 -c][1 ,4]benzodiazepin-3- yl]oxymethyl]phenyl]methoxy]-6-hydroxy-2-methoxy-8-methyl-11-oxo-6a,7-dihydro- 6H-pyrrolo[2,1 -c][1 ,4]benzodiazepine-5-carboxylate (12)
Tetrakisacetonitrile copper(l) triflate (27.1 mg, 0.0719 mmol) was added to a solution of 11 (520 mg, 0.359 mmol) and Stahl TEMPO solution in acetonitrile (0.359 mL, 0.0718 mmol, 0.2 mol/L) in dichloromethane (8.00 mL), under an atmosphere of air (air balloon). The solution was stirred at 34°C for 18h, when completion was observed by LCMS. The solution was loaded directly, and eluted, on a 50g Ultra column with 4/1 DCM/MeOH in DCM, gradient from 15% to 30% Elution around 20 to 22%. The pure fractions were pooled and the volatiles removed under vacuum to give 12 (410 mg, 0.284 mmol, 79.0% Yield) as a white solid. LC-MS (method 2) 1.72 min, ES+ m/z 1446.8 [M+H]. 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 2H), 8.12 (d, J = 6.9 Hz, 2H), 7.69 - 7.49 (m, 5H), 7.49 - 7.37 (m, 3H), 7.32 (s, 1 H), 7.25 - 7.13 (m, 5H), 7.13 - 7.04 (m, 3H), 6.94 (s, 1 H), 6.71 (s, 1 H), 6.63 (p, J = 1.9 Hz, 2H), 6.03 - 5.81 (m, 2H), 5.59 (s, 1 H), 5.40 - 5.21 (m, 2H), 5.18 - 4.76 (m, 10H), 4.58 - 4.28 (m, 6H), 4.02 (s, 2H), 3.89 (dd, J = 8.8, 6.7 Hz, 2H), 3.83 - 3.73 (m, 6H), 3.69 (td, J = 9.7, 3.6 Hz, 1 H), 3.55 (s, 1 H), 3.02 - 2.75 (m, 2H), 2.38 - 2.18 (m, 1 H), 2.05 - 1.85 (m, 2H), 1.79 - 1.66 (m, 6H), 1.27 (s, 6H), 0.85 (dd, J = 18.4, 6.7 Hz, 12H).
(f) [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1- yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop anoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (6S,6aS)-3- [[3-[[(6aS)-5-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1- yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop anoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2- methoxy-8-methyl-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1 -c][1 ,4]benzodiazepin-3- yl]oxymethyl]phenyl]methoxy]-6-hydroxy-2-methoxy-8-methyl-11-oxo-6a,7-dihydro- 6H-pyrrolo[2,1 -c][1 ,4] benzodiazepi ne-5-carboxy late (13)
Compound 12 (200 mg, 0.138 mmol, 100 mass%) was dissolved in a mixture of DCM (6.00 mL) and pyrrolidine (77.3 μL, 0.921 mmol). The atmosphere was purged with argon. A catalytic amount of tetrakis(triphenylphosphine)palladium(0) (3.21 mg, 0.00276 mmol, 99.5 mass%) was added and the reaction allowed to proceed for 30 min, when LCMS showed completion.
The reaction mixture was partitioned between DCM (10 mL), 1 M aqueous ammonium chloride (10 mL). The organic layer was decanted through an isolera cartridge, and the volatiles were removed under vacuum to give the crude deprotected amine (177 mg, 0.139 mmol), which was redissolved in dichloromethane (5.00 mL) and methanol (0.2 mL), followed by addition of mal-amido-peg8-acid (245 mg, 0.405 mmol, 98 mass%) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (78.0 mg, 0.407 mmol, 100 mass%). The reaction was allowed to proceed at room temperature for 45 min when completion was observed by LCMS. The reaction mixture was concentrated (2 mL), loaded on a 3g biotage silica samplet and dried under vacuum.
The samplet was loaded on a 25g Ultra Biotage column, and eluted (gradient 15/90 to 60/40 of 20% MeOH in DCM / DCM in 12CV; Elution from around 50 to 60%; gradient to avoid streaking). All fractions were analysed by TLC (15% MeOH in DCM). The pure fractions were pooled. The solvent was removed by evaporation to give (B, 274 mg, 0.113 mmol, 100 mass%, 81 .5% Yield). The purity was 95.6%. LC-MS (method 3) 7.00 min, ES+ m/z 1214.4 [(M+2H)/2], (method 2) 1.48 min, ES+ m/z 1214.5 [(M+2H)/2]. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 2H), 8.14 (d, J = 6.8 Hz, 2H), 7.99 (t, J = 5.6 Hz, 2H), 7.85 (d, J = 8.7 Hz, 2H), 7.55 (s, 5H), 7.49 - 7.36 (m, 3H), 7.17 (s, 3H), 7.13 - 7.04 (m, 3H), 6.99 (s, 4H), 6.94 (s, 1 H), 6.70 (s, 1 H), 6.62 (d, J = 2.4 Hz, 2H), 5.59 (s, 1 H), 5.04 (dd, J = 33.8, 16.4 Hz, 8H), 4.37 (s, 2H), 4.26 - 4.15 (m, 2H), 4.01 (s, 2H), 3.79 (d, J = 3.8 Hz, 6H), 3.72 - 3.64 (m, 2H), 3.59 (dd, J = 7.9, 6.6 Hz, 8H), 3.53 - 3.41 (m, 56H), 3.36 (t, J = 5.9 Hz, 4H), 3.14 (q, J = 5.8 Hz, 4H), 2.87 (s, 2H), 2.47 - 2.36 (m, 4H), 2.35 - 2.29 (m, 6H), 1.95 (q, J = 6.7 Hz, 2H), 1.78 - 1.69 (m, 6H), 1.27 (s, 6H), 0.84 (dd, J = 15.4, 6.7 Hz, 12H). Example 3
Figure imgf000079_0001
Figure imgf000080_0001
Compound 14 is described in Tiberghien et al., Org. Process Res. Dev. 2018, 22, 9, 1241- 1256. a) [(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3- dihydropyrrol-1 -yl]-(5-methoxy-2-nitro-4-triisopropylsilyloxy-phenyl)methanone (15) Trifluoromethanesulfonic anhydride (8.87 mL, 51 .7 mmol) was added to a solution of 14 (20.0 g, 34.4 mmol) and 2,6-dimethylpyridine (8.1 mL, 69 mmol) in dry toluene (120 mL) at -40°C under argon. The solution was stirred at -35°C for 30 min at which point completion was indicated by LCMS and TLC (EtOAc / hexane 1/3). The solution was diluted with ethyl acetate (100 mL) and water (100 mL). The organic layer was washed further with 0.02 N HCI (100 mL), followed by saturated bicarbonate (100 mL), and brine (50 mL). The organics were dried with magnesium sulfate and concentrated down to around 50 mL under vacuum. The solution was diluted with toluene (100 mL). The solution underwent two vacuum/argon cycles, followed by addition of potassium phosphate dibasic (36.7 g, 206 mmol, 98.0 mass%), 4-methoxyphenylboronic acid (7.01 g, 44.7 mmol), tetrakis(triphenylphosphine)palladium(0) (800 mg, 0.689 mmol), and water (30 mL). The reaction was stirred under argon at room temperature for 1 h, at which point TLC and LCMS indicated reaction completion. The mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL) and brine (50 mL). The organics were dried over magnesium sulfate and concentrated under vacuum.
The residue was dry-loaded on silica and purified by filtration over a bed of silica (gradient EtOAc/Hexane 10/90 up 25/75). The purer fractions were pooled and concentrated under vacuum to give 15 (20.0 g, 29.8 mmol, 86.6% Yield) as a dark yellow foam. LC-MS (method 2) 2.66 min, ES+ m/z 672.0 [M+H]. b) (2-amino-5-methoxy-4-triisopropylsilyloxy-phenyl)-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3-dihydropyrrol-1 - yl]methanone (16)
Zinc (39.7 g, 606 mmol) was added to mixture of water (5.50 mL), acetic acid (5.50 mL, 95.9 mmol) and ethanol (88.0 mL) at 0°C. A solution of 15 (A, 11.0 g, 16.4 mmol) in ethanol (60 mL) was added slowly at 5°C, followed by further stirring at 5°C. After 1 h30, about 20% of hydroxylamine side product was observed. A further 20g of zinc was added, together with another 5 mL of acid and water. The reaction was allowed to warm up to room temperature slowly over 3h, at which point completion was observed. The solids were removed by filtration over a bed of celite. The solution was partitioned between water (300 mL) and ethyl acetate (300 mL), separated, and washed again with water (300 mL), followed by aqueous sodium bicarbonate (150 mL). The organic phase was dried over magnesium sulfate and the volatiles were removed under vacuum. The residue was purified by flash chromatography (340g Ultra Biotage, gradient ethyl acetate / hexane 8/92 to 24/76 in 6 CV. Elution around 24/76) to give 16 (9.3 g, 15 mmol, 89% Yield) as a pale yellow foam. LC-MS (method 2) 2.49 min, ES+ m/z 642.5 [M+H], c) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[2-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3-dihydropyrrole-1 -carbonyl]- 4-methoxy-5-triisopropylsilyloxy-phenyl]carbamate (17)
Triphosgene (1.46 g, 4.87 mmol, 99 mass%) was added to a stirred solution of 16 (8.70 g, 13.6 mmol) in dry dichloromethane (87.0 mL, 99.5 mass%) at -10°C, followed by dry triethylamine (4.18 mL, 29.8 mmol, 99.5 mass%). The mixture was allowed to warm up to room temperature. Solid allyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo- ethyl]carbamoyl]-2-methyl-propyl]carbamate (5.38 g, 14.3 mmol) was added in one portion, followed by triethylamine (2.85 mL, 20.3 mmol) and dichloromethane (87.0 mL). The reaction mixture was allowed to stir for 4h at room temperature. Full dissolution of the starting material was observed. Completion was observed by LCMS and TLC (ethyl acetate / hexane 1/2). The solution was washed with 0.15 N HCI (500 mL), followed by saturated hydrogen carbonate (200 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was dried overnight under vacuum to give 17 (13.87 g, 13.28 mmol, 97.8% Yield) as a solid orange foam. LC-MS (method 2) 2.47 min, ES+ m/z 1045.4 [M+H], d) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[2-[(2S)-2-(hydroxymethyl)-4-(4- methoxyphenyl)-2,3-dihydropyrrole-1-carbonyl]-4-methoxy-5-triisopropylsilyloxy- phenyl]carbamate (18) p-Toluenesulfonic acid (2.24 g, 12.7 mmol) was added to a solution of 17 (13.3 g, 12.7 mmol) in a mixture of acetic acid (2.66 mL), methanol (13.3 mL), water (4.00 mL) and 2- methyltetrahydrofuran (80.0 mL) and stirred at room temperature. The reaction was found to be progressing rapidly and complete after 1h by LCMS.
The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL), followed by saturated aqueous hydrogen carbonate (50 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified with a first chromatography (340 g ultra, EtOAc/Hexanes 35/65 up to 100/0 in 6 CV. Elution from 90/10); The pure fractions were pooled and the volatiles removed under vacuum to give 18 (11.8 g, 12.7 mmol, 99.6% Yield) as a yellow solid. LC-MS (method 2) 2.12 min, ES+ m/z 931.1 [M+H], e) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-2-methoxy-8-(4- methoxyphenyl)-11 -oxo-3-triisopropylsilyloxy-6a,7-dihydro-6H-pyrrolo[2,1 - c][1 ,4]benzodiazepine-5-carboxylate (19)
Diisopropyl azodicarboxylate (5.70 mL, 28.4 mmol) was added to a solution of 18 (12.0 g, 12.9 mmol) and triphenylphosphine (9.00 g, 34.1 mmol) in tetrahydrofuran (200 mL). The reaction was heated at 40°C for 2h, at which point a satisfactory amount of product was formed by LCMS. The volatiles were removed under vacuum and the residue was purified by chromatography (340g Ultra, Biotage, EtOAc/Hexane, gradient from 35% to 75%. Elution around 65% upwards). The pure fractions were pooled and concentrated under vacuum to give 19 (8.50 g, 9.32 mmol, 72.2% Yield) as a pale yellow solid (85% pure by LC).
LC-MS (method 2) 2.15 min, ES+ m/z 913.1 [M+H], f) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-hydroxy-2-methoxy-8-(4- methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5- carboxylate (20)
Lithium acetate (1.3 g, 20 mmol) was added to a solution of 19 (8.40 g, 9.21 mmol) in DMF (42.0 mL, 543 mmol) and water (1.7 mL, 94 mmol). The solution was stirred at 40°C for 1 h and was then partitioned between 2-MeTHF (250 mL) and water (400 mL). The organics were washed with brine (150 mL) and dried over magnesium sulfate. The solution was concentrated under vacuum. The residue was purified by chromatography (100g Ultra, gradient 50/50 EtOAc / Hexane up 100% EtOAc). The pure fractions were concentrated and dried under vacuum. The solids were taken up in diethylether (100 mL), collected by filtration and dried to give 20 (5.0 g, 6.6 mmol, 72% Yield) as an off-white powder.
