NZ751432B2 - Pyrrolobenzodiazepines and conjugates thereof - Google Patents

Pyrrolobenzodiazepines and conjugates thereof Download PDF

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NZ751432B2
NZ751432B2 NZ751432A NZ75143214A NZ751432B2 NZ 751432 B2 NZ751432 B2 NZ 751432B2 NZ 751432 A NZ751432 A NZ 751432A NZ 75143214 A NZ75143214 A NZ 75143214A NZ 751432 B2 NZ751432 B2 NZ 751432B2
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group
formula
conjugate
compound
alkyl
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NZ751432A
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NZ751432A (en
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John A Flygare
Philip Wilson Howard
Thomas Pillow
Binqing Wei
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Genentech Inc
Medimmune Limited
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Priority claimed from NZ710745A external-priority patent/NZ710745A/en
Publication of NZ751432A publication Critical patent/NZ751432A/en
Publication of NZ751432B2 publication Critical patent/NZ751432B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • A—HUMAN NECESSITIES
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    • 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
    • A—HUMAN NECESSITIES
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    • 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
    • A—HUMAN NECESSITIES
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    • 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
    • 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
    • A61K47/6869—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 a cell of the reproductive system: ovaria, uterus, testes, prostate
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • 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
    • C07D487/12—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 three hetero rings
    • C07D487/16—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Abstract

The present invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepines having a linker group connected to a cell binding agent. In one aspect, the present invention provides a conjugate comprising a PBD dimer connected through the dimer bridging portion via a specified linker to a cell binding agent. The present invention is suitable for use in providing a PBD conjugate to a preferred site in a subject. In particular, the present invention provides a conjugate of formula (A), use of a conjugate of formula (A) the manufacture of a medicament, a pharmaceutical composition comprising a conjugate of formula (A), a compound of formula (B), a compound of formula (C), a compound of formula (D), a compound of formula (E) and/or a method of synthesis of a compound. In preferred embodiments, use of a conjugate of formula (A) the manufacture of a medicament for treating cancer and/or a proliferative disease. Conjugate compounds of formula (A): wherein: R2 is, where R36a and R36b 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 R36a and R36b 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', NO2, Me3Sn and halo; R7 is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halo; Y is selected from formulae A1, A2, A3, A4, A5 and A6: L is a linker connected to a cell binding agent; CBA is the cell binding agent; n is an integer selected in the range of 0 to 48; RA4 is a C1-6 alkylene group; either (a) R10 is H, and R11 is OH, ORA, where RA is C1-4 alkyl; or (b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R10 is H and R11 is OSOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R and R' are each independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups, and optionally in relation to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring; wherein R16, R17, R19, R20, R21 and R22 are as defined for R6, R7, R9, R10, R11 and R2 respectively; wherein Z is CH or N; wherein T and T' are independently selected from a single bond or a C1-9 alkylene, which chain may be interrupted by one or more heteroatoms e.g. O, S, N(H), NMe, provided that the number of atoms in the shortest chain of atoms between X and X' is 3 to 12 atoms; and X and X' are independently selected from O, S and N(H). linker to a cell binding agent. The present invention is suitable for use in providing a PBD conjugate to a preferred site in a subject. In particular, the present invention provides a conjugate of formula (A), use of a conjugate of formula (A) the manufacture of a medicament, a pharmaceutical composition comprising a conjugate of formula (A), a compound of formula (B), a compound of formula (C), a compound of formula (D), a compound of formula (E) and/or a method of synthesis of a compound. In preferred embodiments, use of a conjugate of formula (A) the manufacture of a medicament for treating cancer and/or a proliferative disease. Conjugate compounds of formula (A): wherein: R2 is, where R36a and R36b 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 R36a and R36b 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', NO2, Me3Sn and halo; R7 is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halo; Y is selected from formulae A1, A2, A3, A4, A5 and A6: L is a linker connected to a cell binding agent; CBA is the cell binding agent; n is an integer selected in the range of 0 to 48; RA4 is a C1-6 alkylene group; either (a) R10 is H, and R11 is OH, ORA, where RA is C1-4 alkyl; or (b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R10 is H and R11 is OSOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R and R' are each independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups, and optionally in relation to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring; wherein R16, R17, R19, R20, R21 and R22 are as defined for R6, R7, R9, R10, R11 and R2 respectively; wherein Z is CH or N; wherein T and T' are independently selected from a single bond or a C1-9 alkylene, which chain may be interrupted by one or more heteroatoms e.g. O, S, N(H), NMe, provided that the number of atoms in the shortest chain of atoms between X and X' is 3 to 12 atoms; and X and X' are independently selected from O, S and N(H).

Description

PYRROLOBENZODIAZEPINES AND CONJUGATES THEREOF The present invention, and the invention of NZ , from which the present application was divided, relate to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepines having a linker group connected to a cell binding agent.
Background to the invention Pyrrolobenzodiazepines Some obenzodiazepines (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, 795 (1965); uber, et al., J. Am. Chem. Soc., 87, 793 ). 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); Antonow, D. and Thurston, D.E., Chem. Rev. 2011 111 (4), 2815-2864). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), ramycin (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. otics, 29, 2492- 2503 ; Langley and Thurston, J. Org. Chem., 52, 91-97 ), sibanomicin (DC- 102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), mycin (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: N 11 8 H A B 11 a 1 7 N C O 3 They differ in the , 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 (NHCH (OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. Aii oi the known naturai products have an (S)—contiguration at the ohirai Ct ta 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—dimensionai shape for isoheiioity with the minor groove of B~torm DNA, ieadihg to a snug fit at the hinding site (Kohn, in Antibiotics iii. ermVeriag, New York, pp. 341 (1975); Huriey and NeedhammVanDevanter, Ace.
Chem. Res, 19, 230—237 (i986)). Their y to form an adduot in the minor groove, enahies them to interfere with DNA processing, hence their use as antitumour .
A particuiariy advantageous pyrroiohenzodiezepihe compound is described by Gregson at at. (Chem. Common. 19%, 797—798) as compound 1, and by Gregson etai. (J. Med! Chem. 2631, 44, 1161—1174) as compound 4a. This compound, aiso known as fifi, is shown heiow: SJG-13$ cher dimerio PBD compounds, such as those hearing C2 aryi substituents in WC) 2005i085251, have been disoiosed, an exarnpie heing: These compounds have been shown to he highiy usefui cytotoxic agents.
Antibody—drug ates Antibody y has been estabiished tor the targeted treatment of ts with cancer, immunoiogioai and angiogenie disorders (Carter, P. (2006) Nature Reviews immuhoiogy 8:343—357). The use of antibody~drug conjugates (ADC), Le. immunooonjugates, tor the tocai detivery of cytotoxic or cytostatic agents, i.e. drugs to kiti or inhibit tumor ceiis in the treatment of cancer, targets deiivery ot the drug moiety to tumors, and intraoeiiuiar aocumuiation therein, whereas systemic administration of these uneonjugated drug agents may resuit in unacceptahie ieveis of toxicity to normai oeiis as wait as the tumor oeiis sought to be eiimineted (Xie et of (230%) Expert. Opin, Bioi. Titer. 6(3):281—29‘i; Kovtun eta! (2006) Cancer Res. 66(6):3214—3121; Law et at (2008) Cancer Res. 86(4):2328~2337; Wu et ai (2005) Nature Biotech. 23(9):1137—1145; Lambert J. (2005) Current (Join. in Pharmacoi. :543—549; Hamann P. (2005) Expert Coin. Titer. Patents 15(9):1087~1193; Payne, (3. (2003) Cancer Ceti 3207—212; Trait et at (2003) Cancer immunoi. immunother. 52:328m337; Syrigos and Eoenetos (1999) Anticancer Research 19:805—614). t efficacy with minimat toxicity is sought thereby. Efforts to design and refine ADC have focused on the seiecttvity of onat antibodies (mAbs) as weti as drug mechanism of action, drug—iinking, druglantibody ratio (ioading), and drug—reieasing properties (dunutuia, et at, 2308b Nature Biotech.. 26(8):925»932; Dornan etai (2009) Blood 114(13):2721*2729; US 7521541; US 7723485; WO2009/052249; htctionagh (2006) Protein Eng. Design 8: Set. 19(7): 299—307; Doronina et at (2008) Biocont. Chem. —124; Erickson etai (2006) Cancer Res. :1~8; Sanderson et a! (2005) Ciin. Cancer Res. 11:843—852; Jeffrey er a! (2065) J. Med. Chem. 4s‘1358; t-iambiett eta! (2004) Ciin. Cancer Res. 16:?063* '7070). Drug rnoieties rnay impart their cytotoxic and cytostatic effects by mechanisms inciuding tubuiin binding, DNA binding, or topoisomerase tion. Some cytotoxic drugs tend to be inactive or iess active when ated to iarge dies or protein receptor tigands.
PBDS in ADCS Dimeric FEDS have been sed as the drugs in drug conjugates. For examote, in WO 2011/130598, dimer PBD compounds having tinker groups for connection to a ceti binding agent, such as an antibody, are disctosed where the tinker group is attached to one ot the avaitabte N10 positions, and are generaity cieaved by action of an enzyme on the tinker group.
By contrast, in WC 2011/13)st and W0 3061b, dirner PBD compounds having tinker groups for connection to a ceii binding agent, such as an antibody, are disoiosed where the tinker group is attached via an aromatic group at one of the C2 bostions, and are generaiiy d by action of an enzyme on the tinker group. Such antibody drug conjugates are aisc described in Fiygare, d., etai, Chem. Bibi. Drug Des. 8t: 113—121 (2013) which atso describes other types of antibody drug conjugates.
A further approach is described in WC 2007l085930, wherein tomamycin—tike dimers have a tinker group for connection to a ceit binding agent, such as an antibody, where the iinker group is attached to the tether between the tornarnycin unite, and are generaiiy cieayed by action of an enzyme on the tinker group.
The present inventors have ped a nevei approach to forming PBD conjugates with oeii binding agento, and in partiouiar PBD antibody conjugates“ Summary of the invention in a genera! aspect the present invention provides a conjugate comprising a FED dimer compound with a tinker for connecting to a ceii binding agent, n the iinker has a triezoie, oiperazine, prepargyiene or oxime group attached to a ohenyiene or pyriydyiene in the bridge Sinking the two FEED monomers. The ceii binding agent is preterebiy an antibody. in a first aspect, the present invention provides novei conjugate compoundo oi formuia (A): e2 is R” Rm and Rm are independentiy seieoted from H, , where F, (314; ted aikyi, (32-3 aikenyi, whieh aikyi and aikenyi groups are optionaiiy tuted by a group seieoted from GM, aikyi amide and {31-4. aikyi ester; or, when one of R353 and R35” is H, the other is eeieoted from nitrite and 3 (31.4 aikyi ester; n5 and n9 are independentiy Seemed from H, n, on, en, en, en hit-i2, NHR, nee: N02“ iViegSn and hate; R7 is independentiy eeiected from H; R, OHS OR, 8H5 SR, Nita, NHR, NRR’a N02, MegSn and hate; Y is Seiected from fonniiiae A1, A2, A3, A4, A5 and A8: G40 o i in Wvii Lfio \OAL\ HN ( \\ \ N m (At) (A?) i>\—i We HM MW0 (A5) (A6) L is a iinker nennected to a ceii g agent; (EBA is the ceii binding agent; n is an integer seiecied in the range of 0 to 48; R“ is 3 Gina aikyiene group: either (a) R10 is H, and R“ is OH, inn“, where RA is CM aikyi; or (b) R” and R“ form a nitrogenmcarben deubie bond between the nitrogen and carbon atoms to which they are bound; or (e) R10 is H and FR“ ES OSQZM, Where z is 2 or 3 and M is a menoveieni pharmaceutieaiiy acceptabie cation; R and R’ are each independentiy seieeted from optionaiiy substituted (31-4.2 aikyt, €33.20 heterocyciyi and (35.20 aryi groups, and eptionaiiy in reiation to the group NRR’, R and R’ together with the nitrogen atom to which they are ed form an aity suhetituted 4—“ 5w, 8“ or 7—memhered heterocyciic ring; wherein F215, R17, R19, R20, R21 and R22 are as defined for R5, R7, R9, R10; R11 and R2 respectiveiy; wherein Z is CH or N; wherein T and "i“ are independehtiy seiected from a stngie bond or a {31-9 aikyiene, which chain may be interrupted by one or more heteroatorne egg (3, 8, NH), Ni‘vtes ed that the number of atoms in the shortest chain of atoms between X and X’ is 3 to 12 atoms; X and X’ are indeeendentiy setected from O, S and NW), Thus tormuie A is eeiected from the feiiowihg formutee Awt, A—ii, A—Eiia A—iV, A—‘v’ and A—Vi depending on Y: WO 59981 WO 59981 (a): Where ati the groups are as defined in the first aspect of the invention; and Y!’ is seiected from a group of formuiae 81, 82, E33, B4, 85 and {36: (31) (32) (B3) (B4) (85) (86) Where G is a reactive group for connecting to a 083E binding agent.
A third aspect of the t inventian pmvides campounds of formuia (C) which may be used in the praparation 0f the compounds and conjugate newspaunds of the invention: where “r’C is Seieeted from a group of forrnuiae Ct; (32, C3; C4 and C5: H2N+\_ 0 O (‘35) (05) either (a) R30 is H, and R31 is OH, 033’", where RA is (31.4 eikyi; or (h) R30 and R31 term a nitrogenmoarben deuhie bond between the nitrogen and carbon atoms to which they are bound; or (o) R30 is H and R3" is OSQZM, where z. is 2 or 3 and M is e ient phermeceutioeiiy ebie cation; or (d) R30 is a nitrogen protecting group and R31 is OProtO, Where Photo is a hydroxy protecting group; and R40 and R“ are as defined for R30 and R3" r‘espeotiveiy; and eii the remaining groups; are as, d in the first eepeot of the invention.
A fourth aspect of the present invention provides compounds of formuie (D) whieh may be used in the preparation of the compounds of the second and third aspects of the invention: H2N H || LNN3 (D2) <03) HZN—RA4 \ H0 ([34) (06) and ait the remaining groups are as defined in the third aspect of the invention.
A fifth aspect of the present invention provides compounds of tormute (E) which may he used in the preparation of the compounds of the second, third and fourth aspects of the ion: ,X‘ / a. \T' 7 ML/ R17 R / E YE is seieoted from a group of fermniee E1, E2 and E5: WO 59981 RE‘1 | | Mt (En (Es) (ES) where R3 is seiected from H and Tit/is; RE" is seiected from Br, Ci and i; and aii the remaining groups are as defined in the third aspect of the invention.
A sixth aspect of the present invention provides the use of a compound of the first aspect of the ion in a method of medicai treatment. The fourth aspect aiso provides a pharmaceuticai composition comprising a compound of the first aspect, and a pharmaceuticaiiy acceptabie ent.
A seventh aspect of the present invention provides a compound of the first aspect of the ion or a pharmaceuticai composition of the fourth aspect of the invention for use in a method of treatment of a proiiterative disease. The fifth aspect aiso provides the use of a compound of the first aspect in a method of manufacture of a medicament for the treatment of a proiiferative disease, and a method of treating a mammai having a proiiferative disease, comprising administering an effective amount of a compound of the first aspect or a pharmaceuticai composition of the fourth aspect.
An eight aspect of the present invention provides a method of synthesis of a nd of the first aspect of the present invention, comprising the step of conjugating a drug—tinker of the second aspect with a ceii~hinding agent.
The present invention aiso provides the sis of compounds of the second aspect of the invention from compounds of the third, foruth or fifth aspect of the invention by reacting them with suitabie reagents.
Deteiied flescription of the invention The present invention provides a conjugate comprising e PBD dimer connected through the dimer bridging portion via a specified tinker to a ceii binding agent.
The present invention is suitebie for use in providing a PBD conjugate to a preferred site in a subject.
Nitrogen protecting groups Nitrogen protecting groups are weii known in the art. Preferred nitrogen protecting groups for use in the present invention are cerhemete protecting groups that have the generei tormuie: e39"— o n R’” is en optionaiiy substituted eikyi (eg. C149 eikyi), aryi (eg. €35.20 aryi) or heteroaryi (eg. (33-20 heterocyctyi) group.
A iarge number of possihie carhamate nitrogen protecting groups are iisted on pages 7023‘: to 1772 of Greene’s Protective Groups in Organic sis, 4"“ n, John Wiiey & Sons, inc, 2007 (iSBN 978-0—47’i—69754—t), which is incorporated herein by nce.
Particuiariy preferred protecting groups inciude Aiioc, Troo, Teoc, EGG, TCBGC, Fmoo, ’i— Adoc end Z—Adoc.
Hydroxyi protecting groups yi protecting groups are weii known in the art. A ierge number ot suitebie groups are described on pages 24 to 298 of of Greene’s Protective Groups in Qrganic Synthesis, 4‘" Edition, John Witey 8:, Sons, inc, 2007’ (iSBN 471—69?54—t), which is incorporeted herein by nce.
Ciesses of perticuier interest e siiyt ethers, rnethyi ethers, eiityi , benzyi ethers, esters, henzoetes, carbonates, and suifonetes. Perticuieriy preferred hydroxyi protecting groups inciude THP, Preferences The foiiowing preferences may eooty to eii aspects of the invention as described above, or may reiete to e singte aspect. The preferences may be combined together in any combination in some embodiments, R36?“ and R36” are both it. in other embodiments, R“ and R35“ are both methyi. in r embodiments, one of R“ and R“ is it, and the other is ed from CM. saturated aikyi, (32.3 i, which aikyi and aikenyi groups are optionaiiy substituted. in some of these further embodiments, the group which is not H may be seieoted from methyi and ethyi.
R2}? The above preferences for R2 eppty equeiiy to R”. in one embodiment, R5 is independentiy eeieeted from H, R, 0H, OR, SH, SR“ Niig, NHR, NRR’, N02, MegSnm end Haio. in one embodiment, R5 is independentiy seieoted from H, (Di-i, QR, SH, Mir-i2, N02 and Heio. in one embodiment, R5 is independentiy seieoted from H and Hate“ in one embodiment, R5 is independeniiy H. in one embodiment, R6 and R7 together form a group ~O—(CH2)p—Q-, where p is ’i or 2.
These embodiments aiso apoiy to R16.
R7 is independentiy seieo‘ted from H, R, Cit-i, QR“ Sit, SR, NHQ, NHR, NRR’, N02“ M938“ and hate. in one ment, R7 is independentiy OR" in one embodiment, R7 is independentiy OR”, where R” is independentiy optionaiiy substituted 01,5 eiityi. in one embodiment, R” is indeeendentiy optionsiiy substituted saturated C3143 eikyi. in one embodiment, R” is independentiy ootioneiiy substituted C244. eikenyi. in one embodiment, R7A is independentiy Me, in one embodiment, R7" is inoiebenoientigvi onznh. in one embodiment, R” is independentiy eiiyi.
These embodiments eiso sooty to R17, in one embodiment, R9 is independentiy seieoted from H, R, 0H, OR, SH, SR, Niig, NHR, NRR’, N02, iv’ie38nm and Hate, in one embodiment, R9 is independentiy it. in one ment, R9 is independentiy R or OR.
These embodiments eiso sooty to R”.
MTG-CT? in some embodiments, R10 is it, and R11 is OH, QR“, Where RA is (31-..: aikyi. in some of these embodiments, R11 is OH, in others of these ments, R“ is QRA, where RA is CM eikyi. in some of these embodiments, RA is methyi. in some embodiments, R10 and R“ form at nitrogen~oarbon doobie bond between the nitrogen and carbon atoms to which they are bound, in some ments, R10 is it and R“ is 0302M, Where z is 2 or 3 and M is e monoveient phermeoeutioeiiy aeoeotebie cation. in some of these embodiments, M is e monoveient eoeutioaiiy aooeotebie cation, and may be Nah Furthermore, in some embodiments z is 3.
The above ences eppiy eqoeiiy to R20 and R21. in some embodiments, R30 is it, and R31 is OH, ORA, where RA is {31.4, aikyi, in some oi these embodiments, R31 is Git. in others of these embodiments, R31 is ORA, where RA is (31.4 sikyi. in some of these ments, RA is methyi. 2014/025564 in some embodiments, R30 and R31 form a nitrogen—carbon doubie bond between the nitrogen and carbon atoms to which they are bound. in some embodiments, R30 is H and R31 is OSGZM, where z is 2 or 3 and M is a monoveieht pharn‘ieeeutieaiiy aooeptabie cation. in some of these ments, M is a monoyaient phermeeeutieeiiy aeoeptebie cation, and may be i‘xiai~ Furthermore, in some embodiments z is 3. in some embodiments, R30 is a nitro eh roteotin r rou3 . and R31 is , where ProtO is e hydroxy protecting group. in some of these embodiments, the nitrogen ting group may be seieoted trom Aiioo, Troo, Teoe, BOG, TCBQC, Fmoe, ’ieAdoc end 2*Adoo, and more preterabiy be Boo. in some of these embodiments, the nitrogen protecting group may be THF’.
For compounds of formuie D, it may be preferred that R30 and R31 form a nitrogen—carbon dotibie bond between the nitrogen and carbon atoms to which they are bound.
For compounds of tormnie E, it may be preferred that R30 is e nitrogen protecting group and R31 is OProtQ, where i-3rotO is a hydroxy protecting group.
For compounds of tormoia C, where YO is of a ()2, C3 or (34, it may be preferred that R30 and R31 form a nitrogennoarbon doubie bond between the nitrogen and carbon atoms to which they are bound For compounds of tormoie C, where YC is of tormuie Ct or (35, it may be preferred that R30 is a en protecting group and R31 is QProtO, where Protc' is a hydroxy protecting group, The above preferences appiy eoneiiy to R40 and R41, T and T’ Each of T and T’ is independentiy seieeted from a singie bond or a (31-9 eikyiehe group, which chain may be interrupted by one or more heteroatoms, egg. 0, S, N(H) and/or NMe, provided that the number of atoms in the shortest chain of atoms between X and X’ is 3 to 12 atoms. in one embodiment, each eikyiene group of "i" and T’ is optioneiiy interrupted by one or more heteroetoms seieoted from O, S, and Ni‘v'ie, in one embodiment, each of T and T" is independentiy seteoted from a singie bond and e (11,9 eikyiene group. in one embodiment, T is seieoted from a singie bond, {31, Cg, C3 and a C4 eikyiene group and T’ is ted from a singie bond, Cr, (32, Cg, and a C4 ne group. in one embodiment, T is seieoted from a singie bond, Ci, and e C; aiityiene group and T" is seieeted from a singie bond, (31, and a C2 eikyiene group. in one embodiment, T is seieoteo from a singie bond and 3 C1 eikyiene group and T’ is seieoted from e singie bond and a C1 eikyiene group. in one embodiment, T is e singie bond and T" is e singie bond. in one embodiment, T is a C1 ne group and T’ is a C31 he group. in some embodiments, T and T’ are the some, The eikyiene groups iisted above may be optionaiiy interrupted by one or more heteroetoms.
The eikyiene groups iisted above may be unsubstituted iinear aiipnatio eikyiene . in one embodiment, X is seieoted from O, S, or Nr’i-i), Preferapiy, X is O.
Dimers in some embodiments, the groups R22, R15, R”, R”, R20 and R21 are the some as the groups R2, R5, R9, R7, R10 and R“ respeotiveiy. in these embodiments, the PBD monomer units have the same tuents.
Partiouieriy preferred compounds oi the first aspect of the present invention may be of formuie ie: ~ - \ R I 2/ O “- i2 ia L 73 where R”, R“, R”, R21 and Y are as defined above; i1 and t2 ere arr independentiy seieoieci from 0, i and 2 F27“ and Rm are independentiy eeiected from methyi and phenyi, Pariieuiariy preferred compounds of the second aspect of the present invention may be of fermuia iia: ‘\ R11 0 x O H in: i2 e QRWa R730 where R“, R“, R20, R21 and YL are as defined above; t1 and L; are an independentiy seiected from O, ‘i and 2 R73 and Rm ere indepeedeniiy ed from methyi and phenyi.
Pariieeiariy preferred ride of the third aspect of the present invention may be of formuia iiia: Elia R10, R“, R20, R21 and YC are as defined above; ti; and t2 are an independentiy seieoted from O, t and 2 R” and R” are independentiy ed from methyi and onenyi. r: (Y, y?) in some embodiments, n (in Y or Y“) is an integer between 0 and 24, in some embodiments, n (in Y or Y“) is an integer between 0 and 12. in some ments, n (in Y or Y“) is an integer between G and 8. in some embodiments, n (in Y or Y“) is an integer between t) and 6. in some embodiments, n (in Y or Y“) is O. in some embodiments, n (in Y or Y“) is t. in some embodiments, n (in Y or Y“) is 2. in some ments, n (in Y or Y“) is 3. in some embodiments, n (in Y or Y“) is 4. in some embodiments, n (in Y or Y“) is 5. in some embodiments, n (in Y or Y“) is 6. in some embodiments, n (in Y or Y“) is 7. in some embodiments, n (in Y or Y“) is 8. in some embodiments, n (in Y or Y“) is 9. in some embodiments, n (in Y or Y“) is it). in some embodiments, n (in Y or Y“) is it. in some embodiments, n (in Y or Y“) is 12. in some embodiments, n (in Y or Y“) is 13. in some embodiments, n (in Y or Y“) is 14. in some embodiments, n (in Y or Y“) is ’15. in some embodiments when Y is At, or Y“ is 81, n may be seieoted from 3 and b. in some embodiments when Y is A2, or Y“ is i352, n may be seieoted from 4 and 5. in some embodiments when Y is A3, or Y“ is 83, n may be 4. in some embodiments when Y is A4, or Y!’ is 84, n may be 4. in some embodiments when Y is A5, or YL is E35, n may be 11. in some embodiments when Y is A6. or Y" is 86. n may be 2: L and G i. is a tinker connected to the oeii binding agent in the conjugate conmpoundi G is a reactive group for connecting the PBD dirner to the eeii binding agent to form the conjugate compound.