LC-MS (method 2) 1 .53 min, ES+ m/z 756.7 [M+H], g) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3- (bromomethyl)phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11-oxo-6a,7- dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (21) and
[4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[(6aS)-5-[[4-[[(2S)-2- [[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2-methoxy-8-(4- methoxyphenyl)-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1 -c][1 ,4]benzodiazepin-3- yl]oxymethyl]phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11-oxo-6a,7-dihydro- 6H-pyrrolo[2,1 -c][1 ,4]benzodiazepine-5-carboxylate (22)
1 ,3-bis(bromomethyl)benzene (0.698 g, 2.64 mmol) was added to a mixture of 20 (1 .00 g, 1.32 mmol) and potassium carbonate (603 mg, 4.36 mmol) in acetone (10.0 mL).
The mixture was heated at 50°C for 1 .5h, at which point LCMS showed conversion of the phenol to the mono-alkylated and bis-alkylated products. The mixture was decanted, and the supernatant was evaporated to dryness. The residue was loaded on a 10g silica sample!, dried under vacuum, and loaded on top of a 50g Ultra Biotage column. The mixture was purified by chromatography (gradient EtOAc / Hexane 25/75 up to 100% EtOAc, followed by EtOAc / EtOH 90/10). The fractions were pooled and the volatiles were removed under vacuum to give the product 21 (462 mg, 0.492 mmol, 37.2% Yield), and 22 (531 mg, 0.329 mmol, 49.8% Yield), as pale yellow solids.
21 LC-MS (method 2) 1 .85 min, ES+ m/z 940.6 [M+H],
22 LC-MS (method 2) 1.93 min, ES+ m/z 1615.3 [M+H], h) [4-[[(2S)-2-[[(2S)-2-amino-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[(6aS)-5-[[4-[[(2S)-2- [[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxy carbonyl]- 2-methoxy-8-(4-methoxyphenyl)-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1- c][1,4]benzodiazepin-3-yl]oxymethyl]phenyl]methoxy]-2-methoxy-8-(4- methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5- carboxylate (23)
Compound 22 (500 mg, 0.30983 mmol) was dissolved in a mixture of dichloromethane (6.00 mL) and pyrrolidine (65.0 μL, 0.775 mmol). The atmosphere was purged with argon. A catalytic amount of tetrakis(triphenylphosphine)palladium(0) (7.20 mg, 0.00620 mmol) was added and the reaction allowed to proceed for 30 min, when LCMS showed completion.
The reaction mixture was partitioned between DCM (10 mL), 1 M aqueous ammonium chloride (10 mL). The organic layer was decanted through an isolera cartridge, and the volatiles were removed under vacuum to give crude 23 (448 mg, 0.310 mmol, 100% Yield) LC-MS (method 1) 1.44 min, ES+ m/z 1446.9 [M+H], i) [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1- yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop anoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3- [[(6aS)-5-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1- yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop anoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2- methoxy-8-(4-methoxyphenyl)-11 -oxo-6a,7-dihydro-6H-pyrrolo[2,1- c][1,4]benzodiazepin-3-yl]oxymethyl]phenyl]methoxy]-2-methoxy-8-(4- methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5- carboxylate (24)
Mal-amido-peg8-acid (161 mg, 0.266 mmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (51.6 mg, 0.266 mmol) were added to a solution of 23 (175 mg, 0.121 mmol) in dichloromethane (4 mb) at room temperature. The reaction mixture was stirred for 3h when completion was observed by LCMS. The solution was washed with water and decanted through a phase separator. The DCM was removed by evaporation and the residue was purified by flash chromatography (25g Ultra, DCM / DCM/MeOH 80/20; gradient from 15/85 up to 60/40 in 20 CV). The pure fractions were pooled and evaporated. Further purification by preparative reverse phase chromatography, and freeze- drying gave 24 (202.63 mg, 0.078085 mmol, 64.5% Yield) as a yellow solid. Purity 98.1 %. LC-MS (method 1 ) 1.89 min, ES+ m/z 1298.6 [(M+2H)/2J; (method 3) 7.64 min, ES+ m/z 1298.6 [(M+2H)/2J; 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 2H), 8.15 (d, J = 6.9 Hz, 2H), 8.00 (t, J = 5.5 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.62 - 7.52 (m, 5H), 7.52 - 7.38 (m, 9H), 7.28 - 7.09 (m, 8H), 7.00 (s, 4H), 6.95 - 6.87 (m, 4H), 5.19 - 4.83 (m, 8H), 4.38 (t, J = 7.0 Hz, 2H), 4.27 - 3.98 (m, 6H), 3.81 (s, 6H), 3.77 (s, 6H), 3.70 - 3.54 (m, 10H), 3.53 - 3.42 (m, 56H), 3.36 (t, J = 5.9 Hz, 4H), 3.28 - 3.18 (m, 2H), 3.15 (q, J = 5.8 Hz, 4H), 2.75 (d, J = 16.4 Hz, 2H), 2.48 - 2.36 (m, 4H), 2.34 (dt, J = 8.2, 5.5 Hz, 4H), 2.07 - 1 .88 (m, 2H), 1 .28 (d, J = 6.9 Hz, 6H), 0.85 (dd, J = 15.3, 6.8 Hz, 12H).
Example 4 - Alternative Synthesis of 13
Analytical Conditions for this Example High-Performance Liquid Chromatography (HPLC) Method 1
An Agilent 1260 II system was used with a Poroshell 120 PFP (4.6 x 150 mm, 2.7 μm) column with a flowrate of 1 .0 mL/min at a temperature of 40 °C. Mobile phases used were solvent A (H2O with 0.05% TFA) and solvent B (CH3CN with 0.05% TFA). The samples were loaded in CH3CN.
Gradient started with initial composition of 50% B which was held for a 4 minute period, before increasing to 95% B over a further 7 minute period. This was held for 6 minutes before returning to 30% B over 5 seconds and held there for 4 minutes. The total duration of the gradient run was 22 minutes with a sample injection volume of 1 μL and detection at 220 nm.
Method 2
An Agilent 1260 II system was used with a Poroshell 120 PFP (4.6 x 150 mm, 2.7 μm) column with a flowrate of 1 .0 mL/min at a temperature of 40 °C. Mobile phases used were solvent A (H2O with 0.05% TFA) and solvent B (CH3CN with 0.05% TFA). The sample was loaded in DMF. Gradient started with initial composition of 50% B which was held for a 4 minute period, before increasing to 95% B over a further 7 minute period. This was held for 6 minutes before returning to 30% B over 5 seconds and held there for 4 minutes. The total duration of the gradient run was 22 minutes with a sample injection volume of 1 μL and detection at 220 nm.
Method 3
An Agilent 1260 II system was used with a YMC-T riart (50 x 3.0 mm, D. S-3 μm, 12 nm) column with a flowrate of 0.5 mL/min at a temperature of 40 °C. Mobile phases used were solvent A (H2O with 0.05% TFA) and solvent B (CH3CN with 0.05% TFA). The sample was loaded in CH3CN.
Gradient started with initial composition 10% B which increased to 50% B over a 6 minute period, then further increased to 95% over a 2 minute period before returning to 10% B over 5 seconds and held there for 2 minutes. The total duration of the gradient run was 10 minutes with a sample injection volume of 0.1 μL and detection at 220 nm.
Method 4
An Agilent 1260 II system was used with an Atlantis T3 (4.6 x 150 mm, 3 μm) column with a flowrate of 1 .0 mL/min at a temperature of 40 °C. Mobile phases used were solvent A (H2O) and solvent B (CH3CN). The samples were loaded in CH3CN
Gradient started with initial composition 30% B which increased to 60% B over a 4 minute period, then further increased to 95% over an 11 minute period. This was held for 8 minutes before returning to 30% B over 5 seconds and held there for 4 minutes. The total duration of the gradient run was 27 minutes with a sample injection volume of 1 μL and detection at 220 nm.
Ultra-Performance Liquid Chromatography (UPLC)
An Agilent 1260 II system was used with a Waters Acquity (UPLC CSH C18, 1.7μ, 2.1 x 100 mm) column with a flowrate of 0.5 mL/min at a temperature of 50 °C. Mobile phases used were solvent A (H2O with 0.05% acetic acid) and solvent B (CH3CN with 0.05% acetic acid). The sample was loaded in a 1 :1 mixture of CH3CN:H2 O containing 0.05% acetic acid.
Method 1
Gradient started with initial composition of 20% B which was held for a 2 minute period, before increasing to 46% B over a further 14 minute period. This was further increased to 100% B over a 5 minute period, then held for 5 minutes before returning to 20% B over 30 seconds and held there for 4.5 minutes. The total duration of the gradient run was 30 minutes with a sample injection volume of 0.5 μL and detection at 223 nm.
Method 2
Gradient started with initial composition of 10% B which was held for a 4 minute period, before increasing to 30% B over a further 4 minute period. This was further increased to 90% B over a 10 minute period, then held for 3 minutes. The total duration of the gradient run was 21 minutes with a sample injection volume of 0.3 μL and detection at 223 nm.
Method 3
Gradient started with initial composition of 10% B which was held for a 4 minute period, before increasing to 30% B over a further 4 minute period. This was further increased to 90% B over a 10 minute period, then held for 3 minutes, before returning to 10% B over 3 minutes The total duration of the gradient run was 24 minutes with a sample injection volume of 1 μL and detection at 223 nm.
Purification Conditions for this Example
Supercritical Fluid Chromatography (SFC)
Method 1
A GreenSep Basic (3 x 15 cm, 5 μm) column was used with a flowrate of 60 mL/min at a temperature of 35 °C at a pressure of 100 bar. Mobile phases used were A (CO2) and B (MeOH with 0.1 % 2 M NH3-MeOH). An isocratic gradient of 35% B was used with a total gradient duration of 9 minutes with detection at 220 nm.
Preparative High-Performance Liquid Chromatography (Prep-HPLC)
Method 1
A 350 g (3-25 μm, 100 A) column was used with a flowrate of 100 mL/min. Mobile phases used were solvent A (H2O with 0.01 % formic acid) and solvent B (CH3CN with 0.01 % formic acid).
Gradient started with initial composition of 30% B which was held for a 5 minute period, before increasing to 45% B over a further 15 minute period. This was again increase to 50% over a 7 minute period, then ramped to 100% B over a 5 minute period before returning to 30% B over 5 minutes. The total duration of the gradient run was 37 minutes with detection at 220 nm and 254 nm.
Figure imgf000088_0001
19 20
(a) (S)-(2-Amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1 H-pyrrol-1 -yl)methanone (15)
Ethanol (8.0 V), water (0.5 V) and acetic acid (0.5 V) were added to a flask at 20-30 °C and then cooled to between -5 to 0 °C. Zn (37.0 eq.) was added at -10 to 0 °C and then the mixture was stirred for 30 minutes at 5 °C. A solution of compound 14 (1 .0 eq.) in ethanol (2.0 V) was added dropwise at between 0 °C to 5 °C, and then stirred at 5 °C for 30 minutes at which point LCMS showed reaction completion. The reaction mixture was filtered and the cake washed with EtOAc (20 V). The organic phase was washed with saturated aq. NHCO3 (10%, 40 V) and brine (20 V), then dried over Na2SO4. This was then filtered and concentrated to dryness to give 15. 94% yield on 70 g scale. Retention time: 12.06 min (HPLC Method 1 ).
(b) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (2-((S)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4- methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (16)
Compound 15 (1 .0 eq.) was dissolved in DCM (10 V) under argon. The solution was cooled to between -25 °C and -15 °C before triphosgene (0.36 eq.) and triethylamine (1.2 eq.) were added sequentially. The reaction was stirred for 30 minutes at this temperature then activated 4A molecular sieves (2.0 w/w) were added, followed by allyl N-[(1S)-1-[[(1S)-2-[4- (hydroxymethyl)anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (1 .2 eq.) and triethylamine (1.5 eq.). After 10 minutes the reaction was warmed to between 20 °C and 30 °C and stirred for 16 hours at which point LCMS showed reaction completion. The reaction mixture was filtered and the cake washed with DCM (20 V). The organic phase was washed with water (15 V) and brine (15 V), then dried over Na2SO4. This was then filtered and concentrated to dryness and the resulting residue was purified by column chromatography (0-50% EtOAc in petroleum ether). The pure fractions were pooled and the volatiles removed under vacuum to give 16. 89% yield on 65 g scale. Retention time: 12.19 min (HPLC Method 1 ).
(c) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (2-((S)-2-(hydroxymethyl)-4-methyl-2,3- dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (17)
A solution of compound 16 (1 .0 eq.) in THF (6 V) and water (0.3 V) was cooled to 15 °C. p- Toluenesulfonic acid (0.6 eq.) was added and the reaction was warmed to between 20 °C and 30 °C and stirred for 30 minutes at which point LCMS showed reaction completion. EtOAc (10 V) was added and the resultant mixture was washed with water (5 V), sat. NaHCO3 (5 V), and brine (5 V) then dried over Na2SO4. This was then filtered and concentrated to dryness and the resulting residue was purified by column chromatography (0-70% EtOAc in petroleum ether). The pure fractions were pooled and the volatiles removed under vacuum to give 17. 78% yield on 90 g scale. Retention time: 9.37 min (HPLC Method 1 ).
(d) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (S)-7-methoxy-2-methyl-5-oxo-8- ((triisopropylsilyl)oxy)-11,11 a -dihydro-1 H-benzo[e]pyrrolo[1 , 2 -a] [ 1 ,4]diazepine- 10(5H)-carboxylate (18)
Diisopropyl azodicarboxylate (2.2 eq.) was added to a solution of compound 17 (1.0 eq.) and triphenylphosphine (3.0 eq.) in THF (20 V) under a nitrogen atmosphere at 20 °C. The reaction was warmed to 25 °C for two hours at which point LCMS showed reaction completion. The solution was concentrated to give the crude product which was purified by column chromatography (0-80% EtOAc in petroleum ether). The pure fractions were pooled and the volatiles removed under vacuum to give 18. 61% yield on 55 g scale. Retention time: 9.59 min (HPLC Method 1 ).
(e) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (S)-8-hydroxy-7-methoxy-2-methyl-5-oxo- 11,11 a -dihydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine-10(5/7)-carboxylate (19)
LiOAc (0.8 eq.) was added to a solution of compound 18 (1.0 eq.) in DMF (5 V) and water (0.1 V) under a nitrogen atmosphere at between 20 °C and 30 °C. The reaction was stirred at this temperature for 8 hours at which point LCMS showed reaction completion. The solution was diluted with EtOAc (20 V) and cooled to between 0 °C and 10 °C. Aq. citric acid (0.2 M, 10 V) was slowly added and the resultant layers were separated. The aqueous phase was further extracted with EtOAc (10 V x 2). The combined organic layers were washed with brine (5 V) before the brine phase was extracted with EtOAc (5 V). The organic layers were combined and concentrated to dryness. The resultant solid was redissolved in MTBE (15 V) and stirred for 15 minutes, then filtered. The cake was washed with MTBE (5 V) and the solution was concentrated and dried under vacuum at 40 °C to give 19. 90% yield on 33 g scale. Retention time: 2.67 min (HPLC Method 2).
(f) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (S)-8-((3-(bromomethyl)benzyl)oxy)-7- methoxy-2 -methyl -5-oxo-11,11 a -dihydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine- 10(5H )-carboxy late (20)
Potassium carbonate (4.0 eq.) was added to a solution of compound 19 (1 .0 eq.) and α,α'- dibromo-m-xylene (5.8 eq.) in acetone (6.5 V) under a nitrogen atmosphere at between 20 °C and 30 °C. The reaction was stirred at this temperature for 8 hours at which point LCMS showed reaction completion. The reaction mixture was filtered and the cake washed with acetone (5 V) and DCM (5 V). The solution was concentrated to give the crude product which was purified by column chromatography (0-50% EtOAc in petroleum ether). The pure fractions were pooled and the volatiles removed under vacuum to give 20.
61% yield on 10 g scale. Retention time: 5.27 min (HPLC Method 3).
Figure imgf000091_0001
(g) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (2-((S)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-5- hydroxy-4-methoxyphenyl)carbamate (21)
LiOAc (0.8 eq.) was added to a solution of compound 16 (1 .0 eq.) in DMF (5 V) and water (0.1 V) under a nitrogen atmosphere at between 20 °C and 30 °C. The reaction was stirred at this temperature for 8 hours at which point LCMS showed reaction completion. The solution was diluted with MTBE (5 V) and washed with water (5 V x 2). The aqueous phase was extracted with MTBE (5 V) and the combined organic layers were filtered through a silica gel pad and concentrated. Heptane (10 V) was added to the crude product and after 30 minutes at 25 °C the slurry was filtered and the cake was washed with heptane (1.5 V) to give 21. 90% yield on 35 g scale. Retention time: 8.26 min (HPLC Method 1 ).
(h) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (S)-8-((3-((5-((((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-2- methoxyphenoxy)methyl)benzyl)oxy)-7-methoxy-2-methyl -5-oxo-11, 11 a -dihydro-1 H- benzo[e]pyrrolo[1 , 2 -a] [ 1 ,4]diazepine-10(5/7)-carboxylate (10)
Potassium carbonate (2.0 eq.) was added to a solution of compound 20 (1 .0 eq.), compound 21 (1 .0 eq.), and TBAI (0.1 eq.) in acetone (10 V) at between 20 °C and 30 °C. The reaction was stirred at 50 °C for 2 hours at which point LCMS showed reaction completion. Water (6.5 V) and EtOAc (10 V) were added and the resultant layers were separated. The organic phase was further washed with saturated aq. NaHCO3 (2.5 V) and brine (2 V), dried over Na2SO4, and concentrated. The resulting material was purified by column chromatography (0-80% EtOAc in petroleum ether). The pure fractions were pooled and the volatiles removed under vacuum to give 10. 78% yield on 10 g scale.
Retention time: 16.40 min (HPLC Method 4).
(i) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (S)-8-((3-((5-((((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2- (hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-2- methoxyphenoxy)methyl)benzyl)oxy)-7-methoxy-2-methyl -5-oxo-11, 11 a -dihydro-1 H- benzo[e]pyrrolo[1 , 2 -a] [ 1 ,4]diazepine-10(5H)-carboxylate (11 )
A solution of HF. pyridine (70%, 2V) was added to a solution of compound 10 (1 .0 eq.) in THF (20 V) under a nitrogen atmosphere at 0 °C. The reaction was stirred at this temperature for 1 hour at which point LCMS showed reaction completion. Saturated aq. NaHCO3 (150 V) was added to adjust the pH to 7 while maintain a temperature of between 0 °C and 5 °C. The solution was extracted with DCM (20 V x 3) and the combined organic layers were washed with water (10 V x 2), dried over Na2SO4, filtered, and concentrated. The resulting material was purified by SFC (Method 1). The pure fractions were pooled and the volatiles removed under vacuum to give 11. 86% yield on 11 g scale. Retention time: 5.55 min (HPLC Method 4). 1H NMR (400 MHz, DMSO-d6) δ 10.12 - 9.82 (m, 2H), 9.05 (s, 1 H), 8.22 - 8.04 (m, 2H), 7.64 - 7.49 (m, 5H), 7.48 - 7.34 (m, 4H), 7.31 (d, J = 8.3 Hz, 2H), 7.22 (dd, J = 8.9, 3.5 Hz, 3H), 7.09 (d, J = 11.9 Hz, 2H), 6.89 (s, 1 H), 6.67 - 6.59 (m, 1 H), 5.97 (s, 1 H), 5.96 - 5.84 (m, 2H), 5.30 (ddt, J = 16.9, 3.3, 1.7 Hz, 2H), 5.16 (ddd, J = 10.4, 3.5, 1 .8 Hz, 2H), 5.12 - 4.97 (m, 6H), 4.96 - 4.78 (m, 2H), 4.53 - 4.35 (m, 7H), 4.09 - 3.95 (m, 2H), 3.89 (t, J = 7.8 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 3.64 (s, 1 H), 3.53 (s, 2H), 2.83 (d, J = 16.2 Hz, 1 H), 2.71 - 2.58 (m, 1 H), 2.40 (d, J = 16.8 Hz, 1 H), 2.28 (d, J = 16.7 Hz, 1 H), 2.06 - 1 .89 (m, 2H), 1 .74 (s, 3H), 1.59 (s, 3H), 1.35 - 1.21 (m, 6H), 0.95 - 0.75 (m,
12H).
Figure imgf000093_0001
(j) 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (11S,11aS)-8-((3-((((S)-10-(((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)- 7-methoxy-2 -methyl -5-oxo-5,10,11 ,11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-11 -hydroxy-7-methoxy-2 -methyl -5-oxo- 11,11 a -dihydro-1 H-benzo[e]pyrrolo[1 , 2 -a] [ 1 ,4]diazepine-10(5/7)-carboxylate (12) Tetrakisacetonitrile copper(i) triflate (1.0 eq.) was added to a solution of Stahl TEMPO solution (0.2 M/L, 0.4 eq.) and compound 11 (1.0 eq.) in DCM (15.5 V) under an atmosphere of air. The reaction was stirred at 34 °C for 48 hours at which point LCMS showed reaction completion. The reaction mixture was concentrated and the resultant material was purified by column chromatography (0-10% MeOH in DCM). The pure fractions were pooled and the volatiles removed under vacuum to give 12.
86% yield on 9.4 g scale. Retention time: 17.30 (UPLC Method 1 ).
(k) 4-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)benzyl (11S,11aS)-8-((3- ((((S)-10-(((4-((S)-2-((S)-2-amino-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)-7-methoxy-2 -methyl -5-oxo- 5,10,11 ,11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2 -a] [ 1 ,4]diazepin-8- yl)oxy)methyl)benzyl)oxy)-11 -hydroxy-7-methoxy-2 -methyl -5-oxo-11,11 a -dihydro-1 H- benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (12A)
Compound 12 (1.0 eq.) was added to a solution of pyrrolidine (6.7 eq.) in DCM (30 V) at between 20 °C and 25 °C under an atmosphere of argon. Pd(PPh3)4 (0.02 eq.) was added and after 30 minutes LCMS showed reaction completion. DCM (50 V) was added and the solution was extracted with saturated aq. NH4CI (50 V) and brine (20 V). The organic phase was concentrated and the resultant material was purified by column chromatography (0- 10% MeOH in DCM). The pure fractions were pooled and the volatiles removed under vacuum to give 12A. 90% yield on 2.0 g scale. Retention time: 8.53 min (UPLC Method 2).
(I) 4-((2S,5S)-37-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-5-isopropyl-2-methyl-4,7,35- trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (11 S,11 aS)-8-((3-((((S)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-5- isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34- triazaheptatriacontanamido)benzyl)oxy)carbonyl)-7-methoxy-2 -methyl -5-oxo- 5,10,11 ,11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8- yl)oxy)methyl)benzyl)oxy)-11 -hydroxy-7-methoxy-2 -methyl -5-oxo-11,11 a -dihydro-1 H- benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (13)
MAL-dPEG®8-acid (2.93 eq.) and EDCI (2.95 eq.) were added to a solution of compound 12A (1 .0 eq.) in DCM (25 V) and MeOH (1 V) at between 20 °C and 25 °C under an atmosphere of argon in the dark. The reaction was stirred at 50 °C for 2 hours at which point LCMS showed reaction completion. The reaction was concentrated and the resultant material was purified by column chromatography (0-20% MeOH in DCM). The pure fractions were pooled and the volatiles removed under vacuum. The material was purified a second time by prep-HPLC (Method 1 ) and the pure fractions were pooled and the volatiles removed under high vacuum, at a temperature <20 °C and in complete darkness to give 13. 46% yield on 1.0 g scale. Retention time: 12.12 min (UPLC Method 3). 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 2H), 8.14 (d, J = 6.8 Hz, 2H), 7.99 (t, J = 5.6 Hz, 2H), 7.85 (d, J = 8.7 Hz, 2H), 7.55 (s, 5H), 7.49 - 7.36 (m, 3H), 7.17 (s, 3H), 7.13 - 7.04 (m, 3H), 6.99 (s, 4H), 6.94 (s, 1 H), 6.70 (s, 1 H), 6.62 (d, J = 2.4 Hz, 2H), 5.59 (s, 1 H), 5.04 (dd, J = 33.8, 16.4 Hz, 8H), 4.37 (s, 2H), 4.26 - 4.15 (m, 2H), 4.01 (s, 2H), 3.79 (d, J = 3.8 Hz, 6H), 3.72 - 3.64 (m, 2H), 3.59 (dd, J = 7.9, 6.6 Hz, 8H), 3.53 - 3.41 (m, 56H), 3.36 (t, J = 5.9 Hz, 4H), 3.14 (q, J = 5.8 Hz, 4H), 2.87 (s, 2H), 2.47 - 2.36 (m, 4H), 2.35 - 2.29 (m, 6H), 1 .95 (q, J = 6.7 Hz, 2H), 1.78 - 1.69 (m, 6H), 1.27 (s, 6H), 0.84 (dd, J = 15.4, 6.7 Hz, 12H).
Example 5
Herceptin, R347 and 1 c1 antibodies engineered to have cysteine inserted between the 239 and 240 positions were produced following the methods described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI:
10.1021 Zacs.molpharmaceut.6b00995).
HerC239i-7 ADC (ConjA)
A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 13.3 micromoles, 267 μL) to a 9.73 mL solution of antibody (20 mg, 133 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL. 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, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, 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, 2.0 micromoles, 40 μL) in DMSO was added and the reoxidation mixture was allowed to react for 4 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 2 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter- chain cysteine disulfides is observed by UHPLC). 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 .0 mg/mL. Compound 7 was added as a DMSO solution (2.5 molar equivalent/antibody, 333 nanomoles, in 2.0 mL DMSO) to 18 mL of this reoxidised antibody solution (20 mg, 133 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (1.67 micromoles, 16.7 μL at 100 mM) for 30 min at room temperature, then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjA at 280 nm and 330 nm (Compound 7 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 7, consistent with a drug-per-antibody ratio (DAR) of 1 .08 molecules of Compound 7 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjA at 280 nm shows a monomer purity of over 98%. UHPLC SEC analysis gives a concentration of final ConjA at 1.48 mg/mL in 9.8 mL, obtained mass of ConjA is 14.5 mg (73% yield).