Preferabiyq the ’reactive group contains an eiectrophiiic tunctionai group for reaction with a hucieophiiic ‘iunetionai group on the ceii binding agent. Nucieophiiic groups on dies inciude, but are not iirnited to: (i) N~terrninai amine groups, (it) side chain amine groups, eg. iysine, (iii) side chain thioi groups, eg. cysteine, and (iv) sugar hydroxyi or amino groups where the antibody is yiatedu Amine, thioi, and hydroxyi groups are hucieophiiic and capahie of reacting to term nt bonds with eieetroohiiic groups on tinker moieties and tinker reagents inciuding: (i) maieimide groups (ii) activated disuifides, (iii) active esters such as NHS (N—hydroxysuccinimide) esters, HOB”: (N~hydrexybenzotriazoie) , haictormates, and acid haiides; (iv) aikyi and benzyi heiides such as heioacetamides; and (v) aidehydes, ketones, yi. and, some of which are exempiified as foiiovvs: N/\!x /N| s\S/\/lkm/\f S / \ \ o o Br\/\‘ \ “1/wa Br\/lkE/\f Certain antibodies have reducibie interchain disuitides, i.e. cysteine bridges. Antibodies may he made reactive for ation with tinker reagents by treatment with a reducing agent such as DTT (dithiethreitci). Each cysteine bridge wiii thus form; theoreticaiiy, two reactive thioi nucieophiies. Additionai nucieophiiic groups can be introduced into antibodies through the reaction ot iysines with 2~iminothiotane (Traut’e reagent) resuiting in conversion of an amine into a thioi. ve thioi groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine es (eg, preparing mutant antibodies oomprising one or more non—native cysteine amino acid residues). US 7521541 s engineering antibodies by introduction of reactive cysteine amino acids. in some embodiments, a Linker has a reactive nuoteophiiio group which is reactive with an eiectrophitio group present on an antibody. Usefui eiectrophiiic groups on an antibodyi inoiude, but are not iimited to, de and ketone oarbonyi . The heteroatom of a nuoieoohiiic group of a Linker can react with an eieotrophitio group on an antibody and form a nt bond to an antibody unit. Usetui nucieophiiio groups on a Linker inoiude, but are not iimited to, hydrazide, oxime, amino, hydroxyi, hydrazine, thiosemioarbazone, hydrazine oarboxyiate, and aryihydrazide. The eieotrophiiio group on an antibody provides a convenient site for attachment to a Linker. in one embodiment, the group L is: m x where the asterisk indicates the point of attachment to the rest of group Y, the wavy iine indicates the point of attachment to the oeii g agent, and in is an integer seiected from the range 0 to 8. in one embodiment, m is seieoted from 2, 3, 4 and 5. in one embodiment, the connection between the ceii binding agent and L is through a thioi e of the oeti binding agent and a mateimide group of L. in one embodiment, the connection between the oeti binding agent and t. is: xi 0 where the asterisk indicates the point of attachment to the remaining n of the i. group or the remaining portion of the ‘r' group and the wavy iine indicates the point of attachment to the remaining portion of the ceii binding agent. in this embodiment, the 8 atom is typicaiiy d from the ceii binding agent. in each of the embodiments above, an aiternative ttinctidnaiity‘ may be need in piece of the maieimide—derived group ehewn beiow: where the wavy iine indicates the point of ment to the eeii binding agent as before, and the aeterisk indicates the bend to the remaining pertien of the t. group er the remaining portion of the Y greup, in one embodiment, the maieimide—derived group is repiaeed with the group: i it ’* “Mt hi *4 Q where the wavy tine tes point of attachment to the ceii binding agents and the asterisk indicates the bond to the remaining portion of the L group or the remaining n of the Y group. in one ernhodin'tents the maieimide—derived group is repiaced with a group, which aiiy together with the eeii binding agent, is eeieeted from: —C(=Q)NH—, ~C(=Q)O~, ~NHC(=G)~5 —QC(=Q)—, —QC(=Q)O—, “NHCFOKD: —QC(=O)NH~5 *NHCFOWH, —NHC(=Q)NH, ~C(=Q)NHC(=Q}, —S~Sn —CH2C3(=O)— —C(:G>CH2—, PCT/USZOl4/025564 =N—Ni—i—, and Nit—N: in one embodiment, the mateimidenderived group is repiaeed with a group, which optionaiiy together with the oeii binding agent, is Seieoted from: t i ' N No \H/ N“ H N’J N" where the wavy tine indicates either the point of attachment to the oeii binding agent or the hood to the remaining portion of the i. group or the remaining portion of the Y group, and the asterisk indicates the other of the point of attachment to the eeii g agent or the bond to the remaining portion of the t. group or the remaining portion of the Y group. 0ther groups that can be used as i. for ting the remaining portion of the Y group to the eeii binding agent are described in WC) EGGS/082023.
Thus, in embodiments of the present invention, L is of a: “LA"(CH2)m" (Li) Where in is from G to 6; and LA is, eeieoted from: pet; g ........................................................................................................................................................................................................................................... where Ar is CH; erytene group, egt phenytene. in same embodiments Where L is Lt, m may be 2, 3 or 5. in some embodiments where L is Lt; LA may he LA“. in embodiments of the present invention, t. is of formute: iCHflmnG (L2) Where in is from O to 6; and LA is setested from the groups above.
Without wishing to be bound by theory, such a group may be cteeved from the dy such that the cathemete group yieids e terminat amine. in Seine embodiments where L is L2, LA may be LA3'2. in some embodiments where t. is L2, m may be t. in embodimente of the present invention, L is of tormuie: —L’im(eii2)qme—C(=e)mi\iwaiter (L3) Where q is from ’i to 3, and p is from ’i to 3; and LA is seiected from the groups above.
Without wishing to be bound by theory, such a group may he d from the antibody such that the eerbemete group yieide the group: H2N~(CH2)Q~ (L3’). in some embodiments where t. is L35 o may be t, and p may be 2. in eeme embodiments where t. is L3, t.A may he eeieeted from L”, 1 and LAM. in embodiments of the t invention, i. is of ion‘nuie: , H giltLA’Wx1\leNm 0 \ 0‘}; (L4) Where in is from G to 6; X1 and X2 are amino acid groups, eeieoteot from neturei amino acids, which may be modified; LA is eeiected from the groups above.
The naturai amine acids may be eeieeted such that the dipeptide group is oethpesin iahiie. in one embodiment, the group ~X1—X2— is eeieeted from: ~Phe~Lys—, ~Vei~Aiag Mei—Lye, —Aie—Lye—, “VainCitn, —Phe—Cit—, —iie—Cit—, ~Phe~Arg—, ~"ire—Cit“ where Cit is oitruiiine.
Preferahiyq the group —X1nX2w is eeieoted from: fine—Lye: *VahAiag —Vei~Lys—, ~Aia~Lys~, —Vai—Cit—.
Most preferabiy, the group mxi—XT is —Phe~Lys— or ~Vai~Aia—. in some embodiments where t. is L4, m may be in cher dipeptide combinations ma3,i be used, inciuding those described by Dubowchik et at, Bioconjugai‘e Chemistr , 2002, 138554369, which is incorporated herein by reference, in one ment, the amino acid side chain is derivatised, Where appropriate. For exampie, an amino group or carboxy group of an amino acid side chain may be derivatised. in one embodiment, an amino group hit-i2 of a side chain amino acid, such as iysine, is a derivatised form seiected from the group consisting of NHR and NRR’. in one embodiment, a carboxy group CODE-t of a side chain amino acid, such as aspartic acid, is a derivatised form seiected from the group ting of COQR, CQNHZ, CQNHR and CQNRRZ in one embodiment, the amino acid side chain is chemicaity protected, where appropriate.
The side chain protecting group may he a group as discussed heiow in reiation to the group RL. The present inventors have ished that protected amino acid sequences are cieavabie by enzymes, For exampie, it has been estahiished that a ide sequence comprising a Boc side protected Lys residue is hie by cathepsih.
Protecting groups for the side chains of amino acids are weii known in the art and are described in the Novabiochem Cataiog, Additionai protecting group gies are set out in Protective Groups in Qrganic Synthesis, Greene and Writs.
Possibie side chain protecting groups are shown peiow for those amino acids having reactive side chain ‘iuncticnaiity: Arg: Z, Mtr, Tos; Ash: Trt, Xan; Asp: Bzi, t—Bu; Cys: Acm, Bzi, Bzi—Oh’ie, Bzihie, Trt; (3hr: Bzi, t-Bu; Gin: Trt, Xan; His: BOG, an, Toss Trt; Lys: 800, Z~CL Fmoe, Z, Aiiee; Ser: 82!, TBDMS, TEE-DPS; Thr: 82:; Trp: BOG; Tyr: 823, Z, Z—Br.
Thus, in embodiments of the present invention, G is of a: GA'(CH2)W (‘31) Where m is from G to 6; and GA ES seiected from: HaEHfi—é/ Where Hai = E, Br, Ci (N02)- where the NO; group is optionai WO 59981 02N”C"EN where the N02 group is ootionei Where the N132 group is optiehai where Ar ents a C54; eryiene group, eg. phenyiene. in some embodiments where G is (315 m may be 29 3 or 5: in some embodiments where G is Gt, GA may he 6“”. in embodiments of the present invention, G is of terinuia: GA—(CHg)m—G~ (:32) Where m is from 0 to e; and (3" is Seieeteri from the greuee above. in some embodiments where G is (325 GA may be Gm”? in eeme embodiments where G is 62, m may be ’i. in embodiments of the present invention, G ES of iermeie: GA~(CHg)q~O—C(=Q)~NH-(CH2)p- ((33) Where {:3 ie from t to 3, and p is from 1 to 3; and GA is eeiecteo from the groups above. in some embodiments where G is (33, q may be t, and p may be 2. in some embediments where G is GB, GA may he seieeteci from GM and G”. in embodiments of the present invention. G is of e: .i u oil/W><.\ XZ’N‘ O as“ (G4) Where m is from G to 6; X1 and X2 are as defined above for L4; GA is seieoted from the groups above.
R and F?’ in one embodiment, R is independentiy seieoted from optionaiiy Substituted €31-12 eikyi, {33-20 heterooyoiyi and {35-20 aryi groups. These groups are each d in the substituents section beiow. in one embodiment, R is independehtiy‘ optioneiiy substituted (31-12 . in one embodiment, R is ndenti}i optioneiiy substituted (33.20 heteroeyeiyi. in one embodiment R is independentiy optionaiiy substituted (35-20 eryi. in one embodiment R is independentiy optionaiiy substituted (31-12 etkyt.
The preferences for R eppiy eiso to R’. in some embodiments of the invention there is provided a compound having a substituent group *NRR’. in one embodiment. R and R" together with the nitrogen atom to which they are attached form an optionaiiy tuted 4—, 5—, 6— or 'i-membered heterooyoiio ring. The ring may contain a further heteroetom, for exampie N, C) or S. in one embodiment the oyoiio ring is itsei'i substituted with a group R. Where 3 further N heteroetom is present, the substituent may be on the N heteroetom.
RAKE in one embodiment, RM is a (32.4 aikyiene group. in one embodiment, R” is at Cz eikyiene group. in one embodiment, RA4 is a C33 ne group. in one embodiment R“ is an unsubstituted C14,: eikyiene group. in one embodiment, R“ is e iinear €31.53 aikyiene group. in one embodiment, RM is ed from the group consisting of “CHchz', “CHECHZCHZ' and —CHQCH2CH20H2“ Celt Binding Agent A ceii binding agent may be of any itind, and inctude peptides and non—peptides, These can inciude antibodies or a fragment of an antibody that contains at ieast one binding site, iymbhokines, hormones, growth factors, nutrientmtransport moieouies, or any other oeii binding moiecuie or substance.
The term “antibody” herein is used in the broadest sense and sbeoitioaiiy covers n‘ionocionai antibodies, poiycionai antibodies, dimers, muitimers, muitispecific antibodies tag, bisoeoific antibodies), and antibody fragments, so tong as they exhibit the desired bioiogicai activity (Miiier et ai (2003) Jour. ofimmunoiogy 17014854~486t ). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is e of recognizing and binding to a specific antigen. ay, (3., Travers, R, Waiport, hit, Shiomchik (2001) immune gy, 5th Ed, Gariand Pubiishing, New York). A target antigen generaiiy has numerous binding sites, aiso caiied ebitcpes, recognized by CDRs on muitipie antibodies. Each antibody that icaiiy binds to a ditterent e has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody inciudes a fuii—iengtn giobuiin moieouie or an iogicaiiy active portion of a tuiimiength immunogiobuiin moieouie, i.e., a moiecuie that contains an n binding site that in'tmunospecificaiiy binds an antigen of a target of st or part thereof, such targets inciuding but not d to, cancer ceii or ceiis that produce autoimmune antibodies associated with an autoimmune disease. The immunogiobuiin can be of any tyoe (eg. igG, igE, , igD, and igA), ciass (eg. ith, igGE, igC—SS, ingi, igAt and igAZ) or subciass of imrnunogiobuiin moiecuie. The immunogiobuiins can be derived from any species, inciuding human, murine, or rabbit origin.
"Antibody fragments" se a oortion of a fuii iength antibody, generaiiy the antigen g or variabie region thereot. Exampies of antibody fragments inciude Fab, Fab’, ; and Fv fragments; diabodies; iinear antibodies; fragments produced by a Fab expression iibrary, anti—idiotypio id) antibodies, CDR (compiementary determining region), and epitopembinding fragments of any of the above which immunospeciticaiiy bind to cancer ceii antigens, yirai antigens or microbiai antigens, singie~chain antibody moiecuies; and muitisbeoitio antibodies termed from antibody fragments.
The term “monocionai antibody” as used herein refers to an antibody obtained from a popuiaticn of substantiaiiy homogeneous antibodies, is. the duai antibodies comprising the popuiation are identioai except for possibie naturaiiy occurring mutations that may be present in minor amounts. cionai antibodies are highty specific, being directed against a singie antigenic site. Furthermore; in contrast to poiycionai antibody preparations which inciude ent antibodies directed against different determinants (epitopes), each monocionai antibody is directed against a singte determinant on the antigen. in addition to their specificity, the monocionai antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies, The er “rnonocionai” indicates the ter of the antibody as being obtained from a substantiaiiy neous tion of antibodies, and is not to be construed as requiring production of the antibody by any uiar method. For exampie, the monocionat antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohier et ai (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 481656?) The monocionai antibodies may aiso be isoiated from phage antibody iibraries using the techniques described in Ciackson et at (1991) Nature, 352624—628; iviarks et at (1991) .i.
Moi. Biot, 2221581n597.
The monocionai antibodies herein specificaiiy inciude “chimeric” antibodies in which a portion of the heavy and/or tight chain is identicai with or gous to corresponding sequences in antibodies derived from a particuiar species or beionging to a barticuiar antibody ciass or subciass, white the remainder of the chaini’s) is identicai with or homoiogous to corresponding sequences in antibodies derived from another species or beionging to another antibody ciass or subciass, as weii as fragments of such antibodies, so tong as they exhibit the desired bioiogicai activity (US 7; and Morrison et a! (1984) Proc. Natl. Acad. Sci. USA, 1—6855). ic antibodies inciude “primatized” antibodies comprising variabie domain antigen—binding sequences derived from a non- human primate (eg. Oid Worid iv‘ionkey or Ape) and human constant region sequences.
An “intact antibody” herein is one sing a Vt. and Vii domains, as weii as a tight chain constant domain (CL) and heavy chain constant domains. CH1, CH2 and CH3. The nt domains may be native sequence constant domains (eg. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more “effector functions” which refer to those bioiogicai ties attributabie to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Exampies of antibody effector functions e Ctd binding; compiement ent cytotoxicity; Fe receptor binding; antibody~dependent ceii~mediated cytotoxicity (ADCC); phagocytosis; and down reguiation of ceii surface receptors such as B ceii receptor and BCR.
Bepending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “ciasses.” There are five major ciasses of intact antibodies: igA, igD, igE, igG, and igivi, and severai of these may be further d into “subciasses” (isotypes), erg", igG’i, igGZ, igGS, ingt, igA, and igAZ. The heavy—chain constant domains that correspond to the different ciasses of antibodies are caiied o, a, a, y, and p, respectiveiy. The subunit structures and three—dimensionai urations of different s of giobuiins are weii known. es of ceii binding agents inciude these agents described for use in WC) 2007l085930, which is incorporated herein.
The ceii binding agent may be, or comprise, a poiypeptide. The poiypeptide may be a cystic poiypeptide. The ceii binding agent may be antibody. Thus, in one embodiment, the present invention provides an antibody—drug conjugate (ADC).
Drug feeding The drug ioading is the average number of PBD drugs per antibody. Brug ioading may range from t to 8 drugs (i3) per antibody (Ab), ie where t, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covaientiy attached to the antibody" Compositions of ADC e coiiections of antibodies conjugated with a range of drugs, from t to 8. The average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventionai means such as mass oscopy, ELESA assay, eiectrophoresis, and HPLCK The quantitative distribution of ADC in terms of p may aiso be determined. By ELESA, the averaged vaiue of p in a particuiar preparation of ADC; may be determined (Hambiett et ai (2004) Ciint Cancer Res. 19:?963JO7G; Sanderson et at (2005) Ciin. Cancer Res, " 852). However, the distribution of p (drug) vaiues is not discernibie by the antibody—antigen binding and detection iin‘iitation of ELESA. Aiso, ELESA assay for detection of antibody—drug conjugates does not determine where the drug moieties are ed to the antibody, such as the heavy chain or tight chain nts, or the partiouiar amino acid residues. in some instances, separation, cation, and characterization of homogeneous ADC where p is a certain vaiue from ADC with other drug ioadings may be achieved by means such as reverse phase HPLC or eiectrophoresis.
For some antihody~drug conjugates, c may be d by the number of ment sites on the antibody. For exampie, an antibody may have onty one or severai cysteine thioi groups, or may have onty one or severat sutiicientty reactive thioi groups through which a tinker mayi be attached. Higher drug toading, e.g. p >5, may cause aggregation. insotubttity, toxicity, or toss ot cetiuiar cermeabitity of certain antibody-drug conjugates.
Tycicaiiy, fewer than the theoreticat maximum of drug moieties are conjugated to an antibody during a ation reaction, An antibody may contain, tor exambie, many tysine residues that do not react with the drug—tinker intermediate (ELL) or tinker reagent. Onty the most reactive tysine groups may react with an amine—reactive tinker reagent. Aiso, oniy the most reactive cysteine thioi groups may react wtth a thioi—reactive tinker reagent. Generatty, antibodies do not contain many, it any free and reactive ne thioi groups which may be tinked to a drug moiety. Most cysteine thioi residues in the antibodies of the compounds exist as ide bridges and must be reduced with a reducing agent such as dithiothreitoi (DTT) or TCEP, under oarttat or totai reducing conditions. The toadtng (drug/antibody ratio) of an ADC may be centretted in t different manners, inciuding: (i) timiting the motar excess of drug—tinker intermediate (tit—L) or tinker reagent retative to dy, (it) tintiting the conjugation on time or temperature, and (iii) t or iimiting reductive conditions for cysteine thioi modification.
Cysteine amino acids may be engineered at reactive sites in an dy and which do not form intraohain or intermotecutar ide tinkages (Junututa, et at, 2008b Nature Biotech., 26(8izt325—932; Dernan et at (2069) Stand t’téi(‘t3):2721—2729; US 7521541; US 7723485; W02099/052249, Shen et at (2012) Nature Biotech, 30(2)2184wt9‘t; dunutuia et at (2008) dour of tmmun. t‘vtethods 3324162). The engineered cysteine thtots may react with tinker reagents or the inker reagents of the present invention which have thioiwreactive, etectrophiitc groups such as mateimide or atpha-hato amides to form ADC with cysteine engineered dies (ThieiViabs) and the P80 drug moieties. The on ot the drug moiety can thus he designed, controtted, and known. The drug toading can be oontroited since the engineered cysteine thioi groups typicatty react with thioi-reactive tinker reagents or drug—tinker reagents in high ytetci. Engineering an tgG antibody to uce a cysteine amino acid by substitution at a singie site on the heavy or tight chain gives two new cysteines on the symmetricat antibody, A drug ioadtng near 2 can be achieved and near homogeneity of the conjugation product ADC.
Where more than one nucieophiiic or eiectrophiiic group of the antibody reacts with a drug— iiniter intermediate, or tinker reagent toiiowed by drug moiety reagent, then the ing product is a e of ADC compounds with a distribution of drug moieties ed to an antibody, egg. 1. 2, 3, etc. Liquid chromatography methods such as poiymeric reverse phase (PLRP) and hydrophobic interaction (Hit?) may separate compounds in the mixture by drug ioading vaiue. Preparations of ADC with a singie drug ioading vaiue to) may be isoiated, however, these singie ioading vaiue ADCs may stiii be heterogeneous mixtures because the drug moieties may be attached, via the tinker, at different sites on the antibody.
Thus the antibody~drug conjugate compositions of the invention inciude mixtures of antibody—drug ate compounds where the antibody has one or more PBD drug moieties and Where the drug moieties may be attached to the antibody at s amino acid residues. in one embodiment, the average number at dimer obenzodiazepine groups per ceii g agent is in the range ’i to 20. in some embodiments the range is seiected from t to 8,2to8.2to8,2to4,and4t08. in some embodiments, there is one dimer pyrroiobenzodiazepine groups per ceii binding agent.
Peptides in one embodiment, the ceii binding agent is a iinear or cyciic peptide comprising 4-20, preterabiy 6—20, contiguous amino acid residues. in this embodiment, it is preferred that one ceii binding agent is iinked to one monomer or dimer obenzodiazepine compound. in one embodiment the ceii binding agent comprises a peptide that binds integrin ovpg. The peptide may be seiective for cutie over XYS. in one embodiment the ceii binding agent comprises the AEOFi‘viEDV—Cys poiypeptide. The AZOFh’iDV—Cys has the sequence: RGDLQVLAQKVARTC. Aiternativeiy, a variant of the AZQFMDV—Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues is substituted with another amino acid residue. in one embodiment the antibody is a monocionai antibody; chimeric dy; humanized antibody; tuiiy human antibody; or a singie chain dy. One embodiment the antibody is 2014/025564 a fragment of one of these antibodies having bioiogicai activity“ Exampies of such fragments inciude Fabg Fab', F(ab')2 and Fv fragments. in these embodiments, eaoh dy may be iinked to one or severai dimer pyrroiobenzodiazepine groups. The preferred ratios of pyrroiohenzodiazepine to ceii binding agent are given above.
The antibody may be a domain antibody (DAB). in one embodiment, the antibody is a monccionai antibody.
Antibodies for use in the present invention inciude these antibodies described in WC) 2005l082023 which is incorporated herein, Partiouiariy preferred are those antibodies for tumounassociated antigens. Exampies of those antigens known in the art inciude, but are not iimited to, those tumour~associated antigens set out in WC) EDGE/082623. See, for instance, pages 4165.
The conjugates of the invention are designed to target tumour ceiis via their ceii surface antigens. The antigens are usuaiiy normai ceii surface antigens which are either oven expressed or expressed at abnormai times. ideaiiy the target antigen is expressed oniy on profiterative ceiis (preferabiy tumour ceiis), however this is rareiy ed in practise. As a resuits target antigens are usuaiiy ed on the basis of entiai expression between prciiterative and heaith‘yi tissue.
Tumor—associated antigens (TAA) are Known in the art, and can prepared for use in generating antibodies using methods and information which are weii n in the art in ts to er effective ceiiuiar targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise associated poiypeptides that are specificaiiy expressed on the surface of one or more particuiar typets) of cancer ceii as compared to on one or more normai nonmcancerous ceiifs). Often, such tumor—associated pciypeptides are more abundantiy sed on the surface of the cancer ceiis as compared to on the surface of the non—cancerous peiis. The fication of such tumor—associated ceii surface antigen poiypeptides has given rise to the abiiity to specificaiiy target cancer ceiis for ction via antibody—based therapies.
Exantpies of TAA inciude, but are net ‘ed to, TAA (1 H36) iisted beicwn For convenience information reiating to these antigens, aii of which are known in the art, is iisted beiow and incitides names, aiternative names, Genbank accession numbers and primary reference(s), feiiowing nncieic acid and protein sequence identification conventions of the Nationai Center for Biotechnoiogy ation (NOSE). Nucieic acid and protein sequences ponding to TAA (1)»(3fi) are avaiiabie in ouhiic databases such as GenBank. Tumoreassociated antigens targeted by antibodies inciude aii amino acid sequence variants and iscfcrrns possessing at ieast about 70%“ 80%, 85%, 99%, or 95% sequence identity ve to the sequences identified in the cited references, or which exhibit substantiaiiy the same bioiogicai properties or characteristics as a TAA having a ce found in the cited references. For exampie, a TAA having a variant sequence generaiiy is abie to bind specificaiiy to an antibody that binds specificaiiy to the TAA with the corresponding sequence iistedt The sequences and disciosure in the reference specificaiiy recited herein are expressiy incorporated by reference.
TUMOR-ASSQCiATED ANTEGENS (1 H36): (1) BiViPRi i3 (bone genetic protein receptor—type iii, Genhank ion no. 001203)ten Dijkefi, erai Science 264 (5155):tOt—104 (1994), anogene 14 (t ‘i):t3?7-t382 @9977»; W02004/‘0633fi2 (Ciairn 2); \N02003i042t’261 (Ciairn 12); US2003/134790~Ai (Page 38—39); W02002/102235 (Ciaim 13; Page 296); WOZODB/‘fit'55443 (Page 91~92); WOZUtiE/Sig’iZfi ie 2; Page 528n530); W02003/029421 (Ciairn 6); WOZOOBIOZIiSQZ (Ciairn 2; Fig 112); WOZOOZ/QSSSS (Ciaim t; Page 183); W62002l54940 (Page 106—101); WOZOGE/SQWWPage 349—350); WOEOGEBOEfiS (Ciairn 2'7; Page 37%); W02091i48204 (Exampie; Fig 4); NP___Otit’i94 bone morphogenetic n receptor, type EB fpidzNPWOO’i 194,1. Crossnreterences: hiihi:893248; NPWOOt 194.1; AY965994 (2) E16 (LATt, SLC7A5, k accession no. NMfiQBch) m. Bicphys, Res.
Commun. 255 (2), 283—288 (1999), Nature 395 (6699):288—29’i (1998), Gaugitsch, i-i.W., at a! (1992) J. Biol. Chem. 26? (16M ’126741273); /048938 (Examoie 2); /032842 (Exarnpie iV); \N02003/042661 (Ciairn 12); tA192003/016475 (Ciaini i); W02062178524 (Exantpie 2); 02i99874 (Ciaim 19; Page 127429); W02002i86443 (Ciaim 27; Pages 222, 393): WQZQOB/OGBSQS (Ciairn 10; Page 293); WOQOGEIMYQS n 33; Page 9395); WOZGOOi‘idEES (Ciairn 5; Page 133436); 1’224454 (Fig 3); WQEOOSIQZStBS (Ciairn f2; Page 150); NFLOOMW soiute carrier farniiy 7’ (cationic amino acid transporter, y+system), member 5 ioid=NPm0034Y7i3 — Horne saoiens; Cross“ references: MiMfiOQ’iEiZ; NP___003477.3; NM___015923; Ntv'i____t303486___i (3) STEAM (six transmembrane epitheiiai antigen of prostate, Genbank accession no.
NM____012449): Cancer Res. tit (15), 585725860 (2001), Hubert, RS, eta! (ii-399) Pros. Naif.