HerC239i-13 ADC (ConjB)
A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 20 micromoles, 400 μL) to a 14.6 mL solution of antibody (30 mg, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL. 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, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, 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, 3.0 micromoles, 60 μL) in DMSO was added and the reoxidation mixture was allowed to react for 4 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 2 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter- chain cysteine disulfides is observed by UHPLC). 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 .0 mg/mL. Compound 13 was added as a DMSO solution (2.5 molar equivalent/antibody, 0.5 micromoles, in 3.0 mL DMSO) to 27 mL of this reoxidised antibody solution (30 mg, 200 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.5 micromoles, 25 μL at 100 mM) for 30 min at room temperature. Then the conjugation mixture was diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->25% then 25->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min. Drug-to-antibody ratio = 1 (DAR 1 ) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjB at 280 nm and 330 nm (Compound 13 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 13, consistent with a drug-per-antibody ratio (DAR) of 1.07 molecules of Compound 13 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAh HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjB at 280 nm shows a monomer purity of 98.5%. UHPLC SEC analysis gives a concentration of final ConjB at 0.73 mg/mL in 15 mL, obtained mass of ConjB is 10.9 mg (36% yield). R347C239i-7 ADC (ConjC)
A 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 107 micromoles, 2.13 mL) to a 133 mL solution of R347C239i (400 mg, 2.67 micromoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. 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 cm2 surface area, 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, 20 molar equivalent/antibody, 53.3 micromoles, 1 .07 mL) in DMSO was added and the reoxidation mixture was allowed to react for 4 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 3 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). 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 2.0 mg/mL. Compound 7 was added as a DMSO solution (2.5 molar equivalent/antibody, 3.33 micromoles, in 10 mL DMSO) to 90 mL of this reoxidised antibody solution (200 mg, 1.33 micromoles) for a 10% (v/v ) final DMSO concentration. The solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of N- acetyl cysteine (10 micromoles, 100 μL at 100 mM) for 30 min at room temperature. Then the conjugation mixture was sterile filtered, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto 2 x 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP columns, eluting with 0->20% then 20->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 120 mL (12 CV) at 5 mL/min. Drug-to-antibody ratio = 1 (DAR 1) fractions were pooled and buffer exchanged into 25 mM Histidine, 205 mM Sucrose pH 6.0 via TFF using mPES, MidiKros® 30 kDa fiber filter with 115 cm2 surface area, sterile-filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjC at 280 nm and 330 nm (Compound 7 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 7, consistent with a drug-per-antibody ratio (DAR) of 1.10 molecules of Compound 7 per antibody. UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjC at 280 nm shows a monomer purity of 97%. Nanodrop UV-Vis analysis gave a concentration of final ConjC at 3.80 mg/mL in 28.0 mL, obtained mass of ConjC is 106.4 mg (53% yield).
R347C239i-13 ADC (ConjD)
A 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 107 micromoles, 2.13 mL) to a 133 mL solution of R347C239i (400 mg, 2.67 micromoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. 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 cm2 surface area, 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, 20 molar equivalent/antibody, 53.3 micromoles, 1 .07 mL) in DMSO was added and the reoxidation mixture was allowed to react for 4 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 3 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). 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 2.0 mg/mL. Compound 13 was added as a DMSO solution (2.0 molar equivalent/antibody, 2.67 micromoles, in 10 mL DMSO) to 90 mL of this reoxidised antibody solution (200 mg, 1.33 micromoles) for a 10% (v/v ) final DMSO concentration. The solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of N- acetyl cysteine (8 micromoles, 80 μL at 100 mM) for 30 min at room temperature. Then the conjugation mixture was sterile filtered, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto 2 x 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP columns, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 150 mL (15 CV) at 5 mL/min. Drug-to- antibody ratio = 1 (DAR 1 ) fractions were pooled and buffer exchanged into 25 mM Histidine, 205 mM Sucrose pH 6.0 via TFF using mPES, MidiKros® 30 kDa fiber filter with 115 cm2 surface area, sterile-filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 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 (Compound 13 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 13, consistent with a drug-per-antibody ratio (DAR) of 1 .11 molecules of Compound 13 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAh HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjD at 280 nm shows a monomer purity of 98.5%. Nanodrop UV-Vis analysis gave a concentration of final ConjD at 3.59 mg/mL in 27.5 mL, obtained mass of ConjD is 98.8 mg (49% yield).
1 c 1 C239i-7 ADC (ConjE)
A 1 M solution of DL-d ith ioth reitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 33.3 micromoles, 33.3 μL) to a 25 mL solution of antibody (50 mg, 333 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL. 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, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, 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, 5.0 micromoles, 100 μL) in DMSO was added and the reoxidation mixture was allowed to react for 3.5 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 2 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter- chain cysteine disulfides is observed by UHPLC). 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 .0 mg/mL. Compound 7 was added as a DMSO solution (2.5 molar equivalent/antibody, 417 nanomoles, in 2.5 mL DMSO) to 22.5 mL of this reoxidised antibody solution (25 mg, 167 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.08 micromoles, 20.8 μL at 100 mM) for 30 min at room temperature. Then the conjugation mixture was diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 4 mL/min. Drug-to-antibody ratio = 1 (DAR 1 ) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjE at 280 nm and 330 nm (Compound 7 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 7, consistent with a drug-per-antibody ratio (DAR) of 0.97 molecules of Compound 7 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjE at 280 nm shows a monomer purity of 99.5%. UHPLC SEC analysis gives a concentration of final ConjE at 1.32 mg/mL in 3.4 mL, obtained mass of ConjE is 4.5 mg (18% yield).
1c1C239i-13 ADC (ConjF)
A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 20 micromoles, 400 μL) to a 14.6 mL solution of antibody (30 mg, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL. 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, using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, 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, 3.0 micromoles, 60 μL) in DMSO was added and the reoxidation mixture was allowed to react for 4 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 2 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter- chain cysteine disulfides is observed by UHPLC). 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 1.0 mg/mL. Compound 13 was added as a DMSO solution (2.5 molar equivalent/antibody, 0.5 micromoles, in 3.0 mL DMSO) to 27 mL of this reoxidised antibody solution (30 mg, 200 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of N-acetyl cysteine (2.5 micromoles, 25 μL at 100 mM) for 30 min at room temperature. Then the conjugation mixture was diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min. Drug-to-antibody ratio = 1 (DAR 1 ) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjF at 280 nm and 330 nm (Compound 13 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 13, consistent with a drug-per-antibody ratio (DAR) of 1.19 molecules of Compound 13 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjF at 280 nm shows a monomer purity of 99%. UHPLC SEC analysis gives a concentration of final ConjF at 3.10 mg/mL in 2.5 mL, obtained mass of ConjF is 7.7 mg (26% yield). HerC239i-24 ADC (ConjG)
A 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 10.7 micromoles, 213 μL) to a 13.3 mL solution of antibody (40 mg, 267 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. 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 using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing 30 mM Histidine, 30 mM Arginine pH 6.8 and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 30 molar equivalent/antibody, 8.0 micromoles, 160 μL) in DMSO was added and the reoxidation mixture was allowed to react for 3.75 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 3 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). 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 0.5 mg/mL. Compound 24 was added as a DMSO solution (3.0 molar equivalent/antibody, 0.8 micromoles, in 8 mL DMSO) to 72 mL of this reoxidised antibody solution (40 mg, 267 nanomoles) for a 10% (v/v ) final DMSO concentration. The solution left to react at room temperature for 20 hours at +37 °C with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.4 micromoles, 24 μL at 100 mM) for 30 min at room temperature. Then the conjugation mixture was sterile filtered, concentration to ~ 30 mL, diluted ~ 4x with 10 mM sodium phosphate pH 6.0 CHT Buffer A and loaded onto a 5 mL Bio-Scale Mini CHT ceramic hydroxyapatite 40 μm Type II cartridge, eluting with 0->100% 10 mM sodium phosphate, 1 M sodium chloride pH 6.0 CHT Buffer B over 100 mL (20 CV) at 3.5 mL/min. High monomeric purity fractions were pooled, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min. Drug-to-antibody ratio = 1 (DAR 1) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile- filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjG at 280 nm and 330 nm (Compound 24 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 24, consistent with a drug-per-antibody ratio (DAR) of 1 .27 molecules of Compound 24 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjG at 280 nm shows a monomer purity of 99%. SEC-UHPLC analysis gave a concentration of final ConjG at 1.29 mg/mL in 4.9 mL, obtained mass of ConjG is 6.3 mg (16% yield).
R347C239i-24 ADC (ConjH)
A 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 10.7 micromoles, 213 μL) to a 13.3 mL solution of R347C239i (40 mg, 267 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. 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 using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing 30 mM Histidine, 30 mM Arginine pH 6.8 and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 30 molar equivalent/antibody, 8.0 micromoles, 160 μL) in DMSO was added and the reoxidation mixture was allowed to react for 3.75 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 3 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). 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 0.5 mg/mL. Compound 24 was added as a DMSO solution (3.0 molar equivalent/antibody, 0.8 micromoles, in 8 mL DMSO) to 72 mL of this reoxidised antibody solution (40 mg, 267 nanomoles) for a 10% (v/v ) final DMSO concentration. The solution left to react at room temperature for 20 hours at +37 °C with gentle shaking, then the conjugation was quenched by addition of A/-acetyl cysteine (2.4 micromoles, 24 μL at 100 mM) for 30 min at room temperature. Then the conjugation mixture was sterile filtered, concentration to ~ 30 mL, diluted ~ 4x with 10 mM sodium phosphate pH 6.0 CHT Buffer A and loaded onto a 5 mL Bio-Scale Mini CHT ceramic hydroxyapatite 40 μm Type II cartridge, eluting with 0->100% 10 mM sodium phosphate, 1 M sodium chloride pH 6.0 CHT Buffer B over 100 mL (20 CV) at 3.5 mL/min. High monomeric purity fractions were pooled, diluted 3x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0->100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min. Drug-to-antibody ratio = 1 (DAR 1) fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile- filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjH at 280 nm and 330 nm (Compound 24 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains linked by or attached to a single molecule of Compound 24, consistent with a drug-per-antibody ratio (DAR) of 1 .30 molecules of Compound 24 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v ) on a sample of ConjH at 280 nm shows a monomer purity of 99%. SEC-UHPLC analysis gave a concentration of final ConjH at 1.84 mg/mL in 4.5 mL, obtained mass of ConjH is 8.3 mg (21% yield).
Synthesis of HER-F241CP2
Anti-HER2 trastuzumab-derived antibodies were expressed containing a lysine analogue bearing a cyclopentadiene as the reactive group (SCpHK/CP2) at F241 (EU numbering according to Kabat) according to the method described in Roy et al., MAbs 12 (1): 1684749 (doi:10.1080/19420862.2019.1684749).
Figure imgf000106_0001
Her-F241CP2-13 ADC (Coni J)
13 was added as a DMSO solution (1 molar equivalent/antibody, 0.67 micromole, in 2.458 mL DMSO) to 22.72 mL of the Herceptin-F241CP2 antibody solution in PBS, 1 mM EDTA, pH 7.4 (100.0 mg, 666.67 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react overnight at room temperature with gentle shaking. Then the conjugation was quenched by addition of N-acetyl cysteine (3.33 micromoles, 33.3 μL at 100 mM), then purified by preparative HIC chromatography using a HiTrap Butyl HP 5 mL column with Buffer A (1M ammonium sulphate, 25 mM potassium phosphate, pH 6.0) and Buffer B (25 mM potassium phosphate pH 6.0) as elution buffers. The purification was done with 30-70% B after 20CV. Fractions were individually analysed by RP-HPLC and fractions containing > 90% bridged heavy chain with 1 molecule of 13, < 5% of unconjugated heavy chain, and < 5% of heavy chain conjugated to 1 molecule of 13 were pooled and then concentrated and formulated in 20 mM His/His HCI, 240 mM sucrose pH 6.0 by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile- filtered and analysed.
UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADC at 214 nm and 330 nm shows a mixture of unconjugated light chains, light chain dimer, unconjugated heavy chains, heavy chains bridged by one molecule of 13, and non- bridged heavy chains conjugated to one molecule of 13 consistent with a drug-per-antibody ratio (DAR) of 0.95 molecules of 13 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomer purity of 99.5%. UV analysis gives a concentration of final ADC at 3.34 mg/mL in 11.0 mL, obtained mass is 36.7 mg (37% yield). LC-MS analysis on a Exactive Plus EMR mass spectrometer connected to Dionex 3000 HPLC equipment using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a de-glycosylated and reduced sample of ADC shows a mixture of unconjugated light chains, and heavy chains bridged by one molecules of 13 consistent with a drug-per-antibody ratio (DAR) of 1 .0 molecules of 13 per antibody.