Acad. Sci. USA. 96 (25):t4523—’i4528); W02004/065577 (Ciaim t3): VVQZOU4/027049 (Fig 1L); EP1394274 (Exampie it); WOZOM/GifiZZS (Ciaim 2); ‘V’VOZOQSIQ4266i (Ciaim 12); U82063f15?089 (Exampie 5); U82003i18583® (Exampie 5); U82003/‘G64397 (Fig 2); W020ii2i8974? (Exempie 5; Page 618—619); VV02003/022995 (Exempie 9; Fig 13A, Exampie 53; Page 173., Exampie 2; Fig 2A); NPmtiiifiSSi six transmembrane epitneiiai n oi the prostate; reterences: MEM:6Q4415; NP_036581.1; 449_1 (4) 0772? (CAiZS, MUC’ES, Genbank accession no. 86); J. Bioi. Chem. 276 (29)::27371—27375 (2001)); W02904i045553 (Cieim t4); WQEOOZXQEZSBB (Ciaim 6: Fig 12); WOZOOZISBSM (Cieirn 15; Page tie—121); USEDOS/in’iMQ (Exempie 16); Crose references: Gi:3450‘i467; AAKM’EEQB; AF36’i486__’i (5) MPF (MPF, MSLN, SMR, niegakaryocyte potentiating factor, eiin, Genbank accession no. Nixlim005823) Yamagueni, N., et a! Biol”. Chem. 269 (2), 805—808 , Free.
Nail. Aeao‘. Soil USA. Q8 (20):11531—1153§ (1999). Pros. Naif. Acad. Sci. USA. 93 (t):136ei40 (19:96), J. Bioi. Chem. 270 (37):21984—2‘i990 (1995)); WGQQQSIiQiZBS (Ciaim i4); (WQZQOZMWESS (Ciaim 13; Page 2874288); W02002f’301675 (Ciaii‘n 4: Page 308— 309); WOZUOEW’iQQS (Page 3204521): WGQ4I10312 (Page 5257); Croesnrefereneee: MiMfiO’iOfii; NP_005814.2; NM_005823_1 (6) NapiBb BB, NaPiQE, NPTiib, SLC34A2, eointe carrier femiiy 34 m phosphate). member 2. type ii sodiumndependent phosphate transporter 3b., Genbank accession no. Ni\/i_008424) J. Bioi. Chem. 27? (22):19685—19672 (2002), Genomics 82 (2):28’i*284 (i999). Feiid, J.A., et ai (1999) m. Biephys. Res. Commun. 258 (3):578* 582); WOZtiiiti/‘GEEWS (Cieim 2); EPiBQéiZM (Exampie it); WOZGDE/‘tOZQBS (Cieim 13; Page 326); EPOSYSSBQ (Ciaiin ’i; Page 17—19): WQEOOtIEY‘iBS (Ciaim 20; Page 329): WOZOOKHOBESQ (Exarnpie iV); WOZOGi/"I’SWY’ n 24; Page iSQ—MO); Crose references: MEM:6042‘i7; NPMOOfiéiiSJ; NMm006424mi. in certain embodiments. conjugate compounds of the invention comprise anti—NaPiZB antibodies. in one embodiment of the ion. an eEiEB antibody of an ABC of the invention comprise (a) CDR L‘i of SEQ ED NOz’i; (b) CDR L2 oiSEQ ED N052; (o) CDR L3 of SEQ if) N633; (d) CDR Hi of SEQ ED N034; (e) CDR H2 of SEQ ED N95; (1“) (EUR H3 of SEQ ED N013. in one embodiment, the antibody comprises, the VH and Vi. sequences in SEQ iD NO:8 and SEQ iii) NO:?, respectiveiy, inciuding post—transiaiionai ations of thOSG ce& in one embodimem, the antibody comprises the heavy chain and Eight chain sequences in SEQ ED NOW and SEQ ED N029, reapectiveiy, ing post—transiationai modifcationg ofthose SEQUGHCEES.
SEQ ED NOTE RSSETLVHSSGNTYLE SEQ ED N02 RVSNRFS SEQ ED N33 FQGSFNPLT "§'§"<3"5'5"E§a'5§li""" """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" GFSFSDFAMS SEQ ED N915 ATEGRVAFHTWPDSMKG SEQ ED NOR? EARHRGFDVGHFDF SEQ ED NO? SPSSLSASVGDRVTETCRSSETLVHSSGNTYLEWYQQ KPGKAPKLLEYRVSNRFSGVPSRFSGSGSGTDFTLTESSLQPED FATYYCFQGSFNPLTFGQGTKVEEKR SEQ ED NOi8 EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFAMSWVRQAP GKGLEWVATEGRVAFHTWPDSMKGRFTE8RDNSKNTLYLQMN SLRAEDTAVYYCARHRGFDVGHFDFWGQGTLVTVSS SEQ ED N919 DEQMTQSPSSLSASVGDRVTETCRSSETLVHSSGNT‘Y’LEWYQQ KPGKAPKLLEYRVSNRFSGVPSRFSGSGSGTDFTLTESSLQPED FATYYCFQGSFNPLTFGQGTKVEEKRTVAAPSVFEFPPSDEQLK SGTASVVCLLNNFY?REAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR SEQ ED NEW EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFAMSWVRQAP GKGLEWVATEGRVAFHTYYPDSMKGRFTE8RDNSKNTLYLQMN SLRAEDTAVYYCARHRGFDVGHFDFWGQGTLVTVSSCSTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYECNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMS SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNST‘YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTESKA KGQPREPQVY‘FLPPSREEMTKNQVSLTCLVKGFYPSDEAVEW ESNGQPENNYKTTPPVLDSDGSFFL‘Y’SKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (7) Seme 513 (PLJ103?2, K1AA1445, 111111142015, SEMASB, SEMAG, Semaphene 5b Hiog, sema domain, seven eependin repeats (type 1 and type 1-Eike), iransrnembrane domain (TM) and short eytepiesmie domain, (eemapherin) 5E3, Genbank accession no.
A8040878); Nagese 1., ea‘ ei (2000) DNA Res. 7 (2):"143450); i/UOOQQ? (Ciaim 1); IGGBQM (Ciaim 1); WO2OOZIO§339 (Ciaim 1; Page 50); W02001/88133 (Cieim 1; Page 41—43, 48-58); WOEOQS/‘054152 (Ciaim 20); WOEOGBMOMQG (Ciaim 11); Accession: ; EMBL; ABS-40878; BAAQSQGQ1 Genew; HGNC:10737 (8) PSCA hig 50012R1k, CSBQOOSOWREK, RiKEN CDNA 27000506312, RiKEN CDNA 27’00650012 geee, Geebank accession no. AY358628); Rees eta—119602) Cancer Res. 6—2553; 11820113129192 (Ciaim 2); US2004/044180 (Ciaim 12); 4ific14179 (Cieim 11); 11331396961 (Cieim 11); 1182003/232056 (Exameie 5); 111102003/105758 (Ciaim 12); U82003i206§18 (Exampie 5); EP1347046 {Ciaim 1); WOEOOS/QZSMB (Cieim ); Cross—references: GE:3?182378: AAQ88991 .1; AYB58628H1 (9) ETBR (Endotheiin type E5 receptor, Genbenk accession no. AY275463); Nakamute 11/1,, e1 a! Bieehem. Biophys. Res. Commun. 11"], 3469, 1991; anwe Y., ez‘ a! Biechem. Biophys.
Res. Gammun. 178, 2484255, 111191; Arai 11., eta] Jpn, Cine. J. 56, 13034307, 111192; Arai 11., e? a! J. Biei. Chem. 268, 34633470, 1993; Sakamete A, Yanagisawa 1V1., e? a! Biochem.
Biephye. Res. Commun. 178, 656—663, 1991; rbagy NA, eiai J. Biei. Chem. 2368, 387345879, 1993; Haendier 8., eta! J. vasc. Pharmacei. 20, s1~84, 19112: Teutsumi M, e: ei Gene 228, £13419, 111199; S‘Eraueberg R.L., e1 a! Free. Natl. Acad. Sci. USA, 99, 1689946903, 2062: Bourgeois (3., etai J. Ciin. Endeerinei. Metab. 82, 3116—3123, 199?; Qkamete Y., eiai Biei. Chem. 27’2, 21589—21596, 1997; Verheij , ef aiAm. J. Med.
Genet. 108, 2234325, 2002; Hefetra R1V1.W., e1 ei Eur. J. Hum. Genet. 5, 180—185, 1997; Puffenberger E.G., eta] Cell 79, 1257—1266, 1994; Atfie "1",, et ai, Hum. Mei. Genet 4, 2:107“ 2499, 1995; Auricehio A, eta! Hum. Moi. Genet. 5351—354, 1996; Amiei J., eiai Hum. Mei.
Genet. 5, 355—357, 1996; Hefetre .W., eta! Naf. Genet 12, 445—447, 1996; Svenseen 13.1, eiaiHum. Genet 103, 145448, 1998: Fuchs 8., et ai Mei. Med. 7, 115424, 2001; Pingau11V., et a] (2002) Hum. Genet. 111, 11318—206; 111620041’045518 (Ciaim 1); 11.102064104189138 (Exampie 2); W02004/G40000 (Ciairn 151); WQEOQS/‘QSWSS (Ciaim 1); W02093/0164?5 (Cieim 1); W02903l01647’5 (Ciaim 1); WO2OOZ/‘fi1087 (Fig 1); W02093/016494 (Fig 6); 003/‘025138 (Cieim 12; Page 144): W02001:’98351 (Cieim 1; Page 124—125); EP0522868 (Ciaim 8; Fig 2); WGEGQHWWE (Ciaim 1; Page 297—299); eeeeeeimeem; 1186518404 (Fig 3); U85773223 (Ciaim 1a; (.301 3184); WOZOMIGOer (10) M86783 (RNF124, hypotheticei n FLJ20315, k accession no.
NMMQW763); EHMEYE (Ciaim i); W02064m46342 (Examoie 2); tN02003/042661 (Ciaim 12); WOZOGBIGB3074 (Cieim 14; Page 61); WOQOGBIOtSfiZi (Ciaim i); WOZOOS/OMSQE (Cieim 2: Fig 93): ISfi689 (Examcie 6); Cress—references: Locusiflz54894; NP_060233.2; NM_017763_1 (11)STEAP2 (HGNQfifiSQ, iPCA—i, PCANAPt, STAMP’i, , STMP, prestate cancer associated gene 1, prostate cancer associated pretein 1, six transmemcrane epitheiiai antigen of prostate 2, six trensmembrene prostate protein, Genbenk accession no. 38); Lab. invest. 82 (’i 1):1573*‘i582 (2002)); WOEQQS/‘QSYBOe; USZGG3IG64397 (Ciaim “i; Fig i); WQEQOZIYZSQES (Ciaim 13; Page 54—55); WQZQOi/72962 (Ciaim ”i; Fig 4B); W020031’104270 (Cieim it); W02003/19427O (Ciaim 16); USZOM/OOSSQS (Cieim 22); WOZOOBIMZM’! (Cieim i2); USESOS/Ofiflfiiii (Ciaim 12; Fig 10); \Ai02002f26822 (Cieim 23; Fig 2); weecomerize (Ciaim 12; Fig 10); Cross-references: (3322655488; AAN04080.1; AF455138m‘i (12) TrciViK-i- (BR22450. M, TRPM4, TRPM4B, transient receptor potentiai cation channei, eub‘iamiiy M; member 4; k accession no. NM_017836); Xu; X.Z., eta! Pros.
Naif. Acad. Sci. USA. 98 (19):16692-1069? (2091), Get! 109 (3):397~407 (2032), J. Bici.
Chem. 27’8 0813—30820 (2303)): U82003/143557 (Ciaim 4); WOZGDOMOGM (Ciaim 14; Page ’190—103); WOEZUOEHOBSZ (Ciaim "i; Fig 9A); WGZOUB/M‘Zfifii (Ciaim 12); WOZOOEISGZfiS (Cieim 27; Page 391); U82003i219806 (Cieim 4); WGEGQiI82794 (Ciaim 14; Fig iA-D); Cross-references: Miivi:606936; NP_O§0106.2; i'ftiBtSmi (t3) CREPTQ (CR, CR1, CRGF, CRiF’TO, TDGFt, teratecercincmanderived growth factor.
Genbank accession no. NFLOOBEOS er NM_003212); Ciccedicoia. A... et a] EMBQ J. 8 (7):198?~1991 (1989), Am. J. Hum. Genet. 49 (3):?)55665 ); USEDQS/Zzezii‘i (Ciaim i): WQ2003/08304’i (Exempte t); IQMQéfi-{i (Ciaim 12); WOQODE/‘SSWO (Ciaim 2; Page 5263); tNOQGOBiOEdiSEiZ (Ciaim 2; Fig 58); WQZOOEZ/tfitiiB (Ciaim ‘i; Page 94—95, 105); WQZQOZIZZSGS (Ciaim 2; Fig i); U85854399 (Exempie 2; (303 17—18); USSNZMS (Fig 2); references: MiM:i8?395; NPJJQSZGBA; NEVLQQSZ’EEJ (t4) CD21 (CR2 (Compiement receptor 2) cr CBDR (Cfidi’Epstein Barr virus receptor) ct Hs.73792 Genbank accession no. MZSOM); Fujiseku eta] (1989) J. 5350!. Chem. 264 (4):2118~2125); Weis J.J., etaiJ. Exp. Med. 167, 1047—10efi, 1988; Moore M, etaiP‘roc.
Naif. Acad. Sci. USA. 84, 9194—9198, 1987; Beret M, at e! Mei. immuoci. 35, WES—1331, 2014/025564 1998; Weis J...i., ei ai Pres. Naif, Acad. Sci. USA. 83, 56366643; 1666; Sinha S.K.. ei‘ ai (1993) J. immunei. 156, 5311—5326; W02664/‘645526 (Examoie 4); U82664i605536 (Exampie 1); WQ2OQ3/‘062401 (Ciaim 9); VVC32604/045526 (Exameie 4): iii/(2391;625:113 (Fig 9.1—9.9); W020041’026595 (Ciaim 1); Accession: P266253: 6; (214212; EMBL; 5126604; AAA357861. (15) CD795 , CD796, iGb (immunegiebuiinwassociaieci beta). 826, Genbank accession no. NM___660626 or 11038674); Pres. Nat}. Acad. Sci. USA. (2663) 160 (7):!«1126— £1131, Bioeci (2062) 106 (6)306845076, iviuiier et ai (1692) Eur. J, immunoi. 22 (6):1621— 162.5); WOZOOzi/‘O16225 i 2, Fig 140); W02663i68?768, U82604/101874 (oiaim 1, page 102); W02063/662461 (eiaim 9); W02062/78524 (Exampie 2); U82602i166573 (oiaii‘n . page 15); 1135644633; WQEOQS/‘MSZOZ (eiaim 1, pages 306 and 369); WQ 69166658, US6534482 (oiaim 13, Fig 17MB); WO2OQQI55351 (eiaini 11, pages; 146); Creee— references: 66511147245; 511313006171; 066263 (16) FQRHE (EFGP4, iRTA4, SPAP1A<SH2 domain containing phosphatase anchor protein 1a), , SPAP1C, Genbank aeeeseien ne. NM___036764, AY358130); Geneme Res. 13 (10):.22652270 (2003). immunogeneties 54 (2)267—95 (2002), Bieoo’ 99 (8)::2662—2669 (2662), Proo. Natl. Acad. Sci. USA 98 (1?):6772~9777 (2661), Xu; Mi; ei‘ ai (2001) Bieehem. Biophys. Res. Commun. 286 (3):?68-1775; WGQQOM616225 (Ciaini 2); W02063i677636; WOZOO’ii’36496 (Ciaim 5; Fig 18D—1—16D—2); W02603i09?663 (Ciaim 12); W02063/689624 (Ciaini 25); Crosenrefereneee: M1M2606569; NPW11O3912; 764m1 (1?) HERE (1311182, Genbank accession no. M11736); Coueeene 1... e1 ai Science (1985) 236(47’30):1132~1139); Yanienieie T., et ai Nature 319, 230—234, 1966; Semba K, eta! Pres. Nari. Acad. Sci. USA. 82, 6497—6561, 1965; Swiercz J.M.. 6? a! J. Ceii‘ Biol”. 165, 869' 886, 2064; Kuhne J.J., e1 ei J. Bibi. Chem. 274, 36422—36427, 1966; (The H.—e., eta! Nature 421. 756—760, 2663; Eheani A, at e! (1963) Genemiee 15, 4264129; W02664i646938 (Exampie 2); i627049 (Fig 1E); W02664/‘609622; W02063i681210; 6102063689964 (Ciaini 9); W02603i016475 (Ciaim 1); U82063/116592; WOEUGB/GOBSB? (Ciaim 1); W02063/055439 (Ciaim 29; Fig 1A—B); W02003i025228 (Ciaim 37; Fig 5(3); W02002f22636 (Exampie 13; Page 95467); WO2OOZI12341 (Ciaim 66; Fig 7); WO2002f13847 (Page 71—74); WGEQOZ/MSO?) (Page 114~117’); W020011’53463 (Cieim 2; Page 4146); W02601i41767 (Page 15); OiMBQEi (Ciaini 52; Fig 7); iii/02000190579 (Ciaim 3; Fig 2); U35669445 {Ciaim 3; (301 31—38); W06630514 (Ciaim 2.; Page 66—61); EP‘1439393 (Ciaim 7’); W02664/O43361 (main: 7); OEWOQ; 16192061/60244 (Exampie 3; Fig 4); Accession: $304626; EMBL; i‘v’it‘im7; MA35808.1. EMBL; M11761; AAA35808.t. in certain embodiments, conjugate compounds of the invention se anti— HERE antibodies. in one embodiment of the invention, an anti~HER2 antibody of an ADC of the invention ses a zed anti~HER2 antibody, e.g., hnMAo495mt, huMAh4D5~2, hoit/iAthDS—3, itUMAb-‘ii-D5'4, iiUMAbtii-D5v5, nanometre, hoMAMDSJ and huMAbdDS—B, as described in Tahie 3 of US 5821337. Those dies contain human framework regions with the compiementarity—determining regions of a mnrine dy (4D5) that binds to HERE. The humanized antibody hutViAbliDfinS is aieo referred to as trastnznmab, commerciaiiy avaiiabie under the tradenama HERCEPTiN. in another embodiment of the invention, an antieHERZ dy of an ABC of the ion comprises a humanized anti— HERE antibody, eg. humanized 204, as described in USYBEZSW. An exemoiary humanized 2C4 antibody is oeduzumao, commerciaiiy bie under the ame PERJETAK (18) NCA (CEACAi‘i/ifi, Genbank accession no. ); Barnett T., etai Genomics 3, 59— 66, 1988; Tawaragi Y., etai Biochem. Biophys. Res. Commun. tdfi, 89—96, 1988; Strausherg Rim, eta! Proc. Nat}. Aoed. Sci. USA. 99:16899—16933, 2002; WGEOOd/QEBYOQ; EP1439393 (Ciaim T); VVQZOU4/O44t78 (Examoie 4): WQEOO4IO31238; WOSZtiOBiOéiZfidt (Ciaim 12); ITSdZd (Exarnpie 2); WOZOOE/‘86443 (Ciaim 27; Page 427); WOEOQZ/EQSW (Ciaim 2); Accession: P40199; QMQZQ; EMBL; M29541; AAAEQQrtSt EMBL; M18728 (19) MDP (BPEPt, Genhank accession no, BCGt?023); Pros, Naif. Acad. Sci USA. Q9 6899e‘ifi903 (2602)): WG2QO3/‘Ott34?5 (Ciaim t); WOZGGEIMNS (Ciain‘i 33; Page 85* 87’); JP05003790 (Fig 6%); W099l46284 (Fig 9); Cross-references: MEM:179780; AAHWUZBJ; 8001770233 (20) iLEQRa a, ZCYTORY, Genbank accession no.AF18497’t); Ciark HE, eta! Genome Res. 13, 2265—2270, 2003; Mungaii A.d., at al Nature 425, 805-811, 2003; Biumherg it, eta! Ceii 104, 949, 2001; [Juniontier t.., etai J. immunoi. 167’, 3545—3549, 2001; Parrishwi‘tovak J., eta! J. Bioi. Chem. 277. 4?5‘i7~47523, 2002; Pietnev S. et ai (2003) Biochemistry 42:126t?—12624; Shaikh F., eta! (2004) J. immune]. 172, 2006~2010; EP13942M (Examoie tt); 1005320 (Examoie 5); W026031029262 (Page '74—75); W02003/002717 (Ciaim 2: Page 63); W02002l22153 (Page 45—47); USEQQZIMZBSES (Page 20mm); WOZGO’EMSZSt (Page 57—59); WOZOOtMfiZSZ (Page 6&65): W098i37t93 (Ciaim t; Page 555$); Accession: QQUi—ii—Yi; (RESUWAQ; ; EMBL; AF18497’1; AAFOBEOJ. (21) Brevicari (SCAN, REHAB, Genbank accession no. AF229053); Gary 8.0., 91‘ a! Gene 250, 13991417; 2000; Ciark HF; 31 a! Genome Res. 18, 70, 2003; Strausberg R.L., eiaiProc. Naif. Acad. Sci. 11.8.21. 99, 10899—10903, 2002; US2003/180372 (Ciaim 11); US2003/180373 (Ciaim 11); i119131 (Ciaim 1; Fig 52); US2003/119122 (Ciaim 1; Fig 52); 031 19120 (Ciaim 1); US2003I119121 (Ciaim 1; Fig 52); US2003/119129 (Ciaim 1); 03200191 19130 (Ciaim 1): 11 10128 (Ciaim 1; Fig 52); 0320031119125 (Ciaim 1); WO20031’016475 (Ciaim 1); WO2002l02034 (Ciaim 1) (22) EphB2R (DRT, ERK, Heidi EPHTS; TyroS, Genbank accession no, NM_004442); Char1.J. and Watt, MM, ancogene 0 (0), 105791001 1Qi1mgene 10 (5):897~905 , Annu. Rev. Neurosci. 21:309—345 (1998), 1111. Rev. Cytoi. 190217?~244 (2000)); 10102003042061 (Ciaim 12); WO200053210 (Ciaim 1; Page: 41); WO2004005570 {Ciaim 1); WO2004020583 (Ciaim 0); 10102003004529 (Page 128132); WO200053210 (Ciaim 1; Page «12); Cross—references: MiM:600997; NPm0044332; NMm004442m1 (23) ASLGBSQ (87h, Genbank accesgion no. AX092328); US2004/0101899 (Ciaim 2); WO2003104399 (Ciaim 11): WG2004000221 (Fig 3): US2003/105504 (Ciaim 1); US2003/124140 (Exampie 2); US2003i065143 (Fig 00); WG2002I102235 (Ciaim 1‘3; Page 299); US2003/091580 (Exampie 2); WO2002/‘10187 (Ciaim 8; Fig 10); 1002001194041 (Ciaim 12; Fig 7b); WO2002/‘02024 (Ciaim 13; Fig 1A—18); US2002/034749 (Ciaim 54: Page £15410); WO2002i00317 (Exampie 2; Page 320—321, Ciaim 34; Page; ); WO2002I7’1928 (Page 408—400); WO2002i02587 (Exampie 1; Fig 1); WG2001I40269 (Exampie 3; Pages 190—192); W’O2000i3610? (Exampie 2: Page ?); 0920041053079 (Ciaim 12); WO2003/004989 (Ciaim 1): WO2002/71928 (Page 233-234, 452—453); WC) 01:"10318 (24) PSCA ate stem ceii antigen sor, Genbank accessiom r10. AJ20?480); Reiter RE, 61 a! Pres. Natl. Acad. Sci. USA. 95, 1735—1740, 1998; (30 2., 91a! anogene 19, 12884290., 2000; Biachem. Biophys. Res. Commun. (2000) 275(3):?83—788; WO2004i022709; 15131394274 (Exampie 11); US2004l018553 (Ciaim 17); WO20031’008537 (Ciaim 1); 1002002101846 (Ciaim 1; Page 104); ‘W’Q2003/‘003906 (Ciaim 10; Page 288); WO2001I40309 (Exampie 1; Fig 17’); US2001/055?51 ie 1; Fig 1b); WO2000/32752 (Ciaim 18; Fig 1); W098i51805 (Ciaim 17; Page 97); 1824 (Ciaim 10; Page; 94); W008l40403 (Ciaim 2; Fig 1B); Accession: 043853; EMBL; AF043498; AACS900?.1 (25) GEDA (Genhank accession No. 63); AAP14954 iipoma Hi‘t/iGiC fusion—partner— iike protein ipid=AAPt4954.t - Homo sapiens (human); W02003i054152 (Ciaim 20); /000842 (Ciaim ’i); WQ2003/023013 (Exemoie 3 Cieim 20); US2003/194704 (Ciairn 45); Crceewreferencee: Gi130102449; AAP14954.1: AYZBOMIL’} (26) BAFF—R (i3 ceii —activating factor receptor, BLyS receptor 3, 3R3, Genhank accession No. AF‘i 16456); BAFF receptor lpid=NP____443177.t ~ Home e: Thompson, J.S., ei a! Science 293 (5537). 2198—2111 ; WQZOOMOSSSOQ; WOEOtixt/tit’ifit t; W02003/045422 (Examoie; Page 32—33); IQMZQIi (Ciaim 35; Fig 88); WQQOQSIQSS846 (Ciaim ".70; Page 615—616); WOZGOEIQIILSEZ (Cot 136—137); W020621387n‘6 (Ciaim 3; Page 133); W02002!24909 (Examoie 3; Fig 3); Croee—reierencee: i‘v'iii‘v’iiiiiGEfiQ; NPméidiCtt 77.1; Niv’inSQSiziSmi; AF’i 3260i) (27) 0022 (Beceii receptor (39226 isofonn, ESL—CAM, Lyn—8. Lyhtt, SiGLEC—Z, i—“LJ22814, Genhank accession No. AK026437); Wiison etai(t991) J. Exp. Med. 173:137*‘i46; W020ii3i072036 (Cieim i; Fig i); Croce—references: Mitt/i:i€i?266; 001762.1; titi‘iTT’iW‘i. in certain embodiments, conjugate compounds of the invention se anti—CDZ2 antibodies, in one i‘nent of the ion, an i322 antibody of an ADC of the invention comprise comprise three tight chain hypervariabie regions (HVR—L‘i, HVR—LE and HVR—LS) and three heavy chain hypervariabie regions (HVR—Ht, i-iVR-HZ and HVR—HB). according to US 8226945: HVR—L’i RSSQSEVHSVGNTFLE {SEQ iD N011 i) i-iVR-LZ KVSNRFS (SEQ it.) NOiiE) HVR-LB PYT (SEQ it) NOIiB) HVRwHi GYEFSRSWMN (SEQ 3D Nflzizi) HVRmHZ GRiYPGDGDTNYSC—EKFKG (SEQ ii} NG:15) HVRei-iS DGSSWDW‘Y’FDV (SEQ iD NOiifi) (28) CD79a (CD79A, CD79a, imrnunogiohutin—associated aipha, a B ceiimspecific protein that covaientiy interacts with ig beta (CD798) and forms a compiex on the surface with ig EVE ites, transduces a eignai invoived in B—ceii differentiation), pi: 4.84, MW: 25028 TM: 2 {P} Gene Chromosome: 19q132, Genbank accession No. MfiO); W02003/‘088808, U82033i3228319; W02003l062401 (ciaim 9); US2002/150573 (ciaim 4, pages 1344); WOW/58658 (ciairn 13, Fig 16); W092z’07574 (Fig 1); US5644033; Ha et a! (1992) J. immortal. t48(5):i526~153t; i‘viiiiier et a! (1992) Eur. J. immunol.. 2216214825; Heshimoto et ai (1994) immunogenetics 40(4):287*295; Prend‘homme ei a! (1992) Ciin. Exp. immunei, t4t~146; Yu et ai (1992) J. immunei. 148(2) 633—637; Sakagtiehi etai(1§88) EMBCD J. ?(tt):3457*3464 (29) CXCRE (Burkitt’s tympneme receptor 1, a G proteinneonpied receptor that is activated by the CXCL‘ES ehernekine. functions in iyrripneeyte migration and hurnorei defense, pieys a roie in HEVQ infection and perhaps deveiopment of AiDS, iymphen‘ie, myeioma, and Eeukemie); 37’2 ea, pi: 8.54 MW: 4t959 Tit/i: ? {P} Gene Chromosome: “(123.3, Genbenk aecessien Ne. NPWOO‘i 707.1); WQEOOMMGOOO; WOQUtizt/ti’iSliZfi; USEOOBHOSZQZ (Exempie 2); US$555339 (Exempie 2); W02002f§1087 (Fig 1); WOZOOt/‘ST‘ESS (Ciaim 20. page 269); WOZOQWYESQEO (pages 1243); WOZOGO/ZZ’EZQ (Exempie 1, pages 152—153, Exemete 2, pages 6); WOQQIQSxtfiS (eieim 1, page 38); USSMQOZt (Exempie 2, eet 4952); W094/2893’i (pages 5668); WOW/17497 (eieirn 7, Fig 5); Dobner etai (1992) Eur.
J. immunei. 22:2795—279Q; Bareiia et a! (1995) Bieehem. J. 3091773379 (30) HLA~DQB (Beta subunit of MHC eiess it meiecuie (Ea antigen) that binds peptides and presents them to CD4+ T iyrnphoeytes); 273 ea, pi: 6.56, MW: Ti‘v’i: ’i {P} Gene Chremeseme: 6p2‘i.3, Genbenk aeeessien Ne. NPWOOSZT 11.1); "i’enneiie eta] (1985) EMBO J, 2839~284?; Jenssen et a! (t989) imn’iunegeneties 2§(8):4ttm413; Beck et‘ ai (19%) J. Moi. Biei. 228:433—441; Strausherg et a! (2002) Pres. Nat}. Acad. Sci USA 99:1fi899— 16903; Servenius eta! (1987) J. Bibi. Chem. 262:8759—8766; Beck eta! (1996) J. Mei. Biol. 255:1~13; Neruse et ai (2002) Tissue Antigens 592513519; WOW/58658 (eieim 13, Fig 15):, U86153408 (Co! 3538); US$976551 (eei 188—170); S (eei ); Kesehara et‘ a! (1989) immunogenetios 30(1):fifi-68; Larhemmar eta! (1985) J. Bibi. Chem. 260(26):14’itt—14119 (31) P2X5 (Purinergic receptor P2X iigend~gated ion ehannei 5, an ion ehannei gated by extreeeiiuier ATP, may be inveived in synaptic transmission and enesis. deficiency may contribute in the petnephysieiegy of idiopathic detrusor instebiiity): 422 ee), pi: 7.63, NEW: 427206 TM: 1 [Pi Gene some: 3. Genbenk accession No.
SEZQ); Le er a! (1997) FEBS Lett. 418(t~2):1§5—199; WGEGO4IO4774Q; W020031072035 (sieint 10); Tonehmen erei (2000) Genome Res. 10:165473; WOZOfi2/22660 (Cieii‘n 20); W02063i093444 (oieim 1); W02063i087768 (oieim 1); WOQOGB/UZQZW (page 82) (32) CD72 i differentiation antigen CD72, Lth); 359 ea, pi: 8.66, MW: 40225, TM: 1 {P} Gene Chromosome: 9p133, Genhenk ion No. NP____00177’3.1); tit/02004042346 (ciaim 65); WO20031’026493 (pages 51—52, 57—58); WGZGOOH’SfiSS (pages 105—106); Von i-ioegen etai (1990) J. immunoi. 144(t2):48?fi~487?: Strausberg eta! (2002) Proc. Natl, Acad. Sci USA 99:16899—16903. (33) LYfie (Lymphocyte antigen 84 (RPtGS), type i membrane protein of the ieucine rich repeat (LRR) tarniiy, reguiates B~ceii tion and apoptosis, toes of function is associated with increased disease activity in patients with systemic iiipos eryihematosis); 681 aa, pi: 8.20, MW: 7414? TM: 1 {P} Gene Chromosome: 5q12, Genbahk accession No.
NP_005573:1); ussocznesser; WOW/@7198 (ciaim ti, pages 3942); Miora et at (19%) cs 38(3):299—304; Miura et a! (1998) Biooci 922815—2822; i’08304?; 452 (ciaim 8, pages 57—61); WOZOOD/iZ’iBQ (pages 24—25) (34) FcRi-i‘i (Fe receptor—iike protein 1, a putative receptor for the immnhogiohuiin Fe domain that contains CZ type ig-iike and ETAM domains, may have a roie in Beiymphocyte differentiation); 429 aa, pi: 5.28, MW: 46925 TM: 1 {P} Gene Chromosome: io2’ie’io22, Genbank accession No. NP____443170.1); W020031’077836; t/38490 (ciaim 6, Fig 185148—52); Davis et ai (2001) Pros. Nati. Acad. Sci USA 98(17):9772n9777; WOEOOSIQSQtSEe (ciaim 8); 046 (claim 1); W02003l089624 (claim 7) (35) iRTAZ (immuncgiobuiin amiiy receptor transiccaticn associated 2, a putative immuncreceptcr with possibie rates in B ceii pment and iympnomagenesis; iation of the gene by transiocaiion occurs in some 8 ceii ancies); 9?? aa, pi: 6.88, MW: 106468, TM: t {P} Gene Chromosome: ’iq2’i, Gencank accession No.
HumanzAFBtiSGEiZ, AF343663, AF343864, AF343665, AF369794, AF397453, Ami-30423, AK090475, AL834187, AY358085; MousezAKOBQi’fifii AY’iSSOSiO, AY506558; NPw’i 12571.1: WQEOOSIQZAEBEZBZ (ciaim 2, Fig 97’); Nakayar‘na at at (2000) Biochem, Biophys. Res, Commun. 277(1):’i24427; WOZGOS/OY7836; /SMQQ (ciaim 3, Fig 183—14832) (36) TENBE (TMEFFZ, tomoregniim TF’EF, HPP‘E, TR, putative embrahe proteogiycan, reiated to the EGF/hereguiin tamiiy of growth factors and ‘ioiiistatini; 3?4 aa: NCBi Accession: AADSSWG, AAF91397, Qas’i, NCBE RefSeo: NPm057276; NCBE Gene: 23671; QMEM: 695734; SwissProt QQUiKc; Genbank accession No. AFNQZM; AY358907, CAF85723, (307782436; W02004/074320: JP2004113151; W020031’042fi61; WOZOOSIQOQSM; E91295§44 (pages est—70); WOZOOZl30268 (page 32.9); WOZOOtiQOBM; /249130; U82004i022727; WQEQQMQt‘Si-iBSfi; USZGOM’EQT’SZS; U82003i23235i); US2004/005563; U82003i124579; Horie et‘ a! (2000) Genomics 67:146—152; Ucnicia eta! WO 59981 (1999) Biochem. Biophys, Res. Commun, 2§S:593~602; Liang et ai (2000) Cancer Res, 60:490?-12; Giynnesdones et a! (2061) int J Cancer. Oct 15; 94(2):178s84n (3?) CD33 (C333 moiecuie, SEGLEQB, SiGLECB, p67; C033 antigen ); gpdifi myeieid ceii surface n CD33; siaiic acid binding ig—iike iectin 3; siaiic acid—binding ig—iike ); Nucieotide : k accession no“ Mm23197; Genpank version no. Nh’im23197fi Gi:180097;Qenhank record update date: Jun 23, 2019 08:47 AM: Poivgeqtide: Genhank accession no. AAA51948; Genhank n no. AAA519481 Gi:1880§8: Genhank record update date: Jun 239 2010 08:47 AM; s Di, et at J. immunoi 141 (8), 2797~2809 ;_Antihodies .' H195 (Lintuzumebi— Reze A, et at Leuk Lymphoma 2009 Aug:50(8):1336~44; USEJ’SS-Efido (Seattie Genetics/immunomeciics); mAb QKTQ: Sutheriand, DR. et at. Free NatiAcan’ Sci USA 78(7): 451541519 1981, Schneiderfiu et at J Bio! Chem 25?, 85168522 ; mAb E6: Hoogenboomflfir, et at J irnmunoi 144, 321 1, 321? (1990); USEEQGESS (Human Genome Sciences) —ior exampie, SEQ ED NQs: ’i and 2 and ATCC accession no. 97’521; US7,557’,189 (immunogen) —fFor exampie, an antibody or fragment thereof comprising a heavy chain variahie region which comprises three CDRs having the amino acid sequences of SEQ ii) 3 and a tight chain variabie region comprising three CDRs having the amino acid sequences of SEQ ED NOs:4—6. in some embodiments, the anti—(31333 antihcciy comprises (a) H‘v’R—H’i comprising the amino acid sequence of SEQ ED N020; (h) HVR~H2 comprising the amino acid sequence of SEQ ii) N031; (c) HVR~H3 comprising the amino acid sequence of SEQ ED N01222: (d) HVR~L1 comprising the amino acid sequence of SEQ ED NQ:17; (e) H‘v’R—LQ comprising the amino acid sequence of SEQ ED NEWS; and (f) HVR~L3 comprising the amino acid sequence of SEQ it?) none. in some embodiments, the antisCDBi-i antibody comprises the VH and VL ces in SEQ ii) N024 and SEQ it) N023, respectiveiy, inciuding post—transietienai modifications of these sequences. in some embodiments, the anti—CD33 dy comprises (a) HVR~H1 comprising the amino acid sequence of SEQ ED N028; (p) HVR—i-i2 comprising the amino acid sequence of SEQ ED N029; (c) HVRnHB comprising the amino acid sequence of SEQ ED NQ:30: (d) HVR—L1 comprising the amino acid sequence of SEQ ED N025; (e) HVR—LZ comprising the amino acid sequence of SEQ ED NQQE; and (i) H‘v’R—L3 comprising the amino acid sequence of SEQ it) N027. in some embodiments, the anti—(3033 antibody oomprieee the VH and Vi. ces in SEQ ED NQ:32 and SEQ 3D N631, respectiveiy, inoiuding poetntransiationei cations of these sequences, in some embodiments, the anti—CD33 antibody comprises the VH and VL sequences in SEQ ED N034 and SEQ iD N033, respectiveiy, ineiuding poetetreneiationei modifications; of tnoee sequences. in some embodimente, the anti—CD33 antibody comprises the VH and Vi. ces in SEQ ED N636 and SEQ ED NO:35, reeneotiveiy, indenting oost~transietionai modifications of these sequences. in one embodiment, in some embodiments, the anti~Ci333 antibody comprises the VH and VL sequences in SEQ 3D N038 and SEQ ED NO:37’, teepeetiveiy, inoiuding poet~treneietionei modifioatione of those CSS. 15815.33 RSSQSLLHSNGYNYLD SEQ ED NQTH’ HVR—L‘i 15815.33 LGVNSVS SEQ ED NCM 8 HVRILZ 1561533 MQALQTPWT SEQ ED NQYEQ HVR~L3 15815.33 SEQ ED N020 HVR—H’i 15815.33 GTANYAQKFQG SEQ ED NQ:2’i HVR—HZ ....................................................................................................................................................................................................................................................... 15815.33 EWADVFD SEQ ED NQ222 HVRIHCi 151331533 EEVLTQSPLSLPVTPGEPASESCRSSQSLLHSNGYNYL SEQ ED N023 DWYLQKPGQSPQLLEYLGVNSVSGVPDRFSGSGSGT DFTLKESRVEAEDVGVYYCMQALQTPWTFGQGTKVEE ....................................................................................................................................................................................................................................................... 15615.33 SGAEVKKPGSSVKVSCKASGGiFSNHAESWV SEQ ED NQ224 RQAPGQGLEWMGGESPEFGTAN‘Y’AQKFQGRVTETADES VH. TSTAFMELSSLRSEDTAVYYCAREWADVFDEWGQGT MVTVSS SEQ ED N925 903-HVR L2 AASSLQS SEQ ED N026 903—HVR L3 LQHNSYPWT SEQ ED N027 903~HVR H1 GNYMS SEQ ED N028 903-HVR H2 LEYSGDSTWADSVKG SEQ ED NQ:29 ....................................................................................................................................................................................................................................................... 903—HVR H3 DGWVSDMVV SEQ ED NQi3O DEQMTQSESSLSASVGDRVTETCRASQGERNDLGWYQ SEQ ED NO:3’E QKEGKAPKRLEYAASSLQSGVPSEFSGSQSGTEFTLTE DFATYYCLQHNSYPWTFSQGTKLEEK EVQLVESGGALEQPGGSLRLSCVASGFTESGN‘Y’MSWV SEQ ED NQ:32 RQAPGKGLEWVSLEYSGDSTYYADSVKGRFNESRDESK NTVYLQMNSLRVEDTAVWCVRDGYYVSDMWWGKG 903.2 VE. SESSLSASVGDRVTETCRASQGERNDLGWYQ SEQ ED N033 QKEGKAPKRLEYAASSLQSGVPSEFSGSQSGTEFTLTE SSLQEEDFATWCLQHNSYPWTFGQGTKLEEK 903.2 VH EVQLVESGGALEQPGGSLRLSCVASGFTESGNYMSWV SEQ ED NQz34 RQAPGKGLEWVSLEYSGDSTYYADSVKGRFTESRDESK NTVYLQMNSLRVEDTAVWCVRDGYYVSDMWWGKG TTVTVSS 903.3 VE. DEQMTQSESSLSASVGDRVTETCRASQGERNDLGWYQ SEQ ED N035 QKEGKAPKRLEYAASSLQSGVPSEFSGSQSGTEFTLTE SSLQEEDFATWCLQHNSYPWTFGQGTKLEEK 903.3 VH EVQLVESGGALEQPGGSLRLSCVASGFTESGNYMSWV SEQ ED NQz36 RQAPGKGLEWVSLEYSGDSTYYADSVKGRFSESRDESK NTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKG TTVTVSS 903.4 VE. DEQMTQSESSLSASVGDRVTETCRASQGERNDLGWYQ SEQ ED N03? QKPGKAPKRLEYAASSLQSGVPSRFSGSGSGTEFTLTE SSLQPEDFATWCLQHNSYPWTFGQGTKLEEK 903.4 VH EVQLVESGGALEQPGGSLRLSCVASGFTESGN‘Y’MSWV SEQ ED NQ:38 RQAPGKQLEWVSLEYSGDSTYYADSVKGRFAESRDESK NTVYLQMNSLRVEDTAVYYCVRDGYYVSDMW’WGKG TTVTVSS (38) LGREiGERdiQ; tide: Genbank accession no, NMWOUBESW; k version no.
NEVLQOSESEKE Gi124475886; k record update data: diii 22, 2012 93:38 PM; Poiypeptide: Genbank accession no. NPMGO3fi58; Genbank version no. NFLOGBEESJ Gi:4504379; k record update date: Jui 22, 2912 03:38 PM. in some embodiments, the anti~LGR5 antibody comprises (a) HVR~Ht comprising the amino acid sequence of SEQ it) NC): 46; (h) HVR~H2 comprising the amino acid sequence of SEQ it) NO: 47; (c) NVR-Nffi comprising the amino acid sequence of SEQ it) NO: 48; (d) HVR—Lt comprising the amino acid ce of SEQ ED NO: 43; (e) HVR~L2 comprising the amino acid sequence of SEQ ED NC): 44; and (f) HVR—LB comprising the amino acid sequence of SEQ if) NO: 45. in one embodiment, the antibody comprises the VH and Vi. sequences in SEQ it) NO: 49 and SEQ ED NO: 39, tiveiy, inciuding postntransiationai modifications of those SSQUE‘HCBS in some ments, the anti—LGRE comprises (a) i-iVRd-it sing the amino acid sequence of SEQ ED NO: 52; (b) itVR~i~t2 comprising the amino acid sequence of SEQ iD NC): 53; (c) HVR—HB comprising the amino acid sequence of SEQ it) NC): 54; (d) HVR—L’i comprising the amino acid sequence of SEQ ED NO: 49; (e) H‘v’R—LE comprising the amino acid sequence of SEQ it) NO: 50; and (t) NVR-LGS comprising an amino acid sequence seiected from SEQ iD NO: 51. in some embodiments, the anti—LgRE antibody comprises a Vii as in any of the embodiments provided above, and a Vi. as in an}i of the embodiments provided above. in one embodiment the dy comprises the Vii and Vi. sequences in SEQ iD NO:42 and SEQ ii) NC’inttg respectiveiy, inciuding post—transiationai modifications ofthose sequences. sent/L"'r5iitstos“itits'tat?steenttrrtsnttsssvs""""""""""""""""""""s"soreness""""" NYGNSFMHWY QQKPGQPPKL LiYLASNLES GVPDRFSGSG SGTDFTLTiS SLQAEDVAVY YCQQNYEDF’F KVEE KR EVQLVQSGAE VKKPGASVKV SCKASGYTFS SEQ ii) NQ:40 AYWEEWVRQA PGQGLEWiGE SLPGSDSTDY RATF TSDTSTSTVY RSED ARGG HYGSLDYWGQ GTLVTVSS .......................................................................................................................................................................................................................................................
YW353 Vi. DiQMTQSESS LSASVGDRVT ETCRASQDVS SEQ ED NQKi-‘i TAVAWYQQKP GKAPKLLEYS ASFLYSGVPS RFSGSGSGTD FTLTESSLQP EDFAT‘Y’YCQQ SYTTPPTFGQ GTKVEEKR YW353 VH EVQLVESGGG SLRL SCAASGFTFT SEQ iD NQ:42 SYSESWVRQA PGKGLEW‘V’AE EYPPGGYTDY ADSVKGRFTE SADTSKNTAY LQMNSLRAED TAVYYCAKAR LEFDYWGQGT LVTVSS 8E11~HVR L1 RASESVDNYG NSFMH SEQ iD NO:43 SE 1— HVR L3 QQNYEDPFT SEQ ED Nflfifi "§§'t't'3"i¥i§?§"ii"i""""""""EWEEKEWE"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""'5";E5"i5"i§iE§:Et§"""""§ YW353 HVR L’i RASQDVSTAV A SEQ iD NOV-it? YW353 HVR L2 SASFLYS SEQ ED NO:50 YW353 HVR L3 QQSYTTPPT SEQ iD N051 YWSSS HVR Hi GFTFTSYSES SEQ ED NG:52 YWSSB HVR H2 EEYF’PGGYTD YADSVKG SEQ ED N0253 YWBSB HVR H3 ARLFFDY SEQ ED NQ:54 The parent dyi may eiee be a fusion protein comprising an eibornin~bineiing e (ASP) sequence (Dennis et ai. (2002) “Aiburnin Binding As A Generai Strategy For improving The Pharmaeokinetice Of Proteins” J Biol Chem. 3565043; WO 01145746). Antibodies of the invention inoiude fusion proteins with ASP sequences taught by: (i) Dennie et e! (2002) J Biol Chem. 2?7:35035—35043 at Tabiee iii and EV, page 35038; (ii) US 2004113001827 at {0976}; and (iii) W0 01I45746 at pages 1243, and aii of which are incorporated herein by reference in one embodiment, the antibody nae been raised to target specific the tumour reieted antigen ovifig.
The eeii binding agent may be iabeiied, for e to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate. The iabei may be a biotin iabei. in another embodiment, the ceii binding agent may be iabeiied with a radioisotope. tuents The phrase “optionaiiy substituted” as used herein, pertains to a parent group which may he unsubstituted or which may be tuted.
Uniess ise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term ituent” is used herein in the conventionai sense and refers to a ohemicai moiety which is covaientiy attached to, or it appropriate, fused to, a parent group, A wide variety of substituents are weii known, and methods for their formation and introduction into a variety of parent groups are aiso weii known. in a preferred ment, the substituents described herein (which inciude optionai substituents) are ed to those groups that are not reactive to a oeii binding agent. The iinit to the ceii binding agent in the present case is formed from the bridge between the two FEED moieties through a tinker group to the ceii binding agent. Reactive tunetionai groups iocated at other parts of the RED structure may be capabie of forming additionai bonds to the ceii binding agent (this may be referred to as orossiinking), These additionai bonds may aiter transport and bioiogicai activity of the conjugate. Therefore, in some embodiment, the onai substituents are iimited to those tacking reactive functionaiity. in one embodiment, the substituents are seiected from the group consisting of R, OR, SR, NRR’, N02, haio, COgR, COR, CONHQ, CONHR, and CONRR’. in one embodiment, the substituents are seiected from the group consisting of R, OR, SR, NRR’, N62, CQQR, CQR, CQNHZ, CQNHR, and CONRR‘. in one embodiment, the substituents are seiected from the group consisting of R, OR, SR, NRR’, N92, and hate. in one ment, the substituents are seiected from the group consisting of R, QR, SR, NRR’, and N02.
Any one of the ment mentioned above may be eppiied to any one of the substituents described herein. Aiternativeiy, the substituents may be seiected from one or more of the groups iisted beiow.
Examptes of substituents are bed in more detati betow (31-12 aikyi: The term “C142 atkyt” as used herein“ ns to a monovaient moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from ’i to 12 carbon atoms, which may he aiiphatic or iic, and which may he saturated or rated (eg. partiaity unsaturated, tutiy unsaturated). Thus, the term “atkyt” inciudes the subnciassee aikenyti aikynyi, cyoioaikyi, etc, discussed beiow.
Examptes of saturated aikyi groups inciude, but are not timited to, rnethyi (Ct), ethyi (Cg), propyi ((33), hutyi (C34), pentyi (C35), hexyi ((35) and heptyt (Cy).
Exarhptes of ted iinear atkyt groups e, but are not iimited to, rnethyi ((31)a ethyi (Cg), n—propyi (Cg), hebutyi (C4), nepentyi (an‘iyi) (Cg), n—hexyi (C5) and n~heptyi (C7).
Examptes of saturated branched aikyi groups inciude isonoropyt (C3), isombutyi (C4), sec—butyi (CA), tert—butyi (CA), iso—pentyt (C5), and neo—pentyi ((35).
An aikyi group may optionaity be interrupted by one or more heteroatorns seiected from O, hid-t) and 8. Such groups may be referred to as “heteroaikyi”. (3242 Heteroatkyt: The term “(32-12 heteroaikyi” as used herein pertains to a monovaient moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 2 to 12 carbon atoms, and one or more heteroatoms setected from (I), Nth!) and S, abiy O and S.
Examptes of heteroaikyi groups inciude, but are not iirniied to those comprising one or more ethyiene gtycoi units of the type ~(OCHgCi-t2)—. The at of a heteroatkyt group may be the primary term of a heteroatorn, eg. @Hr ~Si~t or “NH; in a preferred embodiment, the terrninai is “CH3: C2242 Atkenyi: The term “(32.12 atkenyi“ as used herein, pertains to an aikyi group having one or more oarhon~carbon doubie bonds.
Exampies of unsaturated aikenyi groups inoiudes but are not iimited to; ethenyi (yinyi, ~CH=CH2), teorooenyi (~CH=CH*CH3), 2—propeny‘t (atiyi, “CH*CH=CH2), isopropeny‘t (t—methytvinyi, -C(CH3)=CH2), butenyt (Cg), oentenyi (C5), and i ((35). (3242 aikynyi: The term “(32-12 aikyrtyi” as used herein, pertains to an aikyi group having one or more oarhon~oarhon tripie bonds.
Exampies of unsaturated aikynyi groups inoiude, but are not iimited to, ethynyi (—CECH) and Emorooynyi rgyi, ECH). (33.12 oyoioatkyt: The term ”C3342 ikyi” as used herein, pertains to an atkyt group which is aiso a cyoiyi grouo; that is, a monoyaient moiety ed by removing a hydrogen atom from an aiioyoiio ring atom of a oyoiie hydrocarbon (oarbooyoiic) compound, which moiety has from 3 to i7 oarbon atoms, inciuding from 3 to '7 ring atoms.
Examoies of cycioaikyi groups inoiude, but are not iimited to, those derived from: saturated monoeyoiio hydrooarbon compounds: oyoioorobane (C3), utane (C4), oyoiobentane (Cg), cyotohexane (CS), oyotoheotane (Cy), methytoyoioprooane (Cit)a dimetnytoyoiobrooane (Cs), ntethyioyciobutane (C5), dimethyioyetobutane (€35), methyioyoiooentane (C6), dimethyioyetooentane (C7) and methyioyoionexane (C7); unsaturated rnonooyoiie hydrocarbon oomoounds: cyoiooropene (€33), oyetobutene (€34), cyoiopentene (C35), oyoiohexene (Ce), methyioyotoorooene (C34), yioyoioorooene ((35), methytoyoiobutene (C5), dimethyicyoiobutene (C5), methyicyciopentene (C5), dimethyioyoiooentene (C7) and rnethyioyeiohexene (C7); and saturated boiycyotio hydrocarbon compounds: noroarane (C7), norpinane (C7), norbornane ((37). (33.20 heterocyeiyi: The term “(3330 heterooyoiyi" as used herein, pertains to a ient moiety ed by removing a hydrogen atom from a ring atom of a heterocy‘oiic compound, which moiety has from 3 to 20 ring atoms, of which from t to ti) are ring heteroatoms. abiy, eaoh ring has from 3 to 7 ring atoms, of which from “i to 4 are ring heteroatomst in this context, the prefixes (eg. (33.20, 03.7, (35.55, eto.) denote the number of ring atoms, or range of number of ring atoms, Whether oarbon atoms or heteroatoms. For exampie, the term “ngheierocyeiyi”; BS used , periaine to e heterocyciyi group having 5 or 6 ring atoms.