UHPLC analysis on a Shimadzu Prominence system using a Proteomix HIC Butyl-NP5, 5 um, non-porous, 4.6x35 mm (Sepax) column eluting with a gradient of 1.5 M ammonium sulphate, 25 mM sodium phosphate, pH 7.4 and 25 mM sodium phosphate, pH 7.4 with 20% acetonitrile (v/v) on a neat sample of ADC at 214 nm shows singly conjugated 13, consistent with a drug-per-antibody ratio (DAR) of 1 .0 molecules of 13 per antibody.
Example 6
In vitro MTS assay
The in vitro activity of ADCs was measured in the Her2-expressing cell line NCI-N87 and the Her2 negative cell line MDA-MB-468.
The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask are measured by trypan blue staining, and counted using the LUNA-II™ Automated Cell Counter. Cells were diluted to 2x105/ml, dispensed (50 μL per well) into 96-well flat-bottom plates.
A stock solution (1 ml) of antibody drug conjugate (ADC) (20 μg/m I) 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 μL into 900 μL of cell culture medium. ADC dilution was dispensed (50 μL per well) into 4 replicate wells of the 96-well plate, containing 50 μL cell suspension seeded the previously. Control wells received 50 μL cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37°C in a CO2 -gassed incubator for the exposure time.
At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 μL per well) into each well and incubated for 4 hours at 37°C in the CO2 -gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope.
ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87. MDA-MB- 468 and NCI-N87 were cultured in RPMI 1640 with Glutamax + 10% (v/v) HyClone™ Fetal Bovine Serum.
Figure imgf000108_0001
In vitro CellTiterGlo® assay
The in vitro activity of ADCs was measured in the Her2-expressing cell line NCI-N87.
The concentration and viability of cells from a sub-confluent (80-90% confluency) T175 flask are measured and counted using the Vi-Cell BLU automated cell viability analyzer. Cells were diluted to 2.5x105/ml, dispensed (80 μL per well) into 96-well, white wall, flat- bottom plates.
A 5x stock solution of antibody drug conjugate (ADC) (200 μg/ml ) was made by dilution of
ADC into cell culture medium. A set of 4-fold dilutions of stock ADC were made in a 96-well
U-bottomed plate by serial transfer of 75 μL into 225 μL of cell culture medium. ADC dilution was dispensed (20 μL per well) into 2 replicate wells of the 96-well plate, containing 80 μL cell suspension seeded the previously. Control wells received 20 μL cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37°C in a CO2-gassed incubator for the exposure time.
At the end of the incubation period, cell viability was measured by CellTiterGlo® assay. CellTiterGlo® (Promega) was prepared according to manufacturers instructions (100 μL per well) into each well and incubated for 15 minutes at 4°C on a plate shaker. Well luminescence was measured using an Envision plate reader. Percentage cell survival was calculated from the mean luminescence in the 2 ADC-treated wells compared to the mean absorbance in the 6 control untreated wells (100%). IC50 was determined from the dose- response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope. ADC incubation times were 6 days for NCI-N87. NCI-N87 were cultured in RPMI 1640 with D-Glucose, HEPES, L-Glutamine, Sodium Bicarbonate, Sodium Pyruvate + 10% (v/v) Gibco™ HI Fetal Bovine Serum.
Figure imgf000109_0001
Example 7
NCI-N87 Xenografted Mice
Female severe combined immune-deficient mice (Fox Chase SCID®, C.B-17 lIcr-Prkdcscid, Charles River) were:
A. ten weeks old with a body weight (BW) range of 16.9 to 21.9 grams on Day 1 of the study;
B. ten weeks old with a body weight (BW) range of 15.7 to 21.7 grams 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'cobs TM 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.
Tumour Cell Culture
Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 25 μg/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. In Vivo Implantation and Tumour Growth
The NCI-N87 cells used for implantation were harvested during log phase growth and Re- suspended in phosphate buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumour implant, each test mouse was injected subcutaneously in the right flank with 1 x 107 cells (0.1 mL cell suspension), and tumour growth was monitored as the average size approached the target range of 100 to 150 mm3. Twenty-five days later, designated as Day 1 of the study, mice were sorted according to calculated tumour size into groups each consisting of ten animals with individual tumour volumes ranging from:
A. 108 to 172 mm3 and group mean tumour volumes of 131 mm3;
B. 100 to 144 mm3 and group mean tumour volumes of 110-115 mm3.
Tumours were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumour Volume
Figure imgf000110_0001
where w = width and I = length, in mm, of the tumour. Tumour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumour volume.
Treatment
Treatment began on Day 1 in groups of 10 mice (n=10) with established subcutaneous NCI-N87 tumours.
A.ConjA (2 mg/kg) and ConjG (5 mg/kg) was administered intravenously once on Day 1 (qd x 1). A vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 51.
B. ConjB (2 and 6 mg/kg) was administered intravenously once on Day 1 (qd x 1 ). A vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 93.
Each mouse was euthanized when its tumour reached the endpoint volume of 800 mm3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse.
Endpoint and Tumor Growth Delay (TGD) Analysis
Tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 800 mm3 or at the end of the study, whichever came first. Animals that exited the study for tumor volume endpoint were documented as euthanized for tumor progression (TP), with the date of euthanasia. The time to endpoint (TTE) for analysis was calculated for each mouse by the following equation:
Figure imgf000111_0001
where TTE is expressed in days, endpoint volume is expressed in mm3, b is the intercept, and m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. The data set consisted of the first observation that exceeded the endpoint volume used in analysis and the three consecutive observations that immediately preceded the attainment of this endpoint volume. The calculated TTE is usually less than the TP date, the day on which the animal was euthanized for tumor size. Animals with tumors that did not reach the endpoint volume were assigned a TTE value equal to the last day of the study. In instances in which the log-transformed calculated TTE preceded the day prior to reaching endpoint or exceeded the day of reaching tumor volume endpoint, a linear interpolation was performed to approximate the TTE. Any animal classified as having died from NTR (non-treatment-related) causes due to accident (NTRa) or due to unknown etiology (NTRu) were excluded from TTE calculations (and all further analyses). Animals classified as TR (treatment-related) deaths or NTRm (non-treatment-related death due to metastasis) were assigned a TTE value equal to the day of death. Treatment outcome was evaluated from tumor growth delay (TGD), which is defined as the increase in the median time to endpoint (TTE) in a treatment group compared to the control group:
TGD = T - C, expressed in days, or as a percentage of the median TTE of the control group:
Figure imgf000111_0002
where:
T = median TTE for a treatment group, and
C = median TTE for the designated control group.
Tumour growth inhibition
Tumor growth inhibition (TGI) analysis evaluates the difference in median tumor volumes (MTVs) of treated and control mice. For this study, the endpoint for determining TGI was:
A. Day 51
B. Day 43 . Study A finished at day 51 . The MTV (n), the median tumor volume for the number of animals, n, on the day of TGI analysis, was determined for each group. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:
Figure imgf000112_0001
The data set for TGI analysis included all animals in a group, except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.
MTV and Criteria for Regression Responses
Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day. The MTV (n) was defined as the median tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume. Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, 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 mm3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm3 for three consecutive measurements during the course of the study.
Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied. An animal with a CR response at the termination of a study was additionally classified as a tumor-free survivor (TFS). Animals were monitored for regression responses.
Toxicity
Animals were weighed daily on Days 1-5, then twice per week until the completion of the study. The mice were observed frequently for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs were recorded when observed. Individual body weight was monitored as per protocol, and any animal with weight loss exceeding 30% for one measurement or exceeding 25% for three consecutive measurements was euthanized as a TR death. Group mean body weight loss was also monitored according to CR Discovery Services protocol. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed. Deaths were classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy. A TR classification was also assigned to deaths by unknown causes during the dosing period or within 14 days of the last dose. A death was classified as non-treatment-related (NTR) if there was no evidence that death was related to treatment side effects. NTR deaths are further categorized as follows: NTRa describes deaths due to accidents or human error; NTRm is assigned to deaths thought to result from tumor dissemination by invasion and/or metastasis based on necropsy results; NTRu describes deaths of unknown causes that lack available evidence of death related to metastasis, tumor progression, accident or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as NTRu.
Statistical and Graphical Analyses
GraphPad Prism 8.0 for Windows was used for all statistical analysis and graphical presentations. Study groups experiencing toxicity beyond acceptable limits (>20% group mean body weight loss or greater than 10% treatment-related deaths) or having fewer than five evaluable observations, were not included in the statistical analysis. The logrank test was employed to assess the significance of the difference between the overall survival experiences of two groups. The logrank test analyzes the individual TTEs for all animals in a group, except those lost to the study due to NTR death. Statistical analyses of the differences between Day 19 median tumor volumes (MTVs) of control and treated groups were accomplished using the Mann-Whitney (U-test. For statistical analyses, two-tailed tests were conducted at significance level P = 0.05. Prism summarizes test results as not significant (ns) at P > 0.05, significant (symbolized by "*") at 0.01 < P ≤ 0.05, very significant ("**") at 0.001 < P ≤ 0.01 , and extremely significant ("***") at P ≤ 0.001 . Because tests of statistical significance do not provide an estimate of the magnitude of the difference between groups, all levels of significance were described as either significant or not significant. Assay A
Figure imgf000114_0001
On Day 51 , the MTV for the groups dosed with ConjA and ConjG were 288 and 379 mm3 which corresponded to significant TGIs of 50 and 34%, respectively, (P < 0.001 , Mann- Whitney (U-test).
Assay B
Figure imgf000114_0002
Groups dosed at 2mg/kg and 6 mg/kg had a median TTE of 93.0 days, which corresponded to maximal TGD of 25.4 days (38%). The group dosed at 2mg/kg had a single CR response, and all were Day 93 survivors with a MTV of 267 mm3 (Table 2 and Appendix A). The group dosed at 6mg/kg had 100% regression responses, consisting of ten CRs all of which remained as TFSs on Day 93. Both regimens resulted in a significant overall survival difference versus controls (P < 0.001 , logrank). On Day 43, the MTV for Groups dosed at 2mg/kg and 6 mg/kg were 126 and 32 mm3 which corresponded to significant TGIs of 72 and 93%, respectively, (P < 0.001 , Mann- Whitney U-test) both of which attained the 60% threshold for potential therapeutic activity.
Example 8
JIMT-1 Xenografted Mice
Female severe combined immunodeficient mice (Fox Chase SCID®, CB17/lcr- Prkdcscidl\co\crCrl, Charles River) were nine weeks old with a body weight (BW) range of 17.2 to 24.3 g 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 fiber. The mice were housed on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in static microisolators 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.
Tumor Cell Culture
JIMT-1 human breast carcinoma cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, 25 μg/mL gentamicin, and 2 mM glutamine. Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO2 and 95% air.
In Vivo Implantation and Tumor Growth
The JIMT-1 cells used for implantation were harvested during log phase growth and resuspended in cold phosphate buffered saline (PBS) containing 50% Matrigel® Matrix (Corning®). On the day of tumor implant, each test mouse was injected subcutaneously in the right flank with 1 x 107 cells (0.1 mL cell suspension), and tumor growth was monitored as the average size approached the target range of 100 to 150 mm3. Fourteen days later, designated as Day 1 of the study, mice were sorted according to calculated tumor size into groups each consisting of ten animals with individual tumor volumes ranging from 108 to 144 mm3 and group mean tumor volume of 122 mm3. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumour Volume
Figure imgf000116_0001
where w = width and / = length, in mm, of the tumour. Tumour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumour volume.
Treatment
Treatment began on Day 1 in groups of 10 mice (n=10) with established subcutaneous JIMT-1 tumours. The agents were administered i.v. via tail vein injection. The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal.
ConjA (4 mg/kg), ConjB (1 and 2 mg/kg) and ConjG (10 mg/kg) were administered intravenously once on Day 1 (qd x 1 ). A vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 81 .
Each mouse was euthanized when its tumour reached the endpoint volume of 1000 mm3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse.
The analsysis of Endpoint, Tumor Growth Delay (TGD), Tumor Growth Inhibition (TGI), MTV, Regression Responses, Toxicity, and Statistical and Graphical Analyses were all calculated as for Example 5. For this study, the endpoint for determining TGI was Day 36.
Figure imgf000116_0002
Figure imgf000117_0001
The group treated with ConjA at 4 mg/kg had a median TTE of 65.4 days, which corresponded to TGD of 22.5 days (52%). All the animals reached the tumor volume endpoint by Day 81. The ConjA regimen resulted in a significant overall survival difference versus controls (P < 0.001 , logrank). On Day 36, the MTV for the group was 327 mm3 which corresponded to significant TGI of 55% (P < 0.001 , Mann-Whitney U test test) but did not attain the 60% threshold for potential therapeutic activity.