Exampies ef moneeyeiic hetereeyciyi groups inciudei but are not iimiied to“ those derived from: N3: aziridihe (C3), ezetidine (C4), pyrreiidine (teirehydrepyrroie) (Cg), pyrroiine (e.g., 3—pyrreiine, 2,5—dihydrepyrreie) (C5), ZH—eyrreie er BH—eyrreie (ieepyrroie, ieeezeie) (C5), piperidine (C5), dihydrepyridine (CS), ydrepyridine ((35), ezepihe (C7); 01: oxirane (Cg), exe‘iane (C4), exeiane (teirahydreiuren) (C5), exeie (dihydre‘furan) (C5), exane (tetrehydrepyren) (C5), dihydropyreh (Cg), pyreh (Cg)a exepin (C7); 531: thiirene (Cg), ne (C4), ihieiene (tetrahydrethiophene) (C5), ihiene (tetrahydre‘ihiepyren) (C5), thiepene (C7); 02: dioxoiane (C5), dioxane (C5), and diexepane (C7); ()3: irioxane (Cg): N2: imidezoiidine ((35), pyrezeiidihe (diezeiidihe) ((35), oiine (C5), pyrezeiine (dihydmpyrezeie) (C5), piperezine (C35); : ieirahydreoxezoie (C35), ooxazoie (C5), ‘ietrehydroieexezoie {35)5 dihydreieoxazeie (Cs), morpheiine (Cg), teirehydmexazine (Cg), dihydroexezihe (Cs), exazine (Cs); N131: ihiezeiine (C35), ihiezeiidine (C5), thiemorphoiine (C5); N201: exadiazihe (C6); 0181: oxathieie (C5) and exaihiene (ihioxehe) (C5); and, NEG/:31: exethiezine (Cg).
Exempiee of tuted menecyciic heierocyeiyi groups inciude those derived from saccharidess in cyciic form, for exampie, furerieeee ((35), such as erebine‘furanose, nyofuraneee, ribo’i‘ureneee, end xyiefurehse, and pyreneses (Cg), such as renoee, eitrepyrenoee, giucepyreneee, manneeyreheee, guiepyreneee, ehoee, geiae‘iepyreneee, and teiopyranoee. (35.20 eryi: The term “(35.20 aryi", as used herein, ns to e monovaieht moiety obtained by removing a hydrogen atom frem an aremetie ring etem Of an aromatic; compound, which moiety has from 3 ‘ie 20 ring atoms. Preferebiy, each ring has from 5 to 7 ring eiems. in this context, the prefixes (eg. (33.20, (35-7, (35-5, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For exempie, the term “(35.5 aryi” as ueeci herein, pertains to an eryi group having 5 or 6 ring atoms.
The ring atoms may be eii carbon atoms, as in “carboaryi groups”.
Exampies of eerboaryi groups inoiude, but are not iimiied to, those derived from e (Le. nitenyi) (C35), naphthaiene (Cm), azuiene (Cm), anthracene (C14), phenanthrene (CM), naphtheeene (C13), and pyrene (C15).
Exempiee of aryi groups which comprise fused rings, at ieaet one of which ie an aromatic ring, inciude, but are not iimited to, groups derived from indene (eg. 2,3wdihydro—ti-i—indene) (Cg), indene (Cg), isoindene (Cg), tetraiine (t,2,3,4ntetrahydronaphthaiene (C310), acenaphihene (Cm), tiuorene (C13), ohenaiene (Cm), acephenanthrene (C15)1 and hrene (C15).
Aiternativeiy, the ring atoms may ineiude one or more heteroatoms, as in “heteroaryi groups”, ee ot monoeyeiic heteroaryi groupe inciude, but are hot iimited to, those derived from: N1: pyrroie (azoie) (CS), pyridine (azine) (Cg); O1: turan (oxoie) (C5); 81: thiopnene e) (C5); N101: oxezoie {(35), ieoxazoie (C5), isoxazihe (Cs); N291: oxadiazoie an) ((35); mm: oxatriazoie (C5); N431: thiazoie (C5), isothiazeie (C5); N2: imidazoie (t,3~diazoie) (C5), pyrazoie (i,2~diazoie) (C5), pyridazine iezine) {C5}, pyrimidine (1,3-diazine) (Cg) (e.g., cytosine, thymine, uracii), pyrazine (1,4-diazine) (CB); N3: triezoie (C5), triazine (C5); and, N4: tetrazeie (C5).
Exampies of heteroaryi which comprise fused rings, ineiude, but are not iirniied to: Cg(with 2 fueeci rings) d from benzoturan (Oi), ieohenzoturan (01), indoie (N1), ieeindoie (N1), indoiizine (N1), indoiine (N1), oiine (N1), purine (N4) (e.g., adenine, guanine), benzimidazoie (N2), indezoie (N2), benzoxazoie , henzisoxazoie , henzodioxoie (02), benzofurazan (N203), benzotriazoie (N3), behzothiofuran (Si), benzothiazoie (N181), benzothiadiezoie (N28); C10 (with 2 fused rings) derived from ohromene (Q1), or‘hehe (O1), ohrornart (Oi), isoohromah (Qt), behzodioxeh ((32), ouihoiine (N1), isoquihoiine (N1), quinoiizine (N1), hehzoxazine (N101), benzodiazine (N2), pyridopyridine (N2), quinoxaiihe (N2), uuinazotine (N2), cinrtoiihe (N2), phthaiazihe (N2), naphthyridihe (N3), pteridine (N4); CH (with 2 fused ridge) derived from hertzodiezepine (N2); Cw (with 3 fused rings) derived from carbazoie (N1), ofuran (Oi), dihenzothiophene (S1), oarhoiine (N2), dihe (N2), pyridoihdoie (N2); and, C1,: (with 3 fused rings) derived from acridihe (N1), xanthene (O1), thioxahthehe (S1), oxahthrehe (€32), phehoxathiih (04,81), phenazine (hi2), ezihe (N101), phenothiazihe (N181), thiahthrehe ($2), phenehthridine (N1), phehanthroiihe (N2), phehazihe (N2).
The aoove groups“ whether atone or part of r substituent, may themseivee optionatiy be tuted with one or more groups ed from themseives and the additionet substituehts Eisted peiow, Haio: “F, —CE, “Br, and vi.
Hydroxy: “OH.
Ether: QR, wherein R is an ether substituent, for exampie, a (31-7 aikyi group (eiso referred to as a Cm aikoxy group, discussed heiow), a {33.20 heterooyctyi group (aiso referred to as a C320 heterocyoiyioxy , or a (35-20 aryi group (atso referred to as a (35-20 aryioxy group), preferapiy a Ct.,-,reikyi group.
Aikoxy: “QR, wherein R is art atkyt group, for exampte, a (31-7 atkyt group. Exampies of (31-7 aikoxy groups inoiude, but are not Eirnited to, @Me (rnethoxy), ~0Et (ethoxy), ~O(hPr) (n~ propoxy), ~Q(iPr) (isopropoxy), u) (rt—butoxy), —Q(sBu) (seobutoxyi, 008d) (isohutoxy), and ~O(ti3u) (tert—hutoxy).
Aeetai: ~CH(OR1)(GR2), n R1 and R2 are independentty aoetai substituertts, for exampie, a C1_yaikyi group, a (33.20 heterocyoiyi group, or a (35.20 aryi group, preferehiy a (31;; aikyi group, or, in the ease of a “oyotio” aoetat group, R1 and R2, taken together with the two oxygen atoms to whioh they are ed, and the carbon atoms to which they are attached, form a heteroeypiie ring having from 4 to 8 ring atoms. Exemptes of aeetei groups irtoiude, but are hot Eimited to, ~CH(Qi\/te)g, -CH(OEt)g, ehd —CH(Oi\/ie)(QEt).
Hemieoetei: ~CH(OH)(GR1), wherein R1 is e hemieoetei euhetituent, for exempie, a (31-7 eikyi group, 3 (33,20 heterooyciyi group, or a C5.3geryi group, preferabiy a C1_yeikyi group.
Exemoies of hemieeetai groups inoiude, but are hot iimited to, —CH(OH)(OMe) and w CH(OH)(QEE).
Ketai: —CR(OR1)(OR2‘, where R1 and R2 are as defined for aoeteis, and R is a ketai substituent other than hydrogen, for exempie, a (31-7aikyi group, a (33-30 heierooyoiyi group, or a (35-33 eryi group, oreferebiy a (31-7 eikyi group, Exemoies ketai groups Eupiude, but are not Eimited to, “C(MG)(OMG)2, —C(Me)(OEt)3, OMeXOEt), ~C(Et)(GMe)3, ~C(Et)(OEr)3, and —C(Et)(QMe)(OEt).
Hemike’rai: —CR(QH)(OR1), where R“ is as defined for hemiaoe’rais, and R is a hemiketei substiiuent other than hydrogen, for exarnpie, a C1_7aikyi group, 3 C330 heterooyoiyi group, or e 05.30 eryi group, preferabiy a (31.7 eikyi group. Exempiee of hemieoetei groups inciude, but are not d to, (QH)(OE\/Ee), —C(Et)(OH)(OMe), —C(Me)(QH)(OEt), end “C(EtXQHXOEr).
Oxo (keto, ~0he): =0.
Throne (thioketone): =53.
Emiho (Ermine): =NR, wherein R is err Ernirro Substituent, for exempie, hydrogen, (31-7 eikyi group, a (33.30 heteroeyoiyi group, or e £35,303ryi group, oreferebiy en or a CH alkyi group. Exemoies of ester grouper inoiude, but are hot iimiteo to, =NH, =NMe, =NEt, and :NPh. i idehyde, oerboxeidehyde): -C(=O)H, Aeyi (hero): ~C(=Q)R, wherein R is en ecyi substituent, for exampie, a Cm eikyi group (aieo referred to as C1,; eikyiaoyi or CH aikenoyi), a (33-30 heterooyoiyi group (eiso referred to as (33.30 heterooyoiyiaoyi), or a (35.30 eryi group (aiso ed to as (35.33 eryieoyi), ehiy a C1,? eikyi group. Exampies of aoyi grouoe ineiude, out are hot Eimiteo to, —C(=O)CH3 (aoetyi), “C(IQ)CH3CH3 (propionyi), “C(EQ)C(CH3)3 (Ewbutyryi), and —C(:O)Ph (benzoyi, ohenone).
Carhoxy xyiio acid): ~C<=O)QH.
Thiooarboxy (thiooerboxyiio acid): —C(=S)SH,, Thioiooarboxy (thioiooarhoxyiio eoio): ~Ci=0§SH.
Thionooarboxy (ihionooarboxyiio acid): OH= imioio acid: —C(=NH)OH.
Hydroxarnio acid: —C(:NOH)OH.
Eeter (eerhoxyiate, oarboxyiie acid ester, oxyoarbonyi): —C(=O)QR, wherein R is; an ester eobstituent, for exampte, a C1; aikyi group, a (33-20 heterooyoiyt group, or a (35,2oaryi group, preferebiy a CH eikyi group: Exampies ot ester groups inoiude, but are not iimited to, ~Ci=O)QCH3, “C(=Q)OCH2CH3, —C(=O)QC(CH3)3, and -or=o>oen.
Aoyioxy (reverse ester): ~OC(=O)R, n R is an aoyiexy substituent, for exampte, a (31-7 aikyi group, a (33-20 heterooyoiyi group, or e C5_ggeryi group, pre‘iarapiy e C1_7atkyt group, Exampios of aoyioxy groups e, but are not iirnited to, *QC(=O)CH3 (aoetoxy), —OC(=O)CH;CH3, —OC(=Q)C(CH3)3, ~OC(=O)Ph, and —OC(=Q)CH2Ph.
Oxyoerpoyioxy: )OR, wherein R is an ester substituent, for exampie, a Cm aikyi group, a (33-20 heterooyoiyi group, or a (35-20am group, preferebiy a (31;; etkyt group, Exampies of ester groups inoiude, but are not iimiteo to, -QC(=Q)QCH3, ~OC(=O)QCH20H3, )QC(CH3)3, and —QC(=O)C§Ph.
Amino: —NR1R2, wherein R1 and R2 are independentiy amino substiiuente, for exempia, hydrogen, a (31.7 aikyi group (atop referred to as Cw aikyiamino or cit—(31.7 aikyiemino), a 03.20 heterooyotyi group, or a (35-20 aryi group, preterabiy H or a €31,7eikyi group, or, in the page of e “oyoiio” amino group, R1 and R2, taken together with the nitrogen atom to whioh they are attached, form a heterooyotio ring having from 4 to 8 ring atoms, Amino groups may be primary (“Ni-i2), secondary (*NHRfi'), or tertiary (wNHWRQ‘, and in oatiohio form, may he nary (n+NR1R2R3). ee of amino groups inoiurte, but are not iimiteo to, ~NH2, “NHCHs, —NHC(CH3)3, “N(CH3)2, —N(CHZCH3)2, and —i\iHPh. Examptes of oyoiio amino groups inotuoe, but are not timited to, aziridino, azetidino, pyrroiidino, piperidino, zino, morphoiino, and thiornorphoiino.
Arnido (oerbamoyi, oarhemyi, eminooarhonyi, oarhoxar‘nide): ~C(=Q)NR1 R2, wherein R1 and R2 are independeniiy amino substituents, as defined for amino groups. Exampies of emido groups inoiude, but are not iimiied to, ~Ci=Q)Ni-i2, —C(=O)NHCH3, —C(=O)N(CH3)2, —C(=O)NHCH2CH3, and "C(=O)N(CH2CH3)2, as weii as amido groups in which R1 and R2, iogeiher wiih the nitrogen atom to which they are ariaohed, form a heterooyoiio iure as in, for exempie, piperidinocarbonyi, morphoiinooarbonyi, thiomorphoiinoeeroonyi, and piperazinooerbonyi.
Thioarnido aroemyi): ~C(=S)NR1R2, wherein R1 and R2 are independeniiy amino substituents, as d for amino groups. Exempies oi amide groups iheiude, out are not iimited to, NH2, -C(=S)NHCH3. ~C(=S)N(CH3)Z, end NHCH2CH3. ido (eoyiemino): —NR1C(:O)R2, wherein R1 is: an amide substituent, for exarnpie, hydrogen, a CH eikyi group, e 03.20 heterooyoiyi group, or a C5.ggeryi group, oreferabiy hydrogen or e C1..‘,raikyi group, and R2 is an aoyi substiiuent, for exampie, a C31_-;aikyi group, a (33-20 heierooyeiyi group. or a C5_ggeryi group, preferebiy hydrogen or a (31-7 eikyi group.
Exempiee of aoyiamide groups: inoiude, but are not iimi‘ied to, ~NHC{=0)CH3 , ~NHC(=Q)CHECH3, end O)Ph. R1 and R3 may together form a cyoiio structure, as in, for exampie, succinimidyi, maieimidyi, and phiheiimidyi: . NVC} OfiNvfl 0%NVO / \ . .-_-_-_-_/ suczcinimidyi idyi phthaiimidyi Arninooerbonyioxy: —OC(=G)NR1R2, wherein R1 and R2 are independentiy amino subsiituenis, as defined for amino groups. Examoies of eminooarbonyioxy groups inoiude, but are not iimiied to, —QC(=O)NH2, )NHMe, —QC(=O)Nivie2, and —OC(=O)NEt2.
Ureido: «Ni'RUCONRQW wherein R2 and R3 are nden‘iiy amino substiiuente, as defined for amino groups, and R1 is a ureido subsii‘iueni, for exernpie, hydrogen, a €1-7eikyi group, a (33-20 heterooyoiyi group, or a 05.20eryi group, preferabiy hydrogen or a €1.7eikyi group. Exampies of ureido groups ineiude, but are not iimiied to, INHCONHE, “NHCONHMe, —NHCONHEL NHCONMez, —NHCONEt2, ~NMeCONH2, ~NMeCONHi‘x/ie, ~NMeCONHE‘i, — NiVieCONMeg, and «NMeCONEtz, WO 59981 Guanidino: —NH—C(=NH)NH2, Tetrazoiyiz a five membered aromatic ring having four nitregen atome and one earben atom, \ H Emino: =NR, wherein R is an imino substituent, fer exampie, for exampie, hydrogen, a {31.7 aikyi group, a (33-20 eyciyi group, or a C5_goaryi group, preferabiy H or a (31-7aikyi group.
Exampiae of Emino groups inciude, but are not iimited to, =NH, =NMe, and =NE1‘, Amidine (amidino): —C(=NR)NR2, wherein each R ie an amidine eubetituent, for exampie, en, a CH aikyi group, a (33-20 heterocyciyi group, or a {35-2oaryi group, preferaeiy H or a C1,»,aikyi group, Exampies of amidina groups inciude, but are not iimited to, —C(=NH)NH2, ~C<=NH>NM32, and ~C(=NMe)NMe2.
Nitro: “N62. o: NO.
Azido: —N3.
Cyano (nitriia, carbouitriia): ~CN.
Esocyano: NC.
Cyanato: ~QCN.
Esoeyanetoz "NCO. ano (thiocyanato): ~SCN, Eeothiocyano (Esothioeyaueto): —NCS.
Suifhydryi (thioi, mercapto): ~SH, her (euifide): «SR, wherein R is e thioeiher euheiitueni, for exampie, a (31-7 eikyi group (aieo referred to as e Ci..-,reikyithio group), 3 (33,20 heterooyciyi group, or e 05.20eryi group, preferehiy a C1; aikyi group. Exempiee of €31-73ikyithio groups inoiude, but are not iimiied to, —SCH3 and “SCHQCH3, Dieuifioe: eSS—R, wherein R is a disuifide substituont, for exempie, e kyi group, a 03,20 heterooyeiyi group, or 3 (25,20 aryi group, preferebiy a CH eikyi group (3530 referred to herein as {31.7 eikyi disuifioe). Exempies of (31-7 eikyi disuifide groups inoiude, out are not iimi’reo to, «SSCHE and —SSCH2CH3, Suifihe (euifihyi, suifoxide): ~S(=O)R, wherein R is a euifihe euheiiiueni, for example, a CH eikyi group, 3 {33,20 heterooyoiyi group, or e (3510 eryi group, epiy e (31-7aikyi group, Exampies of euifiue groups ihoiude, hui are not iimiied to, CH3 and ~S(=O)CH2€H3, Suifone (euifonyi): —S(=O)2R, n R is e euiforie eupeiiiueni, for e, a C,_,-,reii<yi group, a (33-20 heterooyoiyi group, or a {35-2Oaryi group, prefereoiy e {31,7eikyi group, ihciudihg, for e, a fiuorineied or perfiuorineied CW aikyi group. ee of suifooe groups inoiuue, but are pot iimiteei to, ~S(=Q)ZCH3 (metheneeuironyi, meeyi), -S(=Q)ZCF3 (trifiyi), —S(=O)2CIH2CH3 (esyi), —S(=O)ZC4F9 (nooafiyi), *S(=Q)2CH20F3 i), —S(=0)ZCHQCH2NH2 (teuryi), —S(=O)2Ph (phenyieuifonyi, peeyi), 4—meihyiphenyisuifouyi (ioeyi), zit—ohiorophehyisuiforiyi (oioeyi), 4—bromopheuyisuifonyi (brosyi), rophehyi (nosyi), 2—haphthaieneeui'ioneie (napsyi), and 5~dimethyiamino—nephthaiewi~yisuifona‘ie (daneyi).
Suifiriio acid (euifiuo): —S(=O)QH, —SOgi-i.
Suifonio acid (suifo): —S(=Q)QQH, ~803H, Suifihaie (euifinio acid ester): —S(=Q)OR; n R is a euifinate supetiiueot, for exempie, a (31-7 eikyi group, a (33-20 heterooyoiyi group, or a {35.20 aryi group, prefereoiy a {31-7 eikyi group.
Exampies of euifiriaie groups iooiude, but are not iimiied to, ~S(=O)OCH3 (meihoxyeui‘iényi; methyi suifihaio) and —S(=O)OCHZCH3 (ethoxysuifioyi; ethyi suifineio).
Suifonete (suifouio acid ester): —S(=O)2C)R, n R is e suifonere substitueni, for exampie, a (31-7 aikyi group, a (33-20 heierooyoiyi group, or 3 (35,20 aryi group, ebiy e Ci-»,raikyi group. Exampies of euifouete groups ippiucie, but are hot iimited to, —S(=C>)20CH3 xyeuifopyi; meihyi suifooeie) and ~S(=O)2OCH2CH3 (ethoxyeuifonyi; eihyi euifooeie).
Suifihyioxy: ~QS(=O)R, wherein R is e suifioyioxy sups’riiueni, for exempio, a Cr.,-,reii<yi group, a (33-20 heierooyoiyi group, or e €35-20eryi group, preferepiy e (31-7eikyi group. Exempies of ioxy groups irieiucie, but are not iimited to, mOS(:O)CH3 arid ~OS(:Q)CH2CH3.
Suifonyioxy: ~OS(=O)2R, wherein R is a suifonyioxy‘ substitueot, for exampie, e (31;; aiky‘i group, a (33-20 heieroeyoiyi group, or e (35-2Oaryi group, erebi}i e (31,; eikyi group.
Exempies of suifouyioxy groups inoiude, but are not iimi’red to, —OS(:O)2CH3 (mesyieie) and «OS(=Q)ZCHECH3 te).
Suifeie: —OS(=O)20R; wherein R is e suifeie supsiiiueui, for exampie, e Crszaikyi group, a (33-29 heterooyciyi group, or e geryi group, preferebiy a (31,7eikyi group. Exempies of suifeie groups ineiude, pui are not iirniied to, —QSr':O);_QCH3 and ~SQ(=G)2OCH2CH3, yi (suifemoyi; suiiinio eoid amide; suiiinemide): —S(=O)NR"R2, wherein R" and R2 ere udeniiy amino substitueuis, as d for amino groups. Exempies of suifemyi groups ineiude, but are not iimited io, —S(=O)NH3, —S(=G)NH(CH3), mS(=O)N(Ci-i3)2, —S(=O)NH(CHZCH3), N(CHZCH3)Z, and ~Si=O)NHPh.
Suifonemido (suiiirremoyi; suiionie acid amide; suifenamide): —S(=O)ZNR1R2, wherein R1 and R2 are ndeniiy amino subsiiiuenis, as defined for amino groups. Exempies of suiforiemido groups ineiude, but are not iirnited to, —S(=O)2NH2, —S(=G)ZNH(CH3), ~S(=O)2N(CH3)2, ~S(=O)2NH(CH2€H3), 2N(CH20H3)2, end —S(:Q)2NHPh.
Suifemino: —NR18(:O)2C>H, wherein R1 is an amino subsiiiueui, as defined for amino groups.
Exempies of sui‘famino groups inoiude, but are not iimiied io, «NHSFOEQH ehd —N(CH3)S(=O)ZQH. emirio: —NR18(:O)3R, wherein R1 is an amino substituehi, as defined for amino groups, and R is e iemino subsiitueni, for exampie, a (31-7 aikyi group, a (33-20 heterooyeiyi group, or 3 (35,20 aryi group, preferebiy 3 CW aikyi group. Exampies of suiforiemino groups include, but are not d to, ~NHS§=Q)2CH3 arid —N(CH;3)S(=O)2C6H5.
Suifinar‘nino: “NR1S(=O)R, wherein R" is an amino sups‘iitueri‘i, as defined for amino groups, and R is e suifinemino substitueot, for oxampie, a (31;; aikyi group, a (33.20 heisroeyoiyi group, 2014/025564 or e Cwoaryi group, preferabiy a praikyi group, Examoies of suifinamino groups ihoiude, but are not Eimited to, ~NHS§=O)CH3 ahd —N(CH3)S(=O)CgH5.
Phosphino hine): 43%, wherein R is a phosphino substituent, for exampie, —H, a (31.7 aikyi group, a (33-20 heterooyoiyi group, or a (Eugen/i group, prefereoiy ~H, a (31-7 aikyi group, or a C5.goaryi group. Exampies of phosphiho groups inoiude, but are not Eimited to, “PE-£2, -P(CH3)2, -P(CH2CH3 2, —P(t—E3u)2, and —P(Ph)2. o: 'P<:O)g.
Phosphioyi (phosphine oxide): —P(=O)R2, wherein R is a phosphiuyi Substitueht, for exampie, a C1; aikyi group, a (33-20 heterooyoiyi group, or a (35,20 eryi group, apiy a (31-7eikyi group or e C5_zgeryi group. Exempieo of phosphinyi groups o, but are hot iimitod to, ~P(=Q)(CH3)Z, “PFQXCHZCHQZ 3P(=O)(t—Bu)2, and *P(=Q)<Ph)2.
Phosphonio acid (phosphono): ~P(:O)(QH)2. ouete (phosphono ester): —P(=O)(QR)2, Where R is a phosphonate substitueut, for exampie, —H, a (31.3; aikyi group, a (33.20 heterooyoiyi group, or a C5..2oaryi group, preforaoiy -H, a (31-7 alkyi group, or 3 C320 aryi group. es of phoophonato groups Enoiude, but are not Eimited to, —P(=Q)(OCH3)Z, “P(:Q)(OCH2CH3)Q, ~P(:O)(Ont~8u)2, and (0Ph)3.
Phosphoric eoioi (phosphouooxy): )(OH)2.
Phosphate (phoephonooxy ester): —OP(:O)(QR)3, where R is a phosphate substituent, for exampie, ~H, a 01-;7a3kyi group, a (33-30 heterooyoiyi group, or e Cwoaryi group, preferabiy «H, a CH aikyi group, or e (35.20 aryE group, Exampios of phosphate groups e, but are not iimited to, —OP(=Q)(OCH3)2, )(OCH2€H3)3 ~QP(=O)(O—t—Bu)2, and ~OP(=O)(OPh)2.
Phosphorous acid: «SHOW; Phosphite: —OP(OR)2, Where R is a phopphite substitueot, for exempie, —H, e (31.; alkyi group, a C330 heteropyoiyi group, or a (35-20ary5 group, preferebiy "H, a (31-7aikyi group, or a (35-20 aryi group. Exampios of phosphire groups inoiudo, but are hot iimitod to, —OP{QCH3)3, ~0P<QCH20H3)Z, fiflO—tfiup, and ~OP(QPh)2.
Phosphoremidite: —OP(GR1)~NR22, where R1 and R2 are phosphoramidite substituents, tor exampie, —H, a (optionaiiy tuted) (31;; aiityi group, a (33.20 oy‘oiyi group, or a 05.20 aryi group, preterahiy —i-i, a (31-7aiityi group, or a (15,20 aryi group. Exampios of phosphoramidite groups inotude, but are not iirnited to, ~OP(QCH2CH3)IN(CH3)3, —OP(OCH2CH3)—N(i~Pr)g, and HQCHZCN)~N(i—Pr)z, Phosphoramidate:~QP(=O}(OR1)—NR22, where at and R2 are phosphoramidate suhstituehts, for exampie, —H, a (optionaiiy substituted) {31-7 aikyi group, a {33.20 heterooyoiyi group, or a €35-20 aryi group, preferahiy —H, a Cu aikyi group, or a (35-20 aryi group. es oi phosphoran‘iidate groups inoiude, but are not iimited to, s0P(=Q)(OCHZCH3)—N(CH3)2, —OP(=O)(QCHQCH3)—N(i—Pr)g, and —QP($O)(QCH20H2CN)—N(Li—Pr); Aikyiene C342 aikyiene: The term 2 aikyiene”, as used herein, pertains to a pidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different oarhon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (uniess otherwise specified), which may be aiiphatio or aiicyoiio, and which may he saturated, iiy unsaturated, or tuiiy unsaturated. Thus, the term ene” inoiudes the subsoiasses aikenyiene, aikynyiene, cyoioaikyiene, etc., discussed heiow.