The groups treated with ConjB at 1 mg/kg and 2 mg/kg both had a median TTE of 81 .0 days, which corresponded to TGD of 38.1 days (89%). Three of the group treated at 1 mg/kg reached the tumor volume endpoint by end of study leaving seven Day 81 survivors with an MTV of 847 mm3. There were three PR responses recorded in the group treared ar 2 mg/kg. Two of this group attained the 1000 mm3 endpoint by Day 81 leaving eight end of study survivors with an MTV of 500 mm3. Both regimens resulted in a significant overall survival difference versus controls (P < 0.001). On Day 36, the MTV for the 1 mg/kg and 2 mg/kg groups were 188 and 135 mm3 which corresponded to significant TGIs of 74 and 81% (P < 0.001 , Mann-Whitney U test) and attained the 60% threshold for potential therapeutic activity.
The group treated with ConjG at 10 mg/kg had a median TTE of 59.9 days, which corresponded to TGD of 17.0 days (40%). Nine of the treated animals attained the tumor volume endpoint by study end, leaving one end of study survivor with a tumor volume of 936 mm3. The ConjG regimen resulted in a significant overall survival difference versus controls (P < 0.001 , logrank). On Day 36, the MTV for the group was 405 mm3 which corresponded to significant TGI of 44% (P < 0.001 , Mann-Whitney U test). Example 9
PC-3 Xenografted Mice
Female athymic nude mice (Crl:NU(NCr)-Foxn7nu, Charles River) were eight weeks old on Day 1 of the study and had a BW range of 18.5 to 25.8 g. 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 fiber. The mice were housed on irradiated Enrich-o'cobs™ bedding in static microisolators 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.
Tumor Cell Culture
The androgen-independent PC-3 tumor line was derived from a human prostatic cancer metastatic to bone, and displayed the morphology of a poorly-differentiated adenocarcinoma (Kaighn, ME, et al., Invest Urol, 1979; 17(1 ): 16-23). The PC-3 cells were cultured in RPMI-1640 Medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 0.075% sodium bicarbonate, 2 mM glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin sulfate and 25 μg/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.
In Vivo Implantation and Tumor Growth
The PC-3 cells used for implantation were harvested during log phase growth and resuspended in phosphate buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumor implant, each test mouse was injected subcutaneously in the right flank with 1 x 107 cells (0.2 mL cell suspension), and tumor growth was monitored as the average size approached the target range of 100 to 150 mm3. Eight days later, designated as Day 1 of the study, mice were sorted according to calculated tumor size into ten groups each consisting of ten animals with individual tumor volumes ranging from 100 to 162 mm3 and group mean tumor volumes of 126 to 128 mm3. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumour Volume
Figure imgf000118_0001
where w = width and I = length, in mm, of the tumour. T umour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumour volume.
Treatment
Treatment began on Day 1 in groups of 10 mice (n=10) with established subcutaneous PC- 3 xenografts. The agents were administered i.v. via tail vein injection. The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal.
ConjE (6 mg/kg) and ConjF (6 mg/kg) were administered intravenously once on Day 1 (qd x 1). A vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 80.
Each mouse was euthanized when its tumour reached the endpoint volume of 800 mm3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse.
The analsysis of Endpoint, Tumor Growth Delay (TGD), Tumor Growth Inhibition (TGI), MTV, Regression Responses, Toxicity, and Statistical and Graphical Analyses were all calculated as for Example 5. For this study, the endpoint for determining TGI was Day 28.
Figure imgf000119_0001
The group treated with ConjE at 6 mg/kg had a median TTE of 74.4 days, corresponding to TGD of 44.0 days (145%). The group had 100% regression responses consisting of three PRs and seven CRs, two of which remained as TFSs on Day 80. Six animals reached the 800 mm3 tumor volume endpoint leaving four end of study survivors with an MTV of 123 mm3. The treatment produced significant survival benefit compared to vehicle-treated controls (P < 0.001).
On Day 28, the MTV for the group was 6 mm3, which corresponded to TGI of 99%. This result differed significantly versus control (P < 0.001 , Mann-Whitney test) and attained the threshold for potential therapeutic activity.
The group treated with ConjF at 6 mg/kg had a median TTE of 80.0 days, corresponding to TGD of 49.6 days (163%). The group had 100% regression responses consisting of ten CRs, all of which were TFSs on Day 80. The end of study survivors had a Day 80 MTV of 1 mm3. The treatment produced significant survival benefit compared to vehicle-treated controls (P < 0.001).
On Day 28, the MTV for the group was 6 mm3, which corresponded to TGI of 99%. This result differed significantly versus control (P < 0.001 , Mann-Whitney test) and attained the threshold for potential therapeutic activity.
Example 10
NCI-N87 Xenograft Study
Female athymic nude mice (Hsd:Athymic Nude-Foxn1nu, Envigo) were 5-7 weeks old with a body weight (BW) range of 20.1 to 26.6 grams at the start of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and Envigo Teklad 2918 Global Protein Rodent Diet consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on Envigo Teklad 7097 ¼ inch corncob bedding and in Techniplast Greenline GM500 cages on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% humidity. All animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.
Tumor Cell Culture
Human NCI-N87 gastric carcinoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 0.3g/L L-Glutamine. The cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air. In Vivo Implantation and Tumor Growth
The NCI-N87 cells used for implantation were harvested during log phase growth and resuspended in Hank's Balanced Salt Solution (HBSS) containing 50% Cultrex Basement Membrane Extract BME3 (Bio-Techne). On the day of tumor implant, each test mouse was injected subcutaneously in the right flank with 5 x 106 cells (0.2 mL cell suspension). Ten days later, mice were sorted according to calculated tumor size into groups consisting of 6 animals (Untreated group) or 3 animals (treated groups) when tumors were approximately 155mm3 in size. Randomization was performed using the deterministic randomization method built into the Study Log software package (Studylog Systems, Inc., South San Francisco, CA).
Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumor Volume
Figure imgf000121_0001
where w = width and I = length, in mm, of the tumor. T umor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.
Treatment
Treatment began on Day 10 with established subcutaneous NCI-N87 tumors.
Following randomization a single intravenous dose of ConjJ at 0.25, 0.5, 0.75, 1 , 2, 3, or 4 mg/kg was administered in ADC buffer (20mM Histidine, 240mM Sucrose pH6). The Untreated group served as the control group for efficacy analysis. The dosing volume was 0.2 mL per 20 grams of body weight (10 mb/kg), and was adjusted to the body weight of each individual animal. Tumors were measured twice per week until the study was ended on Day 55.
Tumor growth inhibition
Tumor growth inhibition (TGI) analysis evaluates the difference in mean tumor volumes (MTVs) of treated and control mice. For this study, the endpoint for determining TGI was Day 55. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:
Figure imgf000121_0002
The data set for TGI analysis included all animals in a group, except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.
MTV and Criteria for Regression Responses
Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day. The MTV (n) was defined as the mean tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume. Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of its initial volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm3 for three consecutive measurements during the course of the study.
Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied. An animal with a CR response at the termination of a study was additionally classified as a tumor-free survivor (TFS). Animals were monitored for regression responses.
Toxicity
Animals were weighed twice per week until the completion of the study. The mice were observed frequently for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs were recorded when observed. Both group and individual body weight were monitored as per protocol, and any animal with weight loss exceeding 20% for one measurement was euthanized as a TR death. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed. Deaths were classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy. A TR classification was also assigned to deaths by unknown causes during the dosing period or within 14 days of the last dose. A death was classified as non- treatment-related (NTR) if there was no evidence that death was related to treatment side effects. NTR deaths are further categorized as follows: NTRa describes deaths due to accidents or human error; NTRu describes deaths of unknown causes that lack available evidence of death related to metastasis, tumor progression, accident or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as
NTRu.
Statistical and Graphical Analyses
GraphPad Prism 9.0.0 and Microsoft Excel were used for all statistical analysis and graphical presentations. Study groups experiencing toxicity beyond acceptable limits (>20% group mean body weight loss or greater than 10% treatment-related deaths) or having fewer than five evaluable observations, were not included in the statistical analysis. For statistical analyses, two-tailed t-tests were conducted at significance level P = 0.05. Prism summarizes test results as not significant (ns) at P > 0.05, significant (symbolized by "*") at 0.01 < P ≤ 0.05, very significant ("**") at 0.001 < P ≤ 0.01 , and extremely significant ("***") at P ≤ 0.001 . Because tests of statistical significance do not provide an estimate of the magnitude of the difference between groups, all levels of significance were described as either significant or not significant.
Figure imgf000123_0001
Significant tumour growth inhibition (TGI) was observed with ConjJ across all dose levels.
Partial Response (PRs) were observed with the 3 highest dose levels (2, 3 and 4mg/kg).
Example 11
JIMT-1 Xenograft Study
Female athymic nude mice (Hsd:Athymic Nude-Foxn1 nu, Envigo) were 5-7 weeks old with a body weight (BW) range of 20.8 to 26.9 grams at the start of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and Envigo Teklad 2918 Global Protein Rodent Diet consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on Envigo Teklad 7097 ¼ inch corncob bedding and in Techniplast Greenline GM500 cages on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% humidity. All animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.
Tumor Cell Culture
JIMT-1 human breast carcinoma cells were grown in Dulbecco's Modified Eagle's
Medium (DMEM) containing 10% fetal bovine serum, 1 g/L D-Glucose, 0.3g/L L-Glutamine, 25mM HEPES and 110mg/L Sodium Pyruvate. Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO2 and 95% air.
In Vivo Implantation and Tumor Growth
The JIMT-1 cells used for implantation were harvested during log phase growth and resuspended in cold phosphate buffered saline (PBS) containing 50% Cultrex Basement Membrane Extract BME3 (Bio-Techne). On the day of tumor implant, each test mouse was injected subcutaneously in the right flank with 5 x 106 cells (0.2 mL cell suspension). Seven days later, mice were sorted according to calculated tumor size into groups consisting of 6 animals (Untreated group) or 3 animals (treated groups) when tumors were approximately 190mm3 in size. Randomization was performed using the deterministic randomization method built into the Study Log software package (Studylog Systems, Inc., South San Francisco, CA). Tumors were measured twice per week.
Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumor Volume
Figure imgf000124_0001
where w = width and / = length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.
Treatment
Treatment began on Day 7 with established subcutaneous JIMT-1 tumors.
Following randomization a single intravenous dose of ConjJ at 0.25, 0.5, 0.75, 1 , 2, 3, or 4 mg/kg was administered in ADC buffer (20mM Histidine, 240mM Sucrose pH6). The
Untreated group served as the control group for efficacy analysis. The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was adjusted to the body weight of each individual animal.
The analysis of Endpoint, Tumor Growth Inhibition (TGI), MTV, Regression Responses,
Toxicity, and Statistical and Graphical Analyses were all calculated as for Example 5. For this study, the endpoint for determining TGI was Day 42 since some untreated tumors were removed due to ulcerations or excessive volume as the study progressed.
JIMT-1 xenograft results
Figure imgf000125_0001
Statistically significant TGI was observed with the high dose of 4mg/kg ConjJ, but failed to produce any PRs or CRs based on the established criteria.
All documents and other references mentioned above are herein incorporated by reference.
EMBODIMENTS OF DISCLOSURE
1. A conjugate of formula I:
Figure imgf000126_0001
wherein
Ab is a modified antibody having at least one free conjugation site on each heavy chain D represents either group D1 or D2:
Figure imgf000126_0002
the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R2 is selected from the group consisting of:
(ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
(ib) C1-5 saturated aliphatic alkyl;
(ic) C3-6 saturated cycloalkyl;
(id)
Figure imgf000126_0003
, wherein each of R11, R12 and R13 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 R2 group is no more than 5; (ie)
Figure imgf000127_0001
, wherein one of R15a and R15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(if)
Figure imgf000127_0002
, where R14 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,
R2 is selected from H, OH, F, diF and
Figure imgf000127_0003
, where R16a and R16b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl ester;
D' represents either group D'1 or D'2:
Figure imgf000127_0004
wherein the dotted line indicates the optional presence of a double bond between C2' and C3'; when there is a double bond present between C2' and C3', R22 is selected from the group consisting of:
(iia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
(iib) C1-5 saturated aliphatic alkyl;
(iic) C3-6 saturated cycloalkyl; (iid)
Figure imgf000128_0001
, wherein each of R31, R32 and R33 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 R22 group is no more than 5;
(iie) , wherein one of R25a and R25b is H and the other is selected from:
Figure imgf000128_0002
phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(iif) , where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C
Figure imgf000128_0003
2-3 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',
R22 is selected from H, OH, F, diF and
Figure imgf000128_0004
where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo; where R and R' are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo;
R" is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y' are selected from O, S, and NH;
R11a is:
(i) H; or
(ii) OH or ORA, where RA is C1-4 alkyl;
R6'', R7' and R9' are selected from the same groups as R6, R7 and R9 respectively; and RLL1 and RLL2 are linkers connected to the antibody at different sites which are independently
Figure imgf000129_0003
wherein
Q is:
Figure imgf000129_0001
, where Qx is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;
X is:
Figure imgf000129_0002
where a = 0 to 5, b = 0 to 16, c = 0 or 1 , d = 0 to 5; GLL is a linker connected to the antibody.