Exampies of iinear saturated (33-13aikyiene groups inoiude, out are not iintited to, {CH2}; where n is an integer from 3 to 12, for exampie, ~CHgCHgCH2~ (propyiene), ~CH2CHZCH20H2— (butytene), —CH2CH20H2CHQCH2— (pentyiene) and —CH20H2CHZCH— 2€H2~ (heptyiene).
Exampies of branched saturated €33-1geikyierte groups inciude, but are not iirnited to, —CH(CH3)CH2-, -CH(CHg)CHgCHg-, -CH(CH3)CH20HQCH2—, —CHZCH(CH3)CH2—, —CHZCH(CH3)CH2CZH2—, —CH(CH;CH3)—, —CH(CH28H3)CH2—, and —CH20H(CH2CH3)CH2—.
Exampies of iineer iiy rated (33-12 aikyiene groups ((33.12 aikenyiene, and iene groups) inoiude, but are not iirnited to, ~CH=CH*CH2~, "CH2"CH=CH2", ~CH=CH—CHg-CH2—, —CH=CH-CH2—CH2—CH2—, —CH=CH—CH=CH—, —CH2CH—CH=CH~CH2—, — CH=CH~CH2CH€H2~CH22 —CH:CH~CHg—CH:CH~, “CHZCHfiHEICHEnCHECHI, and “CH2— CEC~CH2~, 2014/025564 Exanipias of branched partiaiiy unsaturated (33-12 aikyiene groups ((33.1; aikenyiane and aikyny‘iene groups) e, out are not iimited to, —C(CH3)=CH—, —C(CH3)=CH-CH2—, —CH=CH—CH(CH3)— and —CEC—CH(CH3)-.
Examoies of aiioyciic saturated (33-12 aikyiene grouos (C342 cycioaikyiehes) inoiude, but are not Eimited to, cyciooentyiene (eg. cyoiooent—t,3-yiene), and exyiene (eg. cyciohex—tfi-yiene).
Exanipias of aiicyciic partiaiiy unsaturated szaikyiene groups ((33-12 cyoioaikyianes) inciude, but are not iirnited to, oyciopentenyiene (so. 4~oyciopenten—t ,3—yiens), cyciohexenyiene (eg. 2—cyciohexen-t,4~ytene; ohexen—t ,2—yiene; 2,5—cyciohexadien— 1,4—yiene). inciudes (Ether Forms Uniess otherwise specified, inoiuded in the above are the weii known ionic, sait, sotvate, and protected forms of these substituents. For exampie, a reference to carhoxyiic acid (—COQH) aiso es the anionic (carboxyiate) term («(300), a sait or soivate thereof, as weii as conventionai protected forms. Simiiariy, a reference to an amino group inciudes the ated form (—N+HR1R2), a sait or soivate of the amino group, for exampie, a hioride sait, as wait as conventionai protected forms of an amino group. Simiiariy, a reference to a hydroxyi group aiso inciudes the anionic term (—0"), a sait or soivate thereof, as wait as conventionai protected forms, Saits it may he convenient or desirabie to prepare, purity, and/or handie a corresponding sait ot the active compound, for exampie, a oharmaoautioaiiy~acceotabie sait. Exampies ot oharmaoeuticaiiy acceptahie saits are sed in Barge, start, J. Pharm. Sci, 6%, 1—19 (1977).
For exanipia, it the compound is anionic, or has a functionai group which may he anionic (eg. ~CQOH may be 4390‘), then a sait may he formed with a suitahie . Examoies of suitahie inorganic cations inciude, but are not iimited to, aikaii metai ions such as Na+ and K1 ne earth s such as Ca” and Mg”, and other cations such as Aim, Examoies o'i suitabie organic cations inciude, but are not iimited to, ammonium ion (La, NHJ) and substituted ammonium ions (so. NH3R+, NHQRJ, NHRJ, NRC). Exampies of some suitahie substituted ammonium ions are those derived from: ethyiamihe, diethyiamine, dicyciohexyiamine, trieihyiamihe, hutyiamine, ethyienediamine, ethanoiamine, noiamine, piperazine, henzyiamine, ohenyibenzyiamine, e, megiumine, and tromethamine, ae wait as, amino acids, such as, iyeihe and arginine. Ah exampie of a common quaternary ammonium ion is N(Cii3),f. it the compound is cationic, or has a tuncticnai group which may he cationic (eg. —t\iH2 ma},i he Mir-if), then a eait may he formed with a eoitahie anioh. Exampies of euitahie inorganic anions inciude, but are not iimited to, those derived from the ing inorganic acids: hydroohioric, hydrobromic, hydroiodic, sui‘iurio, onitnrous, nitric, nitrous, phosphoric, and phosphorous.
Examoies of suitahie organic anions inciude, but are not iimited to, those derived from the ‘ioiiowing organic acids: 2—aceiyoxybenzoic, acetic, ic, ic, benzoic, camphorsoiionic, ic, citric, edetic, ethanedietiitcnic, ethanesuitonic, fomaric, giucheptonic, giuconic, giutamic, giycoiic, ymaieic, hydroxyhachtheiene carhoxyiic, ieethionic, iactic, iactobionic, iauric, , maiic, methanesuifonic, mucic, oieic, oxaiic, paimitic, pamoic, pantoihenic, ohenyiacetic, euifonic, propionic, pyruvic, eaiicyiic, stearic, succinic, iiic, tartaric, toiueneeuitohic, tritiuoroacetic acid and \Jateric.
Exampiee of euitahie potymeric organic anions inctude, but are not iimited to, those derived from the toiiowing poiymenc acids: tannic acid, carhcxymethyi ceiinioee.
Soivates it may be convenient or deeirahie to prepare, purify, andfor handie a corresponding eoivate of the active compound. The term “ccivate” ie used herein in the tionai sense to refer to a compiex of eoiute (eg. active compound, sait of active compound) and scivent, it the soiveni is water, the eoivaie may be convenientiy referred to ac a hydrate, for exampie, a monoehydrate, a di—hydrate, a tri~hydrate, etc.
The invention inciudes compounds where a eoivent adds across the imine bond of the FED moiety, which is iiiuetrated heiow where the Soiveni iS \. meter or an i (RAOH, where RA is CM aittyi): 9 1) R R‘ A o H OR x N N V \ H X H HzC Y RAOH V R7 N R2a N 23 7 2a R R N R 6 6 R O R R2b R2b O These forms can he caiied the carhinoiamihe and carbinoiamine ether forms of the PBD (as described in the section reiating to R10 above). The haiance of these equiiihria depend on the ions in which the compounds are found, as wait as the nature of the moiety itseif.
These particuiar compounds may he isoiatett in soiid form, for exampie, by iyophiiisationn isomers Certain compounds of the invention may exist in one or more particuiar geometric, cpticai, enahtiomeric, diasteriomerio epimeric, atropic, stereoisomeric, tautomeric, conformationai, or anomerio forms, ing but not iimited to, cis- and trans—forms; E and waorms; c-, t, and r— forms; endo— and exo—torms; R: 3—, and enns; D— and L—iorms; o— and s; (+) and (n) terms; ketow, enoiw, and enoiatewtorms; syn“ and anti—forms; synciinai— and inai~torrns; o— and fi—forrns; axiai and equatoriai forms; boata chain, tiririst~a enveiope—, and haifchairsforms; and combinations thereof, hereinafter coiiectiveiy referred to as “isomers” (or “isomeric forms”).
The term “chirai” refers to moiecuies which have the property of non~snperimposabiiity of the mirror image partner, white the term “aohirai” refers to meteorites which are superimposahie on their mirror image partner.
The term “stereoisomers” refers to compounds which have identicai chemicai constitution, but differ with regard to the arrangement of the atoms or groups in space“ “Diastereemer” refers to a stereoisomer with two or more centers of chiraiity and whose moiecuies are not mirror images of one another. Diastereemers have different ai properties; eg. meiting points, hoiiing points, spectrai properties, and reactivities: Mixtures of diastereomers may separate under high tion ticai prooedures such as eiectrophoresis and tography.
“Enantiorners” refer to two stereoisomers of a compound which are nonvsuperimposabie mirror images of one another.
Stereochemicai definitions and tions used herein generaiiy foiicw S. P. Parker, Est, McGraw—Hiii Dictionary of Chemise! Terms (1984) McGraw~Hiii Book Company New York; and Eiiei, E. and Wiien, 3., “Stereochemistry of Qrganic Compounds”, John Wiiey & Sons, inc., New York, 1994. The compounds of the invention may contain asymmetric or chirai centers, and therefore exist in different isomeric forms, it is intended that aii stereoisomeric forms of the compounds of the invention, inctuding out not timited to, diastereomers, enantiomers and atropisomers, as weii as mixtures thereof such as recemic mixtures, form part of the present invention. Many organic compounds exist in opticaiiy active terms, i.e., they have the y to rotate the piane o‘t piane~poiarized tight. in describing an opticaiiy active compound, the prefixes D and L, or R and S, are used to denote the absoiute configuration of the moiecute about its chirei centerts). The prefixes d and i or (+) and (—) are empioyed to designate the sign of rotation of pianewpoiarized tight by the compound, with (a) or i g that the compound is ievorotatory, A compound prefixed with (+33 or d is dextrorotatory. For a given chemicai structure, these stereoisomers are identicai except that they are mirror images of one another. A specific stereoisomer may atso he referred to as an enantiomer, and a mixture of such isomers is often caiied an enantiomerio e. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoseiection or stereospeciticity in a chemicai reaction or process. The terms “racemic e” and “racemate” refer to an eouimoiar mixture of two enantiomeric species, devoid of opticai activity.
Note that, except as discussed heiow tor tautomeric forms, icaiiy exciuded from the term “isomers”, as used herein, are structurei (or tutionai) isomers (i.e. isomers which differ in the connections between atoms rather than mereiy by the position of atoms in space). For exampie, a reference to a methoxy group, “QCH-a, is not to be construed as a reference to its structurai isomer, a ymethyi group, CHZOH, Simiiarty, a nce to ortho~chiorophenyi is not to be construed as a reference to its structure! isomer, mete— chiorophenyt. However, a reference to a ciass of structures may weii inciude structuraiiy isomeric forms tatiing within that ctass (eg. (31-7 aikyi inoiudes n—propyt and ooyt; butyi inciudes n~, isom, sec“, and utyi; methoxyphenyi inoiudes ortho~, meta—, and para“ methoxyphenyi).
The ahove exciusion does not pertain to tautomerio forms, for exampie, keto—, enoin, and enoiate—torms, as in, for exampie, the toitowing tautomeric pairs: ketofenoi (iitustrated heiow), imine/‘enamine, amide/imino aicohoi, e/amidine, nitrosoioxime, thioketone/enethioi, N—nitroso/hyroxyazo, and nitroieci—nitro. ——c—c”i O \\ [OH H O a; pro ms— czc’ \ 1’ \ it; / \ keto enoi enoiate The term “tautomer” or “tautomeric form” refers to structurai isomers of different energies which are interconvertibie via a iovv energy barrier. For exampie, proton tautomers (atso known as prototropic tautomers) inciude interconversions via ion of a proton, such as noi and imine—enamine isomerizations. Vaience tautomers inciude interoonversions by nization of some of the g eiectrons.
Note that specificaiiy inciuded in the term “isomer” are compounds with one or more isotopic substitutions, For exampie, H may be in any isotopic form, inciuding 1H, 2H (D), and 3H (T); C may be in any isotopic form, inciuding 12C, 13C, and 14C; 0 may be in any ic form, inciuding 150 and 18C}; and the iike.
Exampies of isotopes that can be orated into compounds of the invention inciude es of en, carbon, nitrogen, oxygen, phosphorous, fiuorine, and ohiorine, such as, but not iimited to 2H (deuterium, D), 3H (tritium), 11C, ”(3, MC, 15M, 13F, ”P, 32f), 358, 356i, and 125i. Various isotopicaiiy iabeied compounds of the present invention, for exampie those into which radioactive isotopes such as 3H, tSC, and MC are incorporated. Such isotopicaiiy iabeiied compounds may be usefui in metaboiic studies, reaction itinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or singie- photon emission computed tomography (SPECT) inciuding drug or substrate tissue distribution assays, or in radioactive treatment of patients. Deuterium iabeiied or substituted therapeutic compounds of“ the invention may have improved Di‘v’iPK (drug metaboiism and pharmacokinetios) ties, reiating to distribution, iism, and excretion (ADME).
Substitution with heavier isotopes such as ium may afford certain therapeutic advantages ing from greater metaboiic stabiiity, tor exampie increased in vivc haif—iife or reduced dosage requirements. An 18F iabeied compound may be usefui for PET or SPECT studies. isotopicaiiy iabeied compounds of this invention and prodrugs thereof can generaiiy be prepared by carrying out the procedures disciosed in the schemes or in the exampies and preparations described beiovv by substituting a y avaiiahie isotopicaiiy iaheied reagent for a non—isotopicaiiy iabeied reagent. Further, tution with heavier isotopes, uiariy deuterium (i.e., 2H or B) may afford certain therapeutic advantages resuiting from greater metaboiic stabiiity, for e increased in vivo hait—tite or reduced dosage requirements or an improvement in therapeutic index. it is understood that deuterium in this context is ed as a substituent. The concentration of such a heavier isotope, specificaiiy deuterium, may be defined by an isotopic enrichment factor. in the compounds of 2014/025564 this invention any atom not specificaiiy designated as a particuiar isotope is meant to represent any stahie isotope of that atom.
Uniess otherwise specified, a reference to a particuiar compound inoiudes aii such isomeric forms, inciuding (whoiiy or partiaiiy) racemic and other mixtures thereof. s for the preparation (eg. tric synthesis) and tion (eg. fractionai crystaiiisation and chromatographic means) of such isomeric forms are either known in the art or are readiiy obtained by adapting the methods taught herein, or known methods, in a known manner, Bioiogicai Activity in vitro ceii groiiteration assays Generaiiy, the cytotoxic or atic activity of an antibody~drug conjugate (ADC) is measured by: exposing mammaiian ceiis haying receptor proteins, eg. HERZ, to the antibody of the ADC in a ceii cuiture medium; cuituring the ceiis for a period from about 6 hours to about 5 days; and measuring ceii Viahiiity. Ceii—hased in vitro assays are used to measure yiahiiity (proiiferation), cytotoxicity, and induction of apoptosis (caspase activation) of an ABC of the ion.
The in vitro potency of antibody-drug conjugates can be measured by a ceii proiiteration assay. The tenGto® Luminescent Ceii Viabitity Assay is a commerciaiiy avaiiapie (Promega Coro, Madison, Wt), homogeneous assay method based on the recombinant expression of Coieoptera rase (US Patent Nos, 5583024; 5674713 and 5700870), This ceii proiiferation assay determines the number of viabie ceiis in cuiture based on duantitation of the ATP present, an indicator of metaboiicaiiy active ceiis h et a! (1993) J. immune}.
Moth. 169:8168: US 6602877). The CeiiTiter—Gio® Assay is conducted in 96 weii format, making it amenahie to automated high~throughput screening (HTS) (Cree et a! {19%) AntiCanoer Qrugs seesaw), The homogeneous assay procedure invoives adding the singie t (Getfitter—(Gin:® Reagent) directiy to ceiis cuitured in serum—suppiemented medium. Ceii washing, remoyai of medium and muitipie pioetting steps are not required. The system detects as few as to oeiisfweii in a ii format in to minutes after adding reagent and mixing. The ceiis may be treated continuousiy with ABC, or they may he treated and separated from ADC. iiy, ceiis treated hriefiy, i.e. 3 hours, showed the same potency effects as continuousiy treated ceiis.
The homogeneous “add—mix-measure” format s in ceii iysis and generation of a iuminescent signai proportionai to the amount of ATP present. The amount of ATP is directiy proportionat to the number of ceiis present in cuiture. The t3eti"i"iter~Gio® Assay generates a “giow-type” iuminesceht signai, produced by the iuciterase reaction, which has a hait-iite genereity greater than five hours, depending on ceii type and medium used. Viehte cetis are reflected in ve iuminescence units (RLU). The substrate, Beetie Luciferin, is oxidativety decarhoxyiated by recombinant tiretiy tuciterase with concomitant conversion of ATP to AMP and generation of photons. in vivo efficacy The in Vii/O efficacy of antibody—drug conjugates (ADC) ot the invention can be measured by tumor xencgraft studies in mice. For exempts, the in vivo efficacy of an antisHERZ ADC of the invention can be measured by a high expressing HERE transgenic t mouse modet.
An aft is ated from the F05 mmtv transgenic mouse which does not d to, or ds pooriy to, HERCEPTiNe‘) therapy, Subjects were treated once with ABC at n dose teveis (mg/kg) and PET.) drug exposure (pg/mg); and ptacebc butter controt (Vehicie) and monitored over two weeks or more to measure the time to tumor doubting, iog ceii kiii, and tumor shrinkage.
The conjugates of the invention may he used to provide a PET) conjugate at a target tocation.
The target on is preferabiy a protiterative ceti poputation. The antibody is an antibody for an antigen present in a proiiteratiye ceit pcouiation. in one embodiment the antigen is absent or present at a reduced tevei in a non~proiiterative ceti popuiation compared to the amount of antigen present in the protiterative cett tion, tor exampie a tumour ceit pcpuiation, The target iocation may be in vitro, in Vii/O or ex vivo.
The antibody—drug conjugate (ADC) compounds of the invention inciude those with y for anticancer activity. in particuiar, the compounds inciude an antibody conjugated, Le. covaientiy attached by a , to a FEED moiety.
At the target iocaticn the tinker may not be cieaved. The antibody~drug conjugate (ADC compounds of the invention may have a cytotoxic ettect without the cieavage of the tinker to reiease a PBD drug moiety, The antibody—drug conjugates (ADC) ot the invention seiectiveiy deiiver cytotoxic agent to tumor tissue whereby greater seiectiyity, i.e. a tower efficacious dose, may be achieved.
Thus, in one aspect, the present invention provides a conjugate nd as described herein for use in therapy. in a further aspect there is aisc provides a conjugate compound as described herein for use in the treatment of a terative disease, A second aspect of the present invention provides the use of a conjugate compound in the manufacture of a medicament tor treating a erative disease.
Qne ct ordinary skiii in the art is readiiy abie to determine whether or not a candidate conjugate treats a proiiferative condition for any particuiar ceii type, For exampie, assays which may convenientiy he used to assess the activity offered by a particuiar compound are described in the es heiow.
The term “proiiterative disease” ns to an unwanted or uncontroiied ceiiuiar proiiteration ot excessive or ai ceiis which is undesired, such as, neoptastic or hyperpiastic growth, whether in vitro or in viva.
Exampies of proiiterative conditions inciude, but are not iirnited to, benign, pre—rnaiignant, and ant ceiiuiar proiiteration, ing but not iirnited to, sms and tumours (eg. histocytorna, giicrna, astrocyoma, csteorna), cancers (eg. iung cancer, smaii ceii iung cancer, gastrointestinai cancer, howei cancer, ccion cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicuiar cancer, iiver cancer, kidney cancer, hiadder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi’s sarcoma, rneianoma), ieukemias, psoriasis, bone diseases, tihroproiiierative disorders (eg. of connective tissues), and atheroscierosis. Cancers of particuiar interest inciude, but are not iimited to, ias and ovarian cancers.
Any type of ceii may be treated, incinding but not ed to, iung, gastrointestinai ding, eg. howei, , breast (mammary), ovarian, prostate, iiver (hepatic), kidney (renai), biadder, pancreas, brain, and skin, in one embodiment, the treatment is of a pancreatic cancer. in one embodiment, the treatment is of a tumour having ovfie integrin on the surface of the ceii. it is contempiated that the antibodyndrug conjugates (ABC) of the present invention may he used to treat various diseases or disorders, eg, characterized by the overexpression of a tumor antigen, Exempiary conditions or hyperoroiiterative disorders e benign or maiignant tumors; ieukemia, haematoiogicai, and iymohcid maiignancies. Others inciude neuronai, giiai, astrocytai, hypothaiamic, gianduiar, macroohagai, epitheiiai, stromai, hiastocoeiic, intiar‘nmatcry, angicgenic and immunoiogic, inciuding autoimmune, disorders, Generaiiy, the e or er to he treated is a hycerproiiterative disease such as cancer. Examoies of cancer to he treated herein inciude, but are not iimited to, carcinoma, ma, hiastoma, sarcoma, and ieukemia or iymohoid maiignancies. More particuiar exampies of such cancers inciude squamous ceii cancer (eg. eoitheiiai squamous ceii cancer), tung cancer inciuding smaii~ceit iung cancer, non—smaii ceti iung cancer, adenocarcinoma of the iung and squamous carcinoma of the iung, cancer of the peritoneum, hepatoceiiuiar cancer, gastric or stomach cancer inciuding gastrointestinai cancer, pancreatic cancer, giiohiastcma, cervicat cancer, ovarian cancer, tiver cancer, hiadder cancer, hepatoma, breast cancer, coion cancer, rectai cancer, coiorectai cancer, endometriai or uterine oma, saiivary giand carcinoma, kidney or renai cancer, prostate cancer, vuivai cancer, thyroid cancer, hepatic carcinoma, anai carcinoma, oeniie carcinoma, as wait as head and neck cancer, Autoimmune diseases for which the ADC nds may he used in treatment inciude toiogic disorders (such as, for e, rheumatoid arthritis, Sjogren‘s syndrome, scieroderma, iuous such as SLE and iupus nephritis, poiymyositisidermatomyositis, crycgiohuiinemia, hosohoiipid antibody syndrome, and osoriatic arthritis), rthritis, autoimmune gastrointestinai and iiver disorders (such as, for examoie, inflammatory bowei diseases (eg, uicerative s and Crohn’s disease), mune gastritis and pernicious anemia, autoimmune hepatitis, primary hiiiary cirrhosis, primary scierosing chciangitis, and ceiiac disease), itis (such as, for exampie, ANCAassociated vascuiitis, inctuding Chord—Strauss itis, Wegener‘s granuiomatosis, and ooiyarteriitis), mune neuroiogicai disorders (such as, for exampie, muitipie scierosis, opsocionus myocionus me, myasthenia gravis, neuromyeiitis optica, Parkinson’s disease, Aizheimer’s e, and autoimmune pciyneurooathies), renai ers (such as, tor exampie, giomeruicneohritis, sture‘s syndrome, and Berger’s disease), autoimmune 2014/025564 dermatoiogic disorders (such as, for exampie, psoriasis, urticaria, hives, gus yuigaris, huiious pemphigoid, and ous iupus erythematosus), hematoicgic disorders (such as, for exampie, thrombocytooenic purpura, thrombotic thrombocytopenic purpura, post~ transfusion ourpura, and autoimmune hemoiytic anemia), atheroscierosis, uveitis, autoimmune hearing diseases (such as, for exampie, inner ear disease and hearing toss), Behcet's disease, Raynaudis syndrome, organ transpiant, and mune endocrine disorders (such as, for examoie, diabetic—reiated mune diseases such as insuiin— dependent diabetes meiiitus Vi), Addison’s disease, and autoimmune thyroid e (eg. Graves’ disease and thyroiditis». More preferred such diseases inciude, for exampie, toid arthritis, tive coiitis, ANCA-associated vascuiitis, iupus, muitipie scierosis, Sjcgren‘s syndrome, Graves’ disease, iDDiv‘i, pernicious anemia, thyroiditis, and giomeruioneohritis.
Methods of ent The ates of the present invention may be used in a method of therapy. Aiso provided is a method of treatment, comprising administering to a subject in need of treatment a therapeuticaiiymetteotive amount of a conjugate compound of the invention. The term “therapeuticaiiy effective amount” is an amount sufficient to show benefit to a patient. Such benefit may he at ieast ameiioraticn ct at ieast one symptom. The actuai amount administered, and rate and time-course of administration, Witt depend on the nature and severity of what is being treated. Prescription of treatment, eg. ons on dosage, is within the responsihiiity ot i practitioners and other medicai doctors.
A compound of the invention may be administered aione or in combination with other treatments, either sirnuitaneousiy or sequentiaiiy ent upon the condition to he treated.
Exampies of ents and therapies inciude, but are not iimited to, chemotherapy (the administration of active agents, inciuding, eg. drugs, such as chemotherapeutics); surgery; and radiation therapy.
A “chemotherapeutic agent" is a chemicai compound usetui in the treatment of cancer, regardiess of mechanism of . Ciasses ot chen‘totherapeutic agents inciude, but are not iimited to: aikyiating agents, antimetaboiites, spindie poison piant aikaioids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents inciude compounds used in “targeted therapy” and conventionai chemotherapy.
Exempies of chemotherapeutic agents ineiude: eriotinih (TARCEVA®, eethSi Pharm.), docetexei (TAXQTEREGQ, —Aventisi S—FU (tiuoroureeii, 5~ftuoreurecit CAS No. 51—21—63), gemcitabine (GEMZARG), Liiiy), PD~0325901 (CA3 No. BQtZtO—tflmg, ), cispiatin (sis—diamineq dichioropietinun‘tiii), CAS No. 15683274), carbopietin (CAS No. 41575—944); paeiitexei (TAXOL®, Bristot—E‘v’iyers Squibb Oneoiogy, Princeton, NJ); trastuzunteb (HERCEPTENQ’RSJ, Gettentecn), tentozoiomide (4wmetnyie5»oxo~ fi,8* pentezebioycio {4.3.0} none—2,7,Q—triene— Q—cerhoxemide, CAS No. 856224334, TEMQBARQ TEMQBALCS), Schering Piough), tamoxifen ((Z)~2w{4—(’iifldiphenyibut—h enyi)phenoxyH‘iflN—oin'tethfyiiethenantinea NOLVADEX®, ESTUBALQR), VALODEXCPIJ), and bicin (ADREAMYCENEQ), Akti~t/2, HPPD, and repamycin.
More exemptes of chemotherapeutic agents inoiude: oxaiipietin (ELOXATiN-C‘s), Senofi), bortezomib (VELCADE®, Miiiennium Phenn), sutent (SUNETENEBQX SU11248q Pfizer), ietrezeie (FEMARA®, Novattis), imatinib mesytete (GLEEVEC®, Nevertis), XL—SiS (Mek inhibitor, Exeiixis, WC) 44515), ARRYe88t‘S (Eviek inhibitor; AZDfi244, Array BiePhernte, Astra Zeneea), SF—t’i26 (PEBK tor, Semafore Phernteceuticeis), BEZQSS K inhibitor, Novartis), XL—tc‘i? (Pi3K inhibitor, Exeiixis), PTK?87IZK 222584 tis), tuivestrant (FASLQDEXGRL AstraZeneea), ieucovorin (toiinic acid)“ rapamycin (simiimus, RAPAMUNECFES Wyeth), iepatinib (TYKERB®, GSKSTEOtfis Giexe Smith Kiine), innaternih (SARASART’J‘, SCH 66336, Sehering Ptough), sorafenib (NEXAVARGQ, BAWiB-Qflfln, Bayer Labs), getitinib (ERESSACE), AstreZeneee), irinotecen (CAMPTOSARG), CPT—i ‘i, Pfizer), tipifemih (ZARNESTRATM, Johnson & n), ABRAXANETM (CremOpnor—free), aihumin— engineered nahopertieie ‘iorrnuieiions of peoiitaxei (American Phermeceutioet rs, Sohaumberg, ii), vandetenib (i‘iNN, , ZACTiMACR), AstraZeneca), nn‘ibucii, AGMYS, AG1571 (SL5 5271; Sugen), ternsiroiii‘nus (TORESEL®, Wyeth), pezonenib (GiaxoSmithKiine), centosfamide (TELCYTACS), , thiotepe and eyoiospnosphamide (CYTOXANCPIJ, NEOSAR®); eikyi suitonetes such as busuiteh, impresuiten and piposuifen; aziridihes such as henzedope, eerhoquone, n‘ieturedopea and uredope; etnyienimines and methyiameiemines incinding eitretemine, triethyienemeiemine, triethyienephosphoremide, yienethiophosphoremicie and trimethyiomeiamine; acetogenins (especiaiiy buiietacin and butteteeinone); e camptotheoin (ineiudihg the synthetic enaieg topoteeen); bryosteiin; caiiystatin; (EC—“i065 (ineiuding its edozeiesin, oarzeiesin and bizeiesin synthetic. anaiogs); cryptophycins (particuieriy cryptophycin ’i and cryptophyein 8); etin; duooarmycin (inciuding the synthetic anaiogs, KWQTBQ and CBi~TiVit ); eieutherohin; pancretistatin; a saroodietyih; spengistetin; nitrogen mnstards such as mhueii, ohiernephezine, chierophosphemide, estrentustine, ifostemide, meohiorethemine, meohiorethemine oxide hydrochioride, meipheien, ichin, terine, mustine, trotosfemide, urecii mustard; hitrcsouraas such as carmustiha, chicrozotccin, 'ictemustihe, icmustina himustina, and ranimnustina; antibiotics such as the ansdiyha antibiotics (cg. caiichaamicin, caiichaamicin gamma’ii, caiichaamicin omagai’i (Angew Chem. int]. Ed. Engi. (ti-394) 332183488); dynamicin, dynamicin A; bisphcsphonatas, such as ciodrchaia; an asperarnicin; as waii as naccarzihcstatin chrcmcphcra anti reiatad chrcmcprciain ancdiyna antibiotic chromophcrss), aciacincmysihs, aciincmy‘cin, authramycih, azassriha, hiaomycihs, cactinomycih, carabicin, carmihomycin, carzinophiiin, chrcmcmycinis, cactincmycih, daunorubicin, datoruhicin, 6ndiaZOISIOXO—thorteucina, morphctinowdcxorubicin, crphciinc—dcxcrubicih, Empyrroiihadcxcrubicin and cxcrubicin); cpirubicin, ascruhioih, idaruhicih, hamcrubicih, marcaiiomycih, mitomycihs such as cih C, mycophenoiic acid, nogaiamycin, oiivcmycins, papicmycin, pon‘ircmycin, purcmycin, queiamycih, rodoruhicin, streptonigrinq straptozocinq tubercidinq UbGi’iimqu zinostatih, zcrubicin; anti~matabciitas such as raxaia anti 5—fiucrcuracii (5~FU); ‘iciic acid anaicgs such as dahcptsrih, mathotraxatc, ptsrcptai‘ih, trimatrsxatc; purine s such as ahina, @mcrcaptopurina, thiamiprihc, thicguanina; pyrimidine ahaicgs such as ahcitahine, azaciiiciiha, G—azauridina, carmoiur, cytarabina, didacxyuridina, doxiiiuridiha, enociiabiha, fioxuridihe; androgans such as caiusterone, dromostahoiona uropionate, apiticsianci, rhepiiicstanaa tastciactcna; antimadranais such as arnincgiutcthimida, mitctana, ti'iicstahe; iciio acid rspicnishsr such as irciihic acid: acsgiatona; aidcphosphamids giyccsida; aminoiavuiihic acid: aniiuracii; amsacrina; bastrabucii; bisantrahc; edatraxata; defofamina; damacoicina; diaziquone; thiha; aiiipiinium a; an apothiioha; atcgiuciu; gaiiiurn nitrate; hydrcxyuraa; iantihan; icniuaihina; maytansinoids such as maytahsina and ahsamitccihs; mitoguazcna; mitoxautroua; hmci; citracrins; pantostatih; phenamat; pirarubicin; htrcna; podophyiiihic acid; ihydrazida; procarhazine; PSK® poiysaccharida x (JHS i Products, Eugene, QR); razcxana; rhizcxin; an; spircgcrn'ianium; tenuazcnic acid; ‘iriaziqucna; 2,222”— ti‘ichicrciriathyiamina; thacanas (sspaciaiiy T~2 toxin, vsrracurih A, rcridin A and anguiciina); urcihan; vihuesina; aziha; ustiha; mitohrcnitci; mitoiactoi; pipohmman; gacytcsiha; arabihoside (“AranC”); cyciophosphamide; thictepa; 6~ihicguahihe; marcaptcpuriha; mathctrexate; piatihum anaicgs such as cispiatin and carbcpiaiin; vinbiastihs; etcpcsids (VP-16); ifcsfamidc; mitcxahircnc; vihcristihc; vincraihina (NAVELBENE-ED); mna; sida; cdatrcxata; daunomycih; aminopiarin; capacitahine (XELODAfia, Roche); ibandronaia; CPTJE ‘i; topoisomarase inhibitor RFS 2000; diiiucrcmathyicrnithina (DMFQ); ratincids such as ratincic acid; and pharmaceuticaiiy ahis saits, acids and derivatives cf ahy cf the above.
Atso ed in the definition of “chemotherapeutic agent” are: (i) anti—hormonai agents that act to regutate or inhibit hormone action on tumors such as anti—estrogens and seiective estrogen receptor moduiators (SEW/is), ing, for examoie, tamoxifen (inciuding NOLVADEX®; tamoxifen citrate), raioxifene, itene, 4—hydroxytamoxiten, tene, keoxitene, LYt t70t8, onaoristone, and FARESTGN® (torei‘ni‘iine citrate); (it) arornatase inhibitors that inhibit the enzyme ase, which regutates estrogen production in the i giancis, such as, for examoie, 4(5)~imidazoies, aminogiutethimide, MEGASE® (megestroi acetate), AROMASiN® (exemestane; Pfizer), formestanie, fadrozoie, RiViSORfié‘x (vorozoie), ® zoie; is), and ARiWiiDEX® (anaetrozote; AstraZeneca); (iii) anti~androgens such as fiutarnide, niiutamide, bicaiutamide, ieuproiide, and gosereiin; as weii as troxacitabine (a t,3—dioxoiane nucteoside cytosine anaiog); (iv) protein kinase inhibitors such as inEK inhibitors (W0 44515); (v) iipid kinase inhibitors; (vi) antisense oiigonucteotides, barticuiariy those which inhibit expression of genes in signaiing pathways irnpticated in aberrant ceii broiiferation, tor exampie, PKG—aipha, Rat and H—Ras, such as obiimersen (GENASENSECRZ), Genta inc); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGEQZYMEQ’E) and HERE expression inhibitors; (viii) vaccines such as gene therapy vaccines, for e, ALLOVECTiN®, LEUVECTiNiE), and VAXED®; PROLEUKEN® rit—2; toboisomerase 1 inhibitors such as LURTOTECAN®; ABARELEX® rmRi—i; (ix) anti— angiogenic agents such as bevacizun‘tab (AVASTENQ Genentech); and bharmaceuticaiiy acceptabie saits, acids and derivatives of any of the above.
Aiso inciudeci in the definition of “chemotherapeutic agent” are therapeutic dies such as tuzurnab (Carnoath), bevacizumab (AVASTENEQ, Genenteeh); cetuxii‘nab UXCR), tmcione); panitumumab (VECTiBtX®, Amgen), rituxintab (RtTUXAN®, GenentechiBiogen ioec), oertuzumab (OMNETARGW, 2C4, Genentech), trastuzumab (HERCEPTiN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug ate, gerntuzumab ozogarnicin (MYLOTARGQ‘Q, Wyeth), Humanized onai dies with therapeutic potentiai as chemotherapeutic agents in combination with the conjugates of the invention inoiude: aiemtuzumab, anoiizumab, asetizumab, atiizurnab, bapineuzumab, zumab, bivatuzurnab rnertansine, cantuzumab mertansine, cedeiizun‘iab, ceitotizurnab pegoi, cidfusituzumab, un‘iab, daciizurnab, ecutizumab, etaiizumab, epratuzumab, eriizurnab, feivizumab, tontoiizumab, gemtuzumab ozogamicin, inotuzuntab ozogamicin, ibiiimumab, iabetuzumab, iintuzumab, matuzuntab, rneooiizumab, rnotavizurnab, motovizurnab, natatizurnab, nirnotuzurnab, hoiovizurnab, humavizumab, ocretizumab, on‘iatizumab, caiivizun‘tab, pascoiizumab, pectusituzumab, pectuzumab, oertuzumab, pexeiizumab, raiivizumab, ranibizumab, resiivizumab, resiizumab, resyvizurnah, roveiizumah, rupiizumah, sihrotuzumah, sipiizumah, sontuzumab, tacatuzumah tetraxetan, tadocizumah, mah, zumab, tooiiizumah, toraiizumah, trastuzumah, tucotuzumah ceimoieukin, tucusituzumah, umavizumap, urtoxazumah, and visiiizumah.
Pharmaceuticai compositions according to the present invention, and for use in accordance with the present invention, ma},i comprise, in addition to the active ingredient, i..e a conjugate compound, a pharmaceuticaiiy acceptahie exoipient, carrier, putter, stahiiiser or other materiais weii known to those d in the art. Such materiais shouid he non—toxic and shouid not interfere with the efficacy of the active ient. The precise nature of the carrier or other materiai Wiii depend on the route of administration, which may he orai, or by injection, eg. cutaneous, subcutaneous, or intravenous.
Pharmaceuticai compositions for orai administration may be in tabiet, capsuie, powder or iiduid form. A tahiet may comprise a soiid carrier or an adjuvant. Liquid pharmaceuticai compositions generaiiy comprise a iiduid carrier such as water, petroieum, animai or vegetahie oiis, minerai oii or synthetic oii. Physioiogicai saiine soiution, dextrose or other saccharide soiution or giycois such as ne giycoi, propyiene giycoi or poiyethyiene giycoi may he ed. A capsuie mag»i comprise a soiid carrier such a geiatin.
For intravenous, cutaneous or suhcutaneous ion, or injection at the site of attiiction, the active ingredient wiii be in the form of a parenteraiiy acceptabie aqueous soiution which is pyrogenstree and has suitahie pi-i, isotonicity and stahiiity'. Those of reievant sitiii in the art are weii acts to prepare suitapie soiutions using, for exampie, isotonic vehicies such as Sodium Chioride ion, ‘s injection, ed Ringer's injection. Preservatives, stabiiisers, butters, antioxidants and/or other additives may be ed, as required, Formuiat‘icns White it is possihie for the ate compound to he used (e.g., administered) aione, it is often preterahie to present it as a composition or tormuiation, in one embodiment, the composition is a pharmaceuticai composition (e.g., tormuiation, preparation, medicament) comprising a ate compound, as descriheci herein, and a pharmaceuticaiiy acceptabie carrier, diiuent, or excipient, in one embodiment, the composition is a pharmaceuticai composition comprising at ieast one conjugate compound, as described herein, together with one or more other pharmaceuticaiiy acceptahie ingredients weti known to those skiiied in the art, inciuding, but not iimited to, pharmaceuticaiiy acceptabie carriers, diiuents, excipients, adjuvants, fitters, butters, preservatives, anti—oxidants, iubricants, stahiiisers, soiuhitisers, tants tog, wetting agents), masking agents, coiouring agents, tiavouring , and sweetening agents. in one ment, the ition further ses other active agents, for exampie, other therapeutic or prophyiaotic .
Suitabie rs, diiuents, excipients, etc. can he found in standard pharmaceuticai texts.
See, for exampie, Handbook of Pharmaceuticat Additives, 2nd Edition (eds. M. Ash and i.
Ash), 20m (Synapse information Resources, inc., Endicott, New York, USA), Remington‘s ceuticai Sciences, 20th edition, pub. Lippincott, ms 8x Wiikins, 2000; and Handbook of ceuticai Excigients, 2nd edition, 1994.
Another aspect of the present invention pertains to methods of making a ceuticai composition sing admixing at ieast one [1icj-radioiabeiied conjugate or conjugatedike compound, as defined herein, together with one or more other pharmaceuticaiiy acceptahie ingredients weii known to those skiiied in the art, e.g., carriers, diiuents, excipients, etc. it tormuiated as discrete units (e.g., tabiets, etc), each unit contains a predetermined amount (dosage) of the active compound.
The term “pharmaceuticaiiy acceptahie,” as used herein, pertains to compounds, ients, materiats, compositions, dosage terms, etc., which are, within the scope of sound medicai judgment, suitabie tor use in contact with the tissues of the subject in question (eg, human) without excessive toxicity, irritation, aiiergic response, or other probiem or compiication, commensurate with a reasonahie benefit/risk ratio. Each carrier, diiuent, exoipient, etc. must aiso he “accep‘tahie” in the sense of being compatihie with the other ients of the tormuiation.
The tormuiations may he prepared hy any methods weii known in the art of pharmacy. Such methods inciude the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. in generai, the ations are prepared by uniformiy and intimateiy ng into association the active compound with carriers (e,g., iiquid rs, tineiy d soiid carrier, etc), and then shaping the product, it necessary.
The formuiation may be prepared to e for rapid or siovv reiease; immediate, deiayed, timed, or sustained reiease; or a combination thereof.
Formuiations suitabie tor parenterai administration (eg, by injection), inoiude aqueous or non—aqueous, isotonic, nmtree. steriie iiduids (eg, soiutions, suspensions), in which the active ingredient is dissoived, ded, or otherwise provided (e.g., in a iiposome or other microparticuiate). Such iiguids may additionai contain other pharmaceuticaiiy aoceptabie ingredients, such as anti—oxidants, butters, preservatives, stabiiisers, bacteriostats, suspending agents, thickening agents, and s which render the ‘iorrnuiation isotonic with the biood (or other reievant bodiiy tiuid) of the intended recipient.
Exampies of excipients inciude, tor exampie, water, is, poiyois, giyceroi, vegetabie oiis, and the iike. Exampies of suitabie isotonic carriers for use in such formuiations inciude Sodium Chioride ion, Ringer‘s Soiution, or Laotated Ringer‘s injection. Typicaiiy, the concentration at the active ingredient in the iiduid is from about ‘i ng/mi to about ‘iti rig/mi, for exampie from about it} ngimi to about i pg/mi. The ‘iorrnuiations may be presented in unit»dose or rnuiti—dose seated containers, for exampie, ampouies and viais, and may be stored in a freeze—dried (iyophiiised) condition ing oniy the addition of the steriie iiquid carrier, for exampie water for injections, immediateiy prior to use. Externporaneous injection soiutions and suspensions may be prepared from steriie powders, granuies, and tabiets.
Dosage it wiii be appreciated by one of siriii in the art that appropriate dosages of the ate compound, and compositions comprising the conjugate compound, can vary from t to patient. ining the i dosage wiii iiy invoive the baiancing of the ievei of therapeutic benefit against any risk or deieterious side effects. The seiected dosage ievei Wiii depend on a variety of factors inoiuding, but not iimited to, the activity of the particuiar compound, the route of administration, the time of adr‘ninistration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materiais used in combination, the severity of the condition, and the species, sex, age, weight, condition, generai heaith, and prior medicai history of the patient. The amount of nd and route of administration wiii uitimateiy be at the discretion of the physician, veterinarian, or ciinician, aithough generaiiy the dosage wiii be seiected to e iocai concentrations at the site of action which achieve the desired effect t g substantiai i or deieterious side-effects.
Administration can be effected in one dose, continuousiy or intermittentiy (e.g., in divided doses at appropriate ais) throughout the course oi treatment. Methods of ining the most effective means and dosage of administration are weii known to those of shit! in the art and Wiii vary with the formuiation used for therapy, the purpose of the therapy, the target ceiits) being treated, and the t being treated. Singie or muitipie administrations can be d outwith the dose ievei and pattern being seiected by the treating physician, veterinarian, or oiinieian. in generai, a suitabie dose of the active compound is in the range of about "iOti ng to about mg (more typioaiiy about i pg to about 10 mg) per kiiogram body weight of the subject per day. Where the active compound is a satt, an ester, an amide, a prodrug, or the titre, the amount administered is oaieuiated on the basis of the parent nd and so the actuai weight to be used is increased proportionateiy. in one embodiment, the active compound is administered to a human patient according to the toiiowing dosage regime: about 106 mg, 3 times daiiy, in one embodiment, the active compound is administered to a human patient according to the toiiovving dosage regime: about tot) mg, 2 times daiiv. in one ment, the active compound is administered to a human patient according to the toiiovving dosage regime: about 200 mg, 2 times daiiy.
However in one embodiment, the conjugate compound is administered to a human patient according to the foiiowing dosage regime: about 50 or about 75 mg, 3 or 4 times daiiy. in one embodiment, the conjugate compound is administered to a human patient ing to the foiiowing dosage regime: about 100 or about t25 mg, 2 times daiiy.
The dosage amounts described above may appiy to the conjugate (inciuding the P80 moiety and the iinker to the antibody) or to the effective amount of PBD compound provided, for exampie the amount of compound that is reieasabie after cieavage of the tinker.
For the prevention or treatment oi disease, the appropriate dosage of an ABC of the invention Witt depend on the type of disease to be treated, as detined above, the severity and course of the disease, whether the moiecuie is administered for preventive or therapeutic es, previous therapy, the patient‘s ciinicai y and response to the antibody, and the discretion of the attending physician. The moiecuie is suitabiy administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease. about ’i ugi’kg to 15 mg/kg (eg. 0.’i~20 mg/kg) of moiecuie is an initiai candidate dosage for administration to the patient, whether, for exampie, by one or more separate administrations, or by continuous on. A i daiiy dosage might range from about i pg/‘kg to 100 mgikg or more, depending on the factors mentioned above. An exempiary dosage of ABC to be administered to a patient is in the range of ahout 0.1 to about it) mgikg of patient weight, For repeated administrations over severai days or , depending on the condition, the treatment is sustained untii a desired suppression of disease symptoms occurs. An ary dosing regimen comprises a course of administering an initiai ioading dose of about 4 mg/irg, toiiowed by additionai doses every week, two weeks, or three weeks of an ABC. Other dosage regimens may he usetui. The progress of this therapy is easiiy monitored by conventionai techniques and assays.
Treatment The term “treatment,” as used herein in the context oi treating a condition, ns generaiiy to treatment and therapy, whether of a human or an animai (e.g., in veterinary appiications), in which some d therapeutic effect is achieved, for exampie, the inhibition of the progress of the condition, and inciudes a reduction in the rate of ss, a hait in the rate oi progress, regression of the condition, ration oi the condition, and cure of the condition. Treatment as a prophyiactic measure (i.e., prophyiaxis, tion) is aiso inciuded.
The term peuticaiiy—e‘itective ,” as used , pertains to that amount of an active compound, or a materiai, composition or dosage trom comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonabie benefit/risk ratio, when administered in accordance with a desired treatment regimen.
Simiiariy, the term "prophyiacticaiiy—eftective amount,” as used herein, pertains to that amount of an active compound, or a materiai, composition or dosage from comprising an active compound, which is effective for ing some desired prophyiactic effect, commensurate with a reasonabie benetitirisk ratio, when administered in accordance with a desired treatment regimen.
Preparation ofAntibody drug conjugates Antibody drug conjugates may be prepared by severai routes, empioying c chemistry reactions, conditions, and reagents known to those skiiied in the art, inciuding: (1) reaction of a nucieophiiic group of an antibody with a bivaient tinker t, to form antibodymiinker intermediate Ao—L, via a covaient bond, foiiovved by reaction with an activated drug moiety reagent ; and (2) reaction of a drug moiety t with a tinker reagent, to form drug~tinker t ELL, via a covaient bond, totiowed by reaction with the nucteophiiic ot an antibody. ation methods (1) and (2) may be empioyed with a variety of antibodies, and iinkers to prepare the dy/"drug conjugates of the invention.
Nucieophiiic groups on antibodies inciude, but are not iimited to side chain tnioi , e.g. cysteine. Thiot groups are nucieophiiic and oapabie of reacting to term covatent bonds with eiectroohiiic groups on tinker moieties such as those of the present invention. Certain antibodies have reduoihie interchain disuitides, i.e. cysteine bridges. Antibodies may he made reactive for conjugation with tinker reagents by treatment with a reducing agent such as DTT (Cieiand's reagent, ditniothreitoi) or TCEP (tris(2~oarboxyethyi)phospnine hydrochioride; Getz et at (1999) Anai. Biochem. ‘v’oi 27373—80; Soitec Ventures, Beveriy, MA). Each cysteine disuitide bridge wiii thus form, theoreticaiiy, two reactive thioi nucteophiies. onai nucieophiiic groups can he introduced into antibodies through the reaction ot iysines with 2—iminothiotane ’s reagent) resuiting in conversion of an amine into a thioi. the Subject/Patient The subject/patient may be an animai, mammai, a piacentai mammai, a marstipiai (eg, kangaroo, wombat), a monotreme (e.g., iied piatypus), a rodent (eg, a guinea pig, a hamster, a rat, a mouse), murine (e.g., a , a iagomorph (e.g., a rabbit), avian (eg, a bird), canine (eg, a dog), teiine (e.g., a cat), equine (e.g., a , porcine (e.g., a pig), ovine (eg, a sheep), bovine (e.g., a cow), a primate, simian (so, a monkey or ape), a monkey (so, marmoset, baboon), an ape (eg, goriiia, chimpanzee, orangutang, ), or a human.
Furthermore, the subject/patient may be any of its torms ot deveiopment, for exampie, a foetus. in one preferred embodiment, the subjectipatient is a human. in one embodiment, the patient is a population where each patient has a tumour having GVSS integrii‘i on the surface at the cell.
Cine possible synthesis route to a dimer intermediate of formula W is shown below: 19 9 N RC /R R R R\ RC X-H HX N N 17 R7 N 2 R \2 R R 6 o R R o a ll Hal \ Hal \T. z T/ RC IR R19 / x. \ \T' z N 17 RZV R o R16 WO 59981 Enteri‘nediate EV can be used to make intermediate Vii RC /R R19 / X. \ \T' Z N 17 2 R O R W Rc FM R19 / N . I X \ \T z N 17 2 R o R RC FN' R19 / N I X' \ \T‘ z N 17 2 R O R W WO 59981 O R R Vii o O R Viii 2014/025564 o o )L N G R19 / N . l / X \ H N 17 R2 R 0 R IX Aiiemeiiveiyg intermediate EV can be ceupied with intermediate X to make intermediate 3X: I I O + OWN/R WO 59981 RC /R 19 R / RC X. \ H \T z N 17 R2 R \R2 O R N + Xi! 0WWVrHO R RC [RN R19 / \T‘ Z N 17 O R X!!! 1‘ Depmtection HO G \n/ XIV OWOWiN G nH R19 / / \ H \T‘ z N 17 R2 R WO 59981 Enteri‘nediate EV can be used to make intermediate XVE R19 / N l I X4 \ H \T' Z N 17 R22 R XV! R O R 2014/025564 Enteri‘nediate EV can be used to make intermediate XEX RC /R R19 / X. \ \T z N 17 R2 2 O R w RC /RN R19 / N . I X \ \T z N 17 R2 2 o R xvas RC /R 19 R R2 2014/025564 One possibie synthesis route to a dimer intermediate of formuia XEV is Shown beiow: RC- /R 19 R R R\ RC X-H HX N N 17 N Rzg/ R R \R2 e O R R O i ii o H Hal \ Hal \T. Z T/ 0 JL N G H / N C' R 19 R R / xx: + xx“: —> / N . I X \ \T' 2 R22/ R17 0 R16 xxav ediate XXE can be used to make intermediate XXVEE: O H RC /R R19 / X. \ \T' z R22 N 0 R16 0 O H RC /R R19 / X. \ \T' z 22 N 17 R R R2 0 R16 H N" O Nwe WN/O R X. \ \T' z N 17 R2 R R2 0 R16 XXV! 1. eotion HO G + \n/ my O N O N G RC [R R19 / X. \ \T. Z 22 N 17 R R 2 O R XXVEE in the above schemes, R“, RN: and RN" each independentiy represent a nitrogen protecting group. RC and RC each independentiy represent OH or OProtO, Where PratO is e hydroxy protecting group. Protecting groups are weti known in the art. R“, RN' and RN" may be, for exampieq 80C. ProtO may be THP, it may he the protection of the NtflwC’it imine bonds is removed at a di‘iternet stage in the synthesis methods to that shown above, dependent on the chemistries empieyed. in i, the compounds and conjugates ean he prepared by first iinking two PBD monomers with a phenyiene or pyridyiene dimer bridge to produce intermediate 3V or XXL The n group on the eryi ring in the dimer bridge of intermediate EV may then he used to form the tether (inciuding iinker group (3 or L) to connect the PEG dimer to the ceii binding agent. in more detaii, two PBD monomers with “Kit and MX’H groups at the (38 position of each PBD monomer (intermediates i and ii, respectiveiy) may be rooted with mT—i-iai and —T’mHai groups on intermediate iii or intermediate XX. Such a method of synthesis aiiows tor the PBS monomers to be different and so the resuiting PBD dimer is asymmetricai. Eduaiiy, the PBD monomers may be the same.
PBD dimer intermediate i‘v’ mag»i be used to provide the compounds and conjugates of the present invention by reacting the aryi haiogen group in the bridge in a number of ways.
First; intermediate N can be used in a Sonogishira coupiing reaction to provide an ene group on the and group ot the dimer bridge. shira crosswcoupiing reactions are weii known in the art for coupiing a terminai aikyne with an aryi haiide in the presence of a paiiadium cataiyst, such as Pd(Ph3)4. a copper cataiyst, such as Cuii and a base, such as diethyiamine.
When acetyiene is to be used as the terminai acetyiene, one side of the acetyiene moiecuie is typicaiiy protected with, for exampie, Tit/i3 in order to prevent iinking of the PBS . Once the Sonogishira reaction is compiete, the This group can be cieaved to provide aikyhe intermediate V. intermediate V can be reacted with an azido compound to form a triazoie tive in an aikyne Huisgen cycioaddition. Such a reaction may he sed by a copper cataiyst.
To form the compounds and conjugates of the present invention, the azide is bonded to an ethyiene group and a variabie number of PEG . The azide may be terminated with an amine group to react further. Reaction of intermediate V with an azide compound wiii provide intermediate Vi.
The tree amine group of intermediate Vi can then be reacted with a carboxyiic acid group of a iinker group for connecting to a ceii binding unit to form the amide group iinking the P30 dimer to the tinker group G or i. to provide compound Vii.
The iinkerfreactive group, G; of intermediate Vii can be conjugated to a ceii binding agent to provide conjugates of the present invention.
As an ative snira reaction, intermediate N can be coupied to an acetyiamine, ouch ae propargyiamine in the ce of paiiadium and copper cataiyste and base. Such a reaction es part of a tether attached to the PBD dimer bridge Where the aciyne group is preserved and a free terminai amine is avaiiabie for r reaction“ For e, the reaction of intermediate EV with propargyiamine provides intermediate Viii.
The terminai amine of ediate Viii can he reacted with, tor e, a carboxyiic acid group attached to a tinker/reactive group G (for connecting to a ceii binding agent) to provide intermediate iX.
As an aiternative synthesis of intermediate iX, tne carboxyiic acid group of intermediate Xi can be reacted with propargyiamine to term intermediate Xiir Reaction of intermediate iV with intermediate Xii in a Sonogoenira reaction yiedie intermediate Xiii.
The protected amine group terminated the variabie PEG chain can be deprotected and reacted with the carboxyiic acid group of intermediate xrv in order to coupie the /reactive group (3‘: onto the PBD dimer and produce intermediate XEVK intermediate i‘v’ ma}i aise used in a cross~ceupiing amination reaction, such as a Buchwaiddtartwig aminaticn. A carhon~nitrcgen bond is formed via a paiiadium—cataiyeed crosemcoupiing of an amine with an aryi haiider A number of paiiadium cataiysts for use in each cross—coupiing reactions are known, such as Pd(Ph3)4 or RuPhoe/Ruphoapd.
Reaction of intermediate iv with a piperizine functioniised with a protected prcpan—‘i—amine provides intermediate XV. The protected amine of intermediate XV can be further reacted with, for exampie, a carhoxy‘iic acid group ed to a tinker/reactive group, G, tor connecting to a ceii binding agent to provide intermediate XVE. coupiing aminaticn reaction, such as a Buchwaidmi’iartwig amination, of intermediate iV with a partiaiiy protected piperazine tciiowed by deprotection (for exampie with tritiueroacetic acid) previdee intermediate XVii.
The deprotected zine amine group of intermediate XVii can be reacted with a carhoxyiic acid group in intermediate XViii to provide intermediate XiX. intermediate XXE can be used to form the oxime intermediate XXEV. For exarnpies a partiaiiy protected PEG~diamine, intermediate XXiig may be reacted with the carboxyiic acid group of ediate XiV. Deprotection yieids intermediate Xiii. on of ediates XXi and XXtii yieids oxime intermediate XXiV. The syn and anti oximes can be resoived using preparative HPLC. intermediate XXE can aiso be used to form the acryiamide intermediate XXV“. For examptei the aidenyde intermediate XXE can be reacted with maionic acid in a Knoevenagei condensation to yieid the acryciic acid intermediate XXV. This can be reacted with a partiaiiy protected PEG—diamine to yieid intermediate XXVi. Deprotectopm and coupiing with intermediate XiV yieid the acryiamide intermediate XXVii.
The synthesis of P80 compounds containing two imine moieties is extensiveiy discussed in the ‘toitowing references; which discussions are incorporated herein by reference: a) W0 Oil/12508 (pages 14 to 30); b) WO 200le23814 {pages 3 to t0); and c) W0 EGGS/085259 (pages 31 to 39).
Exampies Generat mentai Methods Opticai rotations were measured on an ADP 220 meter (Beiiingitam Staniey Ltd.) and concentrations (c) are given in g/tOOmL. iVieiting points were measured using a digitai meiting point apparatus (Eiectrothermai). ER spectra were recorded on a PerkinwEimer Spectrum 19% FT ER Spectrometer. 1H and 13t3 NEVER spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 409 and 100 MHZ, tivetyu Chemicai shifts are reported reiative to Tit/i8 (8 = 0.0 ppm), and signats are designated as s (singiet), d (doubiet; t (tripiet), dt (doubts tripiet) dd (doubtet of doubtets), ddd e doubiet of doubiets) or m (muitiptet); with coupiing constants given in Hertz (Hz). Mass spectroscopy (MS) data were ooiiected using a Waters Mioromass ZQ instrument d to a Waters 2:395 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: ary (W), 3.38; Cone (V) 35; Extractor (V), 3.0: Source temperature (”C), 100; Deseivation ature ("(3), 200; Gone ttow rate (Lin), 50; Demsoivation tiow rate (Lib), 250. High—resoiution mass spectroscopy (HRMS) data were recorded on a Waters Micron‘iass QTQF Giopai in positive W—mode using coated iicate giass tips to introduce the sampies into the instrument. Thin Layer Chromatography (TLC) was performed on siiica get aiuminium piates (Merck 60, F254), and fiash chromatography utiiised siiica gei (Merck 60, 230—400 mesh ASTM). Aii chemicais and soivents were purchased from SigmanAidrich and were used as suppiied without further purification.
Generai LCIMS conditions: The HPLC (Waters Aiiiance 2695) was run using a mohiie phase of water (A) (formic acid 0.1%) and acetonitriie (i3) (formic acid 0.1%). Gradient: initiai composition 5% fit over 1.0 min then 5% B to Q5% 8 within 3 min. The composition was heid for 9.5 min at 95% E3, and then returned to 5% E: in 0.3 s. Totai gradient run time equate 5 min. Fiow rate 3.0 nit/min, 400w. was spiit via a zero dead voiume tee piece which passes into the mass ometer. Waveiength detection range: 220 to 400 nm.
Function type: diode array (535 scans). Coiumn: Phenomehex® Onyx Monoiithic (318 50 X was mm.
The anaiyticai Lit/MS conditions for Exampie 5 to ’it were as toiiows: Positive mode eieotrospray mass spectrometry was med using a Shimadzu Nexera®lProminence® LCMS—ZOZQ. Mobiie phases used were soiyent A (H25) with 0.1% formic acid) and soiyent E3 (CHgCN with 6.1% formic acid). Gradient: initiai composition 5% i3 heid over 0.25 min, then increased from 5% E3 to 100% E3 over a 2 min . The composition was heict for 0.50 min at 10’ % B, then returned to 5% B in 0.95 min and heid or 0.95 min. The totai duration of the nt run was 3.0 min. Fiow rate was 0.8 miJmih. ion was at 2% and 254 nm. Coiumn: Waters Acquity UPLC® BEE-i Shieid RP18 1.77pm 2.1 x 59 mm at 53 “C.
The preparative HPLC conditions for Exampie 5 to 11 were as foiiows: Reversemphase uitra— fast high—perfori‘nahoe iiduid chromatography (UFLC) was carried out on a dzu Prominence® machine using Phenomehext’é} Gemini NX 5;; (318 coiumns (at 50 °C) of the foiiowing dimensions: 150 x 4.6 mm for anaiysis, and tot) x 21.2 mm for preparative work.
Eiuents used were soivent A (H20 with 9.1% formic acid) and soivent B (CH3CN with 9.1% formic acid). Aii UFLC experiments were performed with gradient conditions: From 0 to 30 min the composition of B was increased from O to 100% and heid at 100% B for a further 2 min. The composition of E5 was decreased from 109% to 0% from 32.0 min to 32.1 min and heid at 0% B untii 35.0 min. The totai duration of the gradient run was 35.0 min. Fiow rates used were it) mLfmin for ahaiyticai, and 20,0 le'min for preparative HPLC. {Detection was at 254 and 280 i'ii’i‘i.
CO (I? Exampie ’i (a) (1 1 S, 1 1eS, 1 1 ’3, 1 1e’S,i-di—terrebutyi ({5-he10~1,3* phehyiene)bis(methyiehe))his(oxy))bisq’V—methexv—Z—methyiehe—fi—oxo~1 1-{(tetrahydro—2H- pyranMQ—y.’texy)2,3,11,11etetrahydro 1H—pyrreief2, ,4jhenzediazepinen10{5H,‘i— oerbdxyiate) (2a , 2h, 2e) OTHP THPQ 30 COTHP HO "- a ‘- :a:X22X 2 Sr 222:: X== e: (i) (11$,11aS,1 13,1 1a’S}~di~tert—butyi 8,8’~(((5~iodo— 1,3— phehyiene)bishmethyiehe))bis(oxy))bis(?~methexy—2—methyiene“5*oxo~1 1-((tetrehydroe2H~ pyran-2~yi,ioxy)-2,3,11,11a-tetrahydre—1H—pyrro!e’,2 1——c][1,4]ttehzodiazepine— 1 0(51-1) eerbexyiate) (2a). 1,3-hiet’hromomethyi)-5—iodehehzehe (2.00 g, 5.20 mmei) was added to a stirred edititieh of BeelTi—ipmproteeted PBD g unit 1 (4.75 g, 10.3 mmei), TBAE (190 mg, 0.52 mmoi) and K2003 (1.42 g, 10.3 mmei) in dry DMF (60 mt). The reaction mixture was heated to 60 °C and stirred under an argon atmosphere for 3 hours at which point ahaiyeie by LC/MS reveaied aubstarttiai product formation at retention time 4,15 min (ES+) m/z 1171 (Vt/1+ Nari“, ~10°/l0 reiative ity) The reaction mixture was aiiewed to eeei to room temperature and the DMF wee d by evaporation in vacuo. The resuiting residue was partitioned between water (50 mi.) and EtCiAc (50 mt) and the aqueous phase was extracted with EtOAe (3 x 20 mt). The combined organic iayere were washed with water (2 X 20 mL), brine (50 mL), dried (Mg804), tittered and evaporated in vacue to provide the crude product.
Purification by tieeh chromatography (gradient eiutieh: 50:50 v/v EtOAe/‘hexane to 80:20 vlv hexene) gave the hienether 2a as a white foam (5.42 g, 91% yieid). (it) (1 18, 1 138, 1 1 'S, 1 1a'S)—di~tert-t>utyi 8,8'*(i’(5—bromo— 1,3— phenyiene)bis(methyiehe»{diatom/,1)hisg’V-methexv—Z—methyiehe—S-oxo~1 1—((tetrahydro—2H~ pyran—Q—y.’texy)2,3,11,11etetrahydro 1H—pyrreief2, 1——c[1,4]henzediazepine — oerbdxyiate) (2b) 1-hromo—3,5—hie(brememethyi)benzehe (1,54 9, 4.53 mmoi) was added to a d seiution of BeelTHP—preteeted PBD capping unit 1 (4.20 g, 9.06 mmoi), TBAi (16? mg, 0.45 mmoi) and K2CQ3 (1.25 g, 9.06 mmei) in dry Bit/iiT (52 mL). The reaction mixture was heated to 60 "C and stirred under an argon atmosphere for 5 hours at which point ahaiyeie by LCIMS reveaied substantiai product formation at ion time 41,10 min (88+) m/z 1101 (iii/1+ Hi"; ~70% ve intensity). The reaction mixture was aiiowed to oooi to room temperature and the DMF was removed by evaporation in vacuo. The resuiting residue was partitioned between water (tit) mi.) and EtCiAc (60 mL) and the aqueous phase was extracted with EtOAo (3 x 25 mt). The combined organic iayers were washed with water (30 rnL), brine (50 mL), dried (MgSQ4), tittered and evaporated in vaouo to provide the crude product“ Purification by fiash tography (gradient etution: 50:50 v/v EtOAo/‘hexahe to ttit % EtOAo) gave the bismether 2b as a white foam (3.37 g, 68% . (iii) (1 1 S, 1 1513, 1 1 ”S, 1 1a’S)—di~tert~butyi 8,8’—(((5—Chloro~1,3~ phenyiene)bishrtethyiene))bis(oxy))bis(?~rnethoxy—2—methyiene*5~oxo~1 1 -((tetrahydroe2H~ pyranQ-yijoxyi-Q, 3, 1 1, 1 rahydro—1H—pyrroiof2, 1—c][1,4jbenzodiazepine-10{5H)— carboxyiate) (2:3) 1a3~bis(bromomethyi)-5—ohiorohenzene (1.42 g, 4.86 mmoi) was added to a stirred sotution of Bocth-iP—proteoted PBD capping unit 1 (4.42 g, 9.60 mmoi), TBAi (1?? mg, 0.48 mmoi) and K2033 (1.33 g, 9.80 mrnoi) in dry Bit/iiT (55 mL). The reaction mixture was heated to 60 "C and stirred under an argon atmosphere for 15 hours at which point anaiysis by LCfiViS reveaied substantiai product formation at retention time aft-"O8 min (ES+) 17112 11357 (iii/1+ HF“, ~30% reiative intensity). The reaction mixture was aiiowed to oooi to room temperature and the DMF was removed by evaporation in vacuo. The resuiting residue was partitioned between water (tit) mi.) and EtQAc (60 mL) and the s phase was extracted with EtOAo (3 X 20 mt). The ed organic iayers were washed with water (20 rnL), brine (40 mL), dried (MgSQ4), tittered and evaporated in vacuo to provide the crude product.
Purification by fiash chromatography (gradient eiution: 5035i) vfy EtOAoihexahe to 83:20 v/‘v hexane) gave the hienether 25:: as a white foam (5.10 g, 99% yieid).
WO 59981 (b) (1 1 S, 1 1513‘, 1 1 ’8, 1 1a’8}~di~tert—butyi 8,8’~(((5~ethynvi~1,3— phenyiene)111501113111yiene))bis(oxy))bis(7~methexy—2—methyienea5~oxo~1 1-((te1rehydroa2H~ pyran-Q-yijexy)-2, 3, 1 1, 1 1a-tetrahydre—1H—pyrro!e’,2 1——c][1.4] benzediazepihe 1 W511)— carbexyiate) (4) x H HPO», 30C :\/©\/: P :& iTHPO '.30 COTHP 2a: X==i 3: : 22in X=- B; at: — 2c: X: Ci (1) (1 1S, 1 15:13, 1 1 ’S, 1 1a ’S)—di—tert~butyi 8, 8'—(((5—(1’trimethyisiiyi)ethyriyi)~ 1 , 3~ phenyiene)bis(me1hyiene))t>is(exy)}bis(?—n1ethexy~2~methyiene-S—exe—1 1*((tetrahydre-2H— pyren—2—yi)oxy)—2, 3, 1 1, 1 1a—tetrahydre-1H—pyrreio[2, 1—e}[1,4jbenzediazepme— 10(5111— earbexyiate) ('3) A eataiytie amount of PdtPPh3)4 (15.0 mg, 13.1 timei) was added to a mixture 01 the his- ether 2a (1750 mg, 3.65 mmoi), Th’iS—aeetyiehe (278 tiL, 191 mg, 1.96 mmoi), Out (5.6 mg, 26.1 emei), diethyiamine (1.35 mL, 956 mg, 13.1 mmei) and even—dried 4 A meieeuiar Sieve peiiete in dry DiViF (5.6 mL) in an even—dried seaiabie vessei. The e was degaeed and tiuehed with argon 3 times then heated in a microwave at 100 “C for 31) s at which point anaiyeie by LCIMS reveaied eompiete consumption of starting materiai1nd substantiei product termatien at retention time 4.37 min (ES+) m/z 1142 (iii/1+ Na} ~4Q°'is reiative intensity). Peak at retention time 3.97 min (ES-F) mi: 1069 (tit/1+ Nat"; ~tit)% reiative intensity) eheerved which eerreeponde to Ti‘viS—eieevege under LCli‘v’iS conditions. The reaction mixture was aiiowed t0 eeei to room ature and was then tiitered through a einter to remove the sieves (washed with Di‘viF). The tiitrate was evaporated in vacue and the resetting residue subjected to tiaeh tography (gradient eiution: 50:50 viv EtOAe/hexane to 80:20 Viv EtOAeihexahe) to provide the TMSMaeetyiene 3 as a yeiiew team (691 mg, 95% yieid). (ii) (1 1S, 1 1aS, 1 1 ’S, 1 1a’S1wdinteit—butyi 8,8’—({(‘5—ethynyim1 ,3~ pher:yiene)bisi’methyierie))bis1’oxyflbis1’7—methexy~2~methylene—5—oxo—1 1~g’(tetrahydro—2H— pyrari~2~yi,iex_v)~2, 3, 1 1, 1 1a~1etrahydrd1Hapyrroie[2, 1*c][1, 4] benzediazepine» 1 W511)- carbexyiate) (4) Seiid K2003 (383 mg. 27? mmei) was added to a stirred eeiutieh of the rotected compound 3 (1.55 g, 1.39 mmei) in ivieOi-i (20 mL), After 3 houre ng at room temperature the reaction was deemed to be eemeiete as judged by LCD/MS {desired product peak at retention time 4.00 min (ES+) mlz 104? ([M+ it} ~30?a; reiative intensity)}. The iv’ieOi-i was removed by evaporation in vacuo and the resuiting residue was partitioned between wateritit) mi.) and EtQAc (60 mi.) The iayers were separatedaand the aqueous phase was extracted with EtOAo (3 x 20 mL). The combined c iayers were washed with water (30 mt; brine (30 niL), dried (MQSO4>, tiitered and evaporated in vacuo to provide the crude product. Purification by tiash chromatography (gradient eiution: 50:50 WV EtOAc/hexane to 80:20 Viv EtQAclhexane) gave the aoetyiene 4 as an orange team (1.13 g, 7’ % . (o) (7 7 S, 7 7 as, 7 1’3, 1 7a’S)—di~teri~butyi 8,8’—(({5—(7—(2—(2—(2—(2— aminoethoxy)ethoxxiethoxy)ethyi)— “ii-fl 7 , 2, 3—ff'i320fl4j/IT)“ 7 , 3— ene)bis(mathyiene))bisr’oxy),ihisr’V-methony—methyiene—S-oxo~ 7 trahydro—2H~ pyran—Q—ytioxy)~2, 3, 7 7 , 7 7 a~tetrahydron 7 H— pyrroi‘ofz 7 nejf'7,djbertzodiazepine 7 0(5Hi— carboxyiate} (5) H N2 ’\/o\/\O/\/ R \/\ || N THPO .0 COTHP THPO IO COTHP .. MZJCEBl —* £53; .Zfiéfi 4: n: H Soiid CuSO4.5i-i20 (13.0 mg, 52.0 umoi) and (+)sodium 1.ascorbate (41.0 mg, 0.21 mmoi) were added to a stirred soiuticn ct ‘it—Azido—35§,§~trioxaundecan~t«amine (227 mg, 207’ uL, t.o4 mmoi) and the aikyne 4 (1.09 g, 1.04 mmoi) in tert~Bu0H (6 mL) and H20 (6 mi.) at room temperature. A coiour change from yeiiow to green was observed as the reaction progressed. After stirring for tit hours anaiysis by LC/‘i‘viS reveaied a substantiai of amount of desired product formed ponding to peak at retention time 3.72 min (ES+) ml}: 72.65 (iii/7+ HF, ~‘iQO% reiative intensity). [NOTE On some occasions reaction progress staiied, however, the on was driven to compietion upon addition of further (311304.5H20 (0.05 equivaients) and (+)~sodium Lmascorbate (0.2 equivaientsfi. The reaction mixture was partitioned (without shaking at the ting tunnei) between water (50 mi.) and EtOAc (50 n‘iL). The s phase was extracted with EtQAc (3 x75 mL) and the combined organic iayers were washed with water (30 mt), brine (50 mL), dried (iv‘igSQQ, fiitered and evaporated in vacuo to provide the crude product 5 as a green team (1.32 g, 100% crude yieid). The crude product was carried through to next step without further purification. (d) (1 1S, 1 15-18, 1 1 ’3, 1 1 a ~tert—butyi 8, 8’~((g’5~g’1—g’18~(2,{idiom—2, 5~dihydre~1H~pyrrei— 1—3/1)— 13-exe-3,6,94mm“12~azaectadecyi)-1H—1,2,3~triazoi-4—y1)—1,3- phei1_yiene)bis(meihyiene))bisr’oxy))bisr’7—methexy—Z—meihyiene—fi—oxo— 1 1-{(tetrai1ydre—2H- pyranMQ—y.’toxy)2,3,1 1,1 1atetrahydre 1 H—pyrreieféf, 1——c][1,4jbenzediazepine 1 01/511,!— carboxyiate) (d) HzN’\,o\/\ ¢Nd’\,o N N \ \ THPO IO COTHP THPO’ 53°C BOCOTHP Ha Elsie areH '- N o 1101110 Soiid t3maieimidohexanoie acid thydroxysueeinimide ester (32? mg 1 01.1 mrjmi) was added to a stirred seiution of the primary amine 5 (1.28 g, 1.01 mmei)in dr}i DOM (30 mi.) at room temperature. Progress was monitored by LC/iViS and after 3 days stirring the reactien proceeded no further, a substantiai amount of desired product was observed at retention time 3.65 min (ES+) mi}: 1458 (iii/1+ HP, ~100% reiative intensity) accompanied by unreaeted starting materiei at retention time 3.15 min. The on mixture was treated with siiiea get and the seivent removed by ation in vacue. The resetting residue was subjected to flash chromatography (gradient eiutien: 100% DOM to 97:3 Viv Oi—i) to give the maieimide 6 as a team (658 mg, 45% yieid). (e) N—(Z—(Z—(Z—(Z—(4—(3, 5—bie(((($)methexy—2—metiiyiene-d-QXQQ, 3, 5, 1 1a-te1ra11ydre— 1 H— pyrreioféé, 1—C][1,4jbenzediazepin—8nyi,iexy)methyimheriyi)" 1H— 1, 2, zoi—1 — yijethexy)ethexy)ethoxy)ethyi)—6e(2,361101602,5~dihydro—1Hepyrroi~1~yi)11exanamide (Z). thdfi» £21:th A soiution 0:195:51 v/v TFAfi-i;Q (5 mt.) was added to a sarnpie of the Pprotected compound 6 (428 mg, (129 rnmoi) at G 'C (ieeiaeetene), After stirring at G “C for 1 hour the reaction was deemed te as judged by“ LC/‘MS, desired product peak at retention time 2.72 min (ES+) (21/2 1054 + HY, ~7’O% reiative intensity). The reactien mixture was kept eoid and added drop wise to a ehiiied saturated aqueous soiution of Nat—i003 (100 rrtL). The mixture was ted with DCiVi (3 x 30 mt.) and the combined organic iayers washed with H20 (20 ntL), brine (40 mt), dried (M9804), tittered and evaporated in vacuo to provide the crude product“ Purification by fiash Chromatography (gradient eiution: 190% CHCig to 98:4 vlv CHCtgii‘vieOi-i) gave 1' as an orange foam (183 mg, 53% yieid), Exampie 2 (a) 11aS,11’S, —di—1ert—butyi 8, 8’4“”{5—{3—aminoprop— 1 ~yn— — 1, 3n phenyiene)bisi’methyiene))bis1'oxyfibis1'7—methoxy~2~methylene—5—oxo—1 11119traitydro—ZEH— pyran—Z—yi)oxy)—2, 3, 1 1, 1 1a—i‘etrahydro~1H—pyrrolo[2, 1—c][1 , 4jhenzodiazepine~ 1 W511)— carboxyiate} (3) THPO’ 5306 I.BOCOTHP THPq 5300 I.BOCOTHP v. N O O N H H ‘- N H O O N ’a a 4.
N OMe MeO N N OMe MeO N 0 0 2a O O A cataiytie amount of Pd(i3i3h3)4 (5.0 mg, 4.2 unto!) was added to a mixture of the hisnether 2a (242 mg, 021 mrnoi), propargyiemine (41 pL, 35 mg 0.63 mrnoi), Cui (1.6 mg, 8,4 pmohs diethyiamihe (0.42 mi_, 309 mg, 4.22 rnmoi) and oven-dried 4A moieouiar sieve peiiets in dry DMF (1.8 mi.) in an ovenmdried seaiahie veseei. The mixture was degased and flushed with argon 3 times then heated in a microwave at 100 ”C for 3 s at which point anaiysis by LCiiViS reveeied oompiete consumption of starting ei and substantiei product formation at ion time 3.18 min (ES+) miz 1076 (EA/1+ HY, ~60% reiative intensity). The reaction mixture was aiiowed to eooi to room ature and was then fittered through a sinter to remove the sieves (washed with Di‘viF). The tiitrate was evaporated in vacuo to provide the unstapie crude product 3 which was used imrnediateiy in the next step without purification or anaiysis. (b) (1 1S, 1 1aS, 1 1 ’S, ‘1 1a ’Stndi—teit—buz‘yi 8, 8’n(({5n{1—{2, 5mo’ioxon2, 5ndihydro—1anyrroin1—yi)n 3, 1 9~dioxo— 7’, 1 0,. 13, 1 6—fetraexa~4, 20~diazatrioos—QZ—yn—ZSyLi— 1, 3— phenyiene)bis(rnethyiene))bis(oxy)}bis(?—me1hoxy~2~methyiene-S—oxo—1 1*((tetrahydro-2H— pyran—2—yi)oxy)—2, 3, 1 1, 1 1a—tetrehydro-1H—pyrroie[2, 1 -r;][1, 4] iezepine— 1 W511)— Carboxyiate) ('9) »Owe/VHWN? NH2 HN O O THPQ IBOG IBOGOTHP EEOC H," N THPQ |IBOCOTHP fijfiOMeo Meomfio N a fifionfleH:- N o “Micaho N H o 8 O o 9 O EG®4~aoid (88 mg, (321 mrnoi) was added to a stirred soiution of EDCi (41 mg, 0.21 mmoi) and the crude primary amine 8 in dry DCit/i (4 mi.) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at whieh point anaiysis hy LC/i‘v’iS showed a substantiai amount of d product at retention time 358 min (ES+) rat/z 1475 (iii/1+ HF, ~‘i % ve intensity), 1498 (tit/1+ Nah ~5% reiative intensity) accompanied by a side t at retention time 3.85 min. The reaction mixture was diiuted with DCivi (30 mi.) and washed with H20 (3 x 10 mL), brine (20 mt)“ dried sm), fiitered and evaporated in vacuo to provide the crude product Purification py tiash chromatography (gradient eiution: .% DCi‘v‘i to 96:4 Viv DCM/‘MeQi—i) gave the ide 9 as a foam (1.17 mg, 22% yieid over 2 steps). ((3) N~(3~(3, 5~bis({((8)—7—methony—methyiena—d—oxo~2, 3, 5, 1 1a—tetrahydro~1H—pyrroio[2, 1 — c][1, 4] benzodiazepin—8—yi)oxyjime{ht/tiphenyi