2. A conjugate according to statement 1 , wherein both Y and Y' are O.
3. A conjugate according to either statement 1 or statement 2, wherein R" is C3-7 alkylene.
4. A conjugate according to either statement 1 or statement 2, wherein R" is a group of formula:
Figure imgf000129_0004
where r is 1 or 2.
5. A conjugate according to any one of statements 1 to 4, wherein R9 is H. 6. A conjugate according to any one of statements 1 to 5, wherein R6 is H.
7. A conjugate according to any one of statements 1 to 6, wherein R7 is selected from H, OH and OR.
8. A conjugate according to statement 7, wherein R7 is a C1-4 alkyloxy group.
9. A conjugate according to statement 8, wherein R7 is a methoxy group.
10. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a double bond between C2 and C3, and R2 is a C5-7 aryl group.
11. A conjugate according to statement 10, wherein R2 is phenyl.
12. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a double bond between C2 and C3, and R2 is a C8-10 aryl group.
13. A conjugate according to any one of statements 10 to 12, wherein R2 bears one to three substituent groups.
14. A conjugate according to any one of statements 10 to 13, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl- piperazinyl, morpholino and methyl-thiophenyl.
15. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a double bond between C2 and C3, and R2 is a C1-5 saturated aliphatic alkyl group.
16. A conjugate according to statement 15, wherein R2 is methyl, ethyl or propyl.
17. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a double bond between C2 and C3, and R2 is a C3-6 saturated cycloalkyl group.
18. A conjugate according to statement 17, wherein R2 is cyclopropyl. 19. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a double bond between C2 and C3, and R2 is a group of formula:
Figure imgf000131_0001
20. A conjugate according to statement 19, wherein the total number of carbon atoms in the R2 group is no more than 4.
21. A conjugate according to statement 20, wherein the total number of carbon atoms in the R2 group is no more than 3.
22. A conjugate according to any one of statements 19 to 21 , wherein one of R11, R12 and R13 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
23. A conjugate according to any one of statements 19 to 21 , wherein two of R11, R12 and R13 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
24. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a double bond between C2 and C3, and R2 is a group of formula:
Figure imgf000131_0002
25. A conjugate according to statement 24, wherein R2 is the group:
Figure imgf000131_0003
26. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a double bond between C2 and C3, and R2 is a group of formula:
Figure imgf000131_0004
27. A conjugate according to statement 26, wherein R14 is selected from H, methyl, ethyl, ethenyl and ethynyl.
28. A conjugate according to statement 27, wherein R14 is selected from H and methyl.
29. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a single bond between C2 and C3, and R2 is H.
30. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a single bond between C2 and C3, R2 is and R16a and R16b are both H.
Figure imgf000132_0001
31. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a single bond between C2 and C3, R2 is , and R16a and R16b are both methyl.
Figure imgf000132_0002
32. A conjugate according to any one of statements 1 to 9, wherein D is D1 , there is a single bond between C2 and C3, R2 is
Figure imgf000132_0003
, one of R16a and R16b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
33. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1 , there is a double bond between C2’ and C3’, and R22 is a C5-7 aryl group.
34. A conjugate according to statement 33, wherein R22 is phenyl.
35. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1 , there is a double bond between C2’ and C3’, and R22 is a C8-10 aryl group.
36. A conjugate according to any one of statements 33 to 35, wherein R22 bears one to three substituent groups. 37. A conjugate according to any one of statements 33 to 36, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl- piperazinyl, morpholino and methyl-thiophenyl.
38. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a double bond between C2' and C3', and R22 is a C1-5 saturated aliphatic alkyl group.
39. A conjugate according to statement 38, wherein R22 is methyl, ethyl or propyl.
40. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a double bond between C2' and C3', and R22 is a C3-6 saturated cycloalkyl group.
41. A conjugate according to statement 40, wherein R22 is cyclopropyl.
42. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a double bond between C2' and C3', and R22 is a group of formula:
Figure imgf000133_0001
43. A conjugate according to statement 42, wherein the total number of carbon atoms in the R22 group is no more than 4.
44. A conjugate according to statement 43, wherein the total number of carbon atoms in the R22 group is no more than 3.
45. A conjugate according to any one of statements 42 to 44, wherein one of R31, R32 and R33 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
46. A conjugate according to any one of statements 42 to 44, wherein two of R31, R32 and R33 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. 47. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a double bond between C2' and C3', and R22 is a group of formula:
Figure imgf000134_0001
48. A conjugate according to statement 47, wherein R22 is the group:
Figure imgf000134_0002
49. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a double bond between C2' and C3', and R22 is a group of formula:
Figure imgf000134_0003
50. A conjugate according to statement 49, wherein R24 is selected from H, methyl, ethyl, ethenyl and ethynyl.
51. A conjugate according to statement 50, wherein R24 is selected from H and methyl.
52. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a single bond between C2' and C3', and R22 is H.
53. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a single bond between C2' and C3', R22 is
Figure imgf000134_0004
and R26a and R26b are both H.
54. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a single bond between C2' and C3', R22 is , and R26a and R26b are both methyl.
Figure imgf000134_0005
55. A conjugate according to any one of statements 1 to 32, wherein D' is D'1 , there is a single bond between C2' and C3', R22 is , one of R26a and R26b is H, and the
Figure imgf000134_0006
other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
56. A conjugate according to any one of statements 1 to 55, wherein R11a is H.
57. A conjugate according to any one of statements 1 to 55, wherein R11a is OH.
58. A conjugate according to any one of statements 1 to 55, wherein R11a is ORA, where RA is C1-4 alkyl.
59. A conjugateaccording to statement 58, wherein RA is methyl.
60. A conjugate according to any one of statements 1 to 59, wherein R6' is selected from the same groups as R6, R7' is selected from the same groups as R7, R9' is selected from the same groups as R9 and Y is selected from the same groups as Y.
61 . A conjugate according to statement 60, wherein R6' is the same group as R6, R7' is the same group as R7, R9' is the same group as R9 and Y is the same group as Y.
62. A conjugate according to any one of statements 1 to 61 , wherein R22 is the same group as R2.
63. A conjugate according to statement 1 , which is of formula la-1 , la-2 or la-3:
Figure imgf000135_0001
Figure imgf000136_0001
where the dotted line represents the possible presence of a double bond between C2 and C3 and C2' and C3'; where there is no double bond between C2 and C3 and C2' and C3', R2a and R22a are the same and are selected from:
(a) H; and
(b)
Figure imgf000136_0002
where there is a double bond between C2 and C3 and C2' and C3' R2a and R22a are the same and are selected from:
Figure imgf000136_0003
Figure imgf000137_0001
R1a is selected from methyl and benzyl; and
RLL1, RLL2 and R11a are as defined in statement 1 .
64. A conjugate according to any one of statements 1 to 63, wherein Q is an amino acid residue selected from Phe, Lys, Vai, Ala, Cit, Leu, lie, Arg, and Trp.
65. A conjugate according to any one of statements 1 to 63, wherein Q is a dipeptide residue selected from:
CO-Phe-Lys-NH,
CO-Val-Ala-NH,
CO-Val-Lys-NH,
CO-Ala-Lys-NH,
CO-Val-Cit-NH,
CO-Phe-Cit-NH,
CO-Leu-Cit-NH,
CO-lle-Cit-NH,
CO-Phe-Arg-NH, and
CO-Trp-Cit-NH.
66. A conjugate according to statement 65, wherein Q is selected from CO-Phe-Lys-NH,
CO-Val-Cit-NH and CO-Val-Ala-NH.
67. A conjugate according to any one of statements 1 to 63, wherein Q is a tripeptide residue selected from:
NH-Glu-Val-Ala-C=O NH-Glu-Val-Cit-C=O NH-αGlu-Val-Ala-C=O NH-αGlu-Val-Cit-C=O
68. A conjugate according to any one of statements 1 to 63, wherein Q is a tetrapeptide linkers selected from:
NH -Gly-Gly-Phe-GlyC=O; and
NH -Gly-Phe-Gly-GlyC=O.
69. A conjugate according to any one of statements 1 to 68, a is 0 to 3.
70. A conjugate according to statement 69, wherein a is 0.
71. A conjugate according to any one of statements 1 to 70, wherein b is 0 to 12.
72. A conjugate according to statement 71 , wherein b is 0 to 8.
73. A conjugate according to any one of statements 1 to 72, wherein c is 0.
74. A conjugate according to any one of statements 1 to 72, wherein c is 1 .
75. A conjugate according to any one of statements 1 to 74, wherein d is 0 to 3.
76. A conjugate according to statement 75, wherein d is 2.
77. A conjugate according to any one of statements 1 to 68, wherein a is 0, c is 1 and d is 2, and b is from 0 to 8.
78. A conjugate according to statement 77, wherein b is 0, 4 or 8.
79. A conjugate according to any one of statements 1 to 78, wherein GLL is selected from:
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0003
where CBA represents the point of connection to the modified antibody Ar represents a C5-6 arylene group, e.g. phenylene and X1 represents C1-4 alkyl.
80. A conjugate according to statement 79, wherein Ar is a phenylene group.
81. A conjugate according to either statement 79 or statement 80, wherein GLL is selected from GLL1-1 and GLL1-2.
82. A conjugate according to statement 81 , wherein GLL is GLL1-1.
83. A conjugate according to either statement 79 or statement 80, wherein GLL is GLL1- 1 A
84. A conjugate according to statement 1 of formula Id:
Figure imgf000140_0001
where m is an integer from 2 to 8, and X is selected from: and
Figure imgf000140_0002
Figure imgf000141_0001
85. A conjugate according to statement 1 of formula le:
Figure imgf000141_0002
where m is an integer from 2 to 8, and X is selected from:
Figure imgf000141_0003
86. The conjugate according to any preceding statement wherein the modified antibody having at least one free conjugation site on each heavy chain is an IgG 1 , lgG2, lgG3 or lgG4 antibody.
87. The conjugate according to statement 86 wherein the modified antibody having at least one free conjugation site on each heavy chain is a human antibody.
88. The conjugate according to statement 87 wherein the modified antibody having at least one free conjugation site on each heavy chain is a humanized antibody. 89. The conjugate according to statement 88, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.
90. The conjugate according to statement 89 wherein the antibody or antibody fragment is an antibody which binds to one or more tumor-associated antigens or cell- surface receptors selected from (1 )-(89):
(1) BMPR1B;
(2) E16;
(3) STEAP1 ;
(4) 0772P;
(5) MPF;
(6) Napi3b;
(7) Sema 5b;
(8) PSCA hlg;
(9) ETBR;
(10) MSG783;
(11) STEAP2;
(12) TrpM4;
(13) CRIPTO;
(14) CD21 ;
(15) CD79b;
(16) FcRH2 ;
(17) HER2;
(18) NCA;
(19) MDP;
(20) IL20R-alpha;
(21) Brevican;
(22) EphB2R;
(23) ASLG659;
(24) PSCA;
(25) GEDA;
(26) BAFF-R;
(27) CD22;
(28) CD79a;
(29) CXCR5;
(30) HLA-DOB; (31) P2X5;
(32) CD72;
(33) LY64;
(34) FcRH1 ;
(35) IRTA2;
(36) TENB2;
(37) PSMA - FOLH1 ;
(38) SST;
(38.1 ) SSTR2;
(38.2) SSTR5;
(38.3) SSTR1 ;
(38.4)SSTR3;
(38.5) SSTR4;
(39) ITGAV;
(40) ITGB6;
(41) CEACAM5;
(42) MET;
(43) MUC1 ;
(44) CA9;
(45) EGFRvlll;
(46) CD33;
(47) CD19;
(48) IL2RA;
(49) AXL;
(50) CD30 - TNFRSF8;
(51) BCMA - TNFRSF17;
(52) CT Ags - CTA;
(53) CD174 (Lewis Y) - FUT3;
(54) CLEC14A;
(55) GRP78 - HSPA5;
(56) CD70;
(57) Stem Cell specific antigens;
(58) ASG-5;
(59) ENPP3;
(60) PRR4;
(61) GCC - GUCY2C; (62) Liv-1 - SLC39A6;
(63) 5T4;
(64) CD56 - NCMA1 ;
(65) CanAg;
(66) FOLR1 ;
(67) GPNMB;
(68) TIM-1 - HAVCR1 ;
(69) RG-1/Prostate tumor target Mindin - Mindin/RG-1 ;
(70) B7-H4 - VTCN1 ;
(71) PTK7;
(72) CD37;
(73) CD138 - SDC1 ;
(74) CD74;
(75) Claudins - CLs;
(76) EGFR;
(77) Her3;
(78) RON - MST1 R;
(79) EPHA2;
(80) CD20 - MS4A1 ;
(81) Tenascin C - TNC;
(82) FAP;
(83) DKK-1 ;
(84) CD52;
(85) CS1 - SLAMF7;
(86) Endoglin - ENG;
(87) Annexin A1 - ANXA1 ;
(88) V-CAM (CD106) - VCAM1;
(89) ASCT2 (SLC1A5).
91. The conjugate according to any one of statements 86 to 90, wherein the native interchain cysteine residues have been substituted for amino acid residues lacking thiol groups.
92. The conjugate according to statement 91 , comprising at least one additional substititions in each heavy chain of an amino acid residue comprising a reactive group suitable for conjugation to a linker. 93. The conjugate according to statement 92, wherein the additionally substituted amino acid is a cysteine or a non-natural amino acid.
94. The conjugate according to statement 92 or 93 wherein the position that is substituted is selected from those set forth below:
Figure imgf000145_0001
95. The conjugate according to any one of statements 86 to 90, comprising at least one insertion in each heavy chain of an amino acid residue comprising a reactive group suitable for conjugation to a linker. 96. The conjugate according to statement 95, wherein the inserted amino acid is a cysteine or a non-natural amino acid.
97. The conjugate according to statement 96, wherein the inserted amino acid is a cysteine.
98. The conjugate according to statement 97, wherein the inserted amino acid is CP2-
NNAA :
Figure imgf000146_0001
99. A compound of formula II:
Figure imgf000146_0002
and salts and solvates thereof, wherein D, R2, R6, R7, R9, R11a, Y, R", Y', D', R6', R7', R9', and R22 (including the presence or absence of double bonds between C2 and C3 and C2' and C3' respectively) are as defined in any one of statements 1 to 62;
RL1 and RL2 are linkers for connecting to a cell binding agent, which are independently
Figure imgf000146_0003
where Q and X are as defined in any one of statements 1 and 64 to 78 and GL is a linker for connecting to an antibody. 100. A compound according to statement 99, wherein GL is selected from:
Figure imgf000147_0001
Figure imgf000148_0002
where Ar represents a C5-6 arylene group, e.g. phenylene, and X1 represents C1-4 alkyl.
101. A compound according to statement 100, wherein Ar is a phenylene group.
102. A compound according to either statement 100 or statement 101 , wherein GL is selected from GL1-1 and GL1-2.
103. A compound according to statement 102, wherein GL is GL1-1.
104. A compound according to statement 99, wherein the compound is of formula lid:
Figure imgf000148_0001
where m is an integer from 2 to 8, and X is selected from: and
Figure imgf000148_0003
Figure imgf000149_0001
105. The conjugate according to any one of statements 1 to 98, for use in therapy.
106. A pharmaceutical composition comprising the conjugate of any one of statements 1 to 98 and at least one pharmaceutically acceptable diluent, carrier or excipient.
107. The conjugate according to any one of statements 1 to 98 or the pharmaceutical composition according to statement 106, for use in the treatment of a proliferative disease in a subject.
108. The conjugate for use according to statement 107, wherein the disease treated is cancer.
109. Use of a conjugate according to any one of statements 1 to 98 or a pharmaceutical according to statement 106 in a method of medical treatment.
110. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 106.
111. The method of statement 110 wherein the method of medical treatment is for treating cancer.
112. The method of statement 111 , wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.
113. Use of a conjugate according to any one of statements 1 to 98 in a method of manufacture of a medicament for the treatment of a proliferative disease.
114. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate according to any one of statements 1 to 98 or a pharmaceutical composition according to statement 106.
115. A method of synthesis of a conjugate according to any one of statements 1 to 98 comprising conjugating a compound according to any one of statements 99 to 104 with a modified antibody. 116. A method of making a compound of formula IV:
Figure imgf000150_0001
from a compound of formula V:
Figure imgf000150_0002
using the by Mitsunobu reaction; where R8 is selected from:
(a) OMe, OCH2Ph, OH and -Y'-R"-Hal;
(b)
Figure imgf000150_0003
Figure imgf000151_0001
R6, R7, R9, R11a , R6' , R7' , R9' , Y, R", Y', D and D' are as defined in any one of statements 1 to 62;
Hal is a halogen;
RL2pre is a precursor to RL2;
RL1pre is a precursor to RL1; and
ProtO is a hydroxyl protecting group.
117. The method according to statement 114, where Hal is Br.
118. The method according to either statement 114 or statement 115, where RL1pre and RL2pre are independently:
Figure imgf000151_0002
where Q is as defined in any one of statements 1 and 64 to 68 and ProtN is an amine protecting group.
119. The method according to statement 118, where ProtN is a carbamate.
120. The method according to statement 119, where ProtN is Alloc.
121. The method according to any one of statements 116 to 120, where ProtO is a silyl ether.
122. The method according to statement 121 , where ProtO is TIPS. 123. The method according to any one of statement 116 to 122, wherein the reaction is carried out using triphenylphosphine and an azodicarboxylate.
124. The method according to claim 123, wherein the azodicarboxylate is diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD).

Claims

1. A conjugate of formula I:
Figure imgf000153_0001
wherein
Ab is a modified antibody having at least one free conjugation site on each heavy chain D represents either group D1 or D2:
Figure imgf000153_0002
the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R2 is selected from the group consisting of:
(ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
(ib) C1-5 saturated aliphatic alkyl;
(ic) C3-6 saturated cycloalkyl;
(id) , wherein each of R11, R12 and R13 are independently selected from H,
Figure imgf000153_0003
C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5; (ie) , wherein one of R15a and R15b is H and the other is selected from:
Figure imgf000154_0001
phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(if) , where R14 is selected from: H; C1-3 saturated alkyl; C
Figure imgf000154_0002
2-3 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,
R2 is selected from H, OH, F, diF and , where R16a and R16b are
Figure imgf000154_0003
independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl ester;
D' represents either group D'1 or D'2:
Figure imgf000154_0004
wherein the dotted line indicates the optional presence of a double bond between C2' and C3'; when there is a double bond present between C2' and C3', R22 is selected from the group consisting of:
(iia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
(iib) C1-5 saturated aliphatic alkyl;
(iic) C3-6 saturated cycloalkyl; (iid)
Figure imgf000155_0001
, wherein each of R31, R32 and R33 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 R22 group is no more than 5;
(iie)
Figure imgf000155_0002
, wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(iif)
Figure imgf000155_0003
where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2' and C3',
R22 is selected from H, OH, F, diF and
Figure imgf000155_0004
, where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo; where R and R' are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo;
R" is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y' are selected from O, S, and NH;
R11a is:
(i) H; or
(ii) OH or ORA, where RA is C1-4 alkyl;
R6'', R7' and R9' are selected from the same groups as R6, R7 and R9 respectively; and RLL1 and RLL2 are linkers connected to the antibody at different sites which are independently
Figure imgf000156_0003
wherein
Q is:
Figure imgf000156_0001
, where Qx is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;
X is:
Figure imgf000156_0002
where a = 0 to 5, b = 0 to 16, c = 0 or 1 , d = 0 to 5; GLL is a linker connected to the antibody.
2. A conjugate according to claim 1 , wherein:
(a) both Y and Y' are O; and/or
(b) R" is
(b-i) C3-7 alkylene; or
(b-ii) a group of formula:
Figure imgf000156_0004
, where r is 1 or 2.
3. A conjugate according to either claim 1 or claim 2, R9 and R6 are both H.
4. A conjugate according to any one of claims 1 to 3, wherein R7 is a C1-4 alkyloxy group.
5. A conjugate according to any one of claims 1 to 4, wherein D is D1 , there is a double bond between C2 and C3, and R2 is selected from:
(a) optionally substituted phenyl, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl;
(b) optionally substituted C8-10 aryl group, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl;
(c) methyl, ethyl or propyl;
(d) cyclopropyl;
(e) a group of formula
, wherein the total number of carbon atoms in the R2 group is no more than
Figure imgf000157_0001
4;
Figure imgf000157_0002
(g) a group of formula:
, wherein R14 is selected from H and methyl.
Figure imgf000157_0003
6. A conjugate according to any one of claims 1 to 4, wherein D is D1 , there is a single bond between C2 and C3, R2 is and:
Figure imgf000157_0004
(a) R16a and R16b are both H; or
(b) R16a and R16b are both methyl; or
(c) one of R16a and R16b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
7. A conjugate according to any one of claims 1 to 6, wherein D' is D'1 , there is a double bond between C2' and C3', and R22 is selected from: (a) optionally substituted phenyl, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl;
(b) optionally substituted C8-10 aryl group, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl;
(c) methyl, ethyl or propyl;
(d) cyclopropyl;
(e) a group of formula
, wherein the total number of carbon atoms in the R2 group is no more than
Figure imgf000158_0001
4;
Figure imgf000158_0002
(g) a group of formula:
, wherein R24 is selected from H and methyl.
Figure imgf000158_0003
8. A conjugate according to any one of claims 1 to 6, wherein D' is D'1 , there is a single bond between C2' and C3', R22 is
Figure imgf000158_0004
and:
(a) R26a and R26b are both H; or
(b) R26a and R26b are both methyl; or
(c) one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
9. A conjugate according to any one of claims 1 to 8, wherein R11a is H or OH.
10. A conjugate according to any one of claims 1 to 9, wherein R6'' is the same group as R6, R7' is the same group as R7, R9' is the same group as R9, Y is the same group as Y and R22 is the same group as R2.
11. A conjugate according to claim 1 , which is of formula la-1 , la-2 or la-3:
Figure imgf000159_0001
where the dotted line represents the possible presence of a double bond between C2 and C3 and C2' and C3'; where there is no double bond between C2 and C3 and C2' and C3', R2a and R22a are the same and are selected from:
(a) H; and
Figure imgf000159_0002
where there is a double bond between C2 and C3 and C2' and C3' R2a and R22a are the same and are selected from:
Figure imgf000160_0001
R1a is selected from methyl and benzyl; and
RLL1, RLL2 and R11a are as defined in claim 1.
12. A conjugate according to any one of claims 1 to 11 , wherein Q is:
(a) an amino acid residue selected from Phe, Lys, Vai, Ala, Cit, Leu, lie, Arg, and Trp;
(b) a dipeptide residue selected from CO-Phe-Lys-NH, CO-Val-Cit-NH and CO-Val-Ala-NH;
(c) a tripeptide residue selected from: NH-Glu-Val-Ala-C=O, NH-Glu-Val-Cit-C=O, NH-αGlu-Val- Ala-C=O and NH-αGlu-Val-Cit-C=O;
(d) a tetrapeptide linkers selected from: NH -Gly-Gly-Phe-GlyC=O and NH -Gly-Phe-Gly-Gly C=O
13. A conjugate according to any one of claims 1 to 12, wherein a is 0, c is 1 and d is 2, and b is from 0 to 8.
14. A conjugate according to any one of claims 1 to 13, wherein GLL is selected from:
Figure imgf000161_0001
Figure imgf000162_0003
where CBA represents the point of connection to the modified antibody, Ar represents a C5- 6 arylene group, e.g. phenylene and X1 represents C1-4 alkyl.
15. A conjugate according to claim 14, wherein GLL is GLL1-1 or GLL1-1A.
16. A conjugate according to claim 1 of formula Id:
Figure imgf000162_0001
where m is an integer from 2 to 8, and X is selected from:
(
( and
(
Figure imgf000162_0002
17. A conjugate according to claim 1 of formula le:
Figure imgf000163_0001
where m is an integer from 2 to 8, and X is selected from:
Figure imgf000163_0002
18. A compound of formula II:
Figure imgf000163_0003
and salts and solvates thereof, wherein D, R2, R6, R7, R9, R11a, Y, R", Y', D', R6'', R7'', R9'', and R22 (including the presence or absence of double bonds between C2 and C3 and C2' and C3' respectively) are as defined in any one of claims 1 to 11 ; RL1 and RL2 are linkers for connecting to a cell binding agent, which are independently
Figure imgf000164_0001
where Q and X are as defined in any one of claims 1 , 12 and 13 and GL is a linker for connecting to an antibody.
19. A compound according to claim 18, wherein GL is selected from:
Figure imgf000164_0002
Figure imgf000165_0002
where Ar represents a C5-6 arylene group, e.g. phenylene, and X1 represents C1-4 alkyl
20. A compound according to claim 19, wherein GL is GL1-1.
21. A compound according to claim 18, wherein the compound is of formula lid:
Figure imgf000165_0001
where m is an integer from 2 to 8, and X is selected from:
Figure imgf000166_0002
22. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 17 and at least one pharmaceutically acceptable diluent, carrier or excipient.
23. A method of making a compound of formula IV:
Figure imgf000166_0001
from a compound of formula V:
Figure imgf000166_0003
using the by Mitsunobu reaction; where R8 is selected from:
(a) OMe, OCH2Ph, OH and -Y'-R"-Hal;
(b)
Figure imgf000167_0001
R6, R7, R9, R11a , R6'', R7'', R9'', Y, R", Y', D and D' are as defined in any one of claims 1 to 11 ;
Hal is a halogen;
RL2pre is a precursor to RL2;
RL1pr e is a p recursor to RL1; and
Prot0 is a hydroxyl protecting group.
24. The method according to claim 23, where RL1pre and RL2pre are independently:
Figure imgf000167_0002
where Q is as defined in either claim 1 or claim 12 and ProtN is an amine protecting group.
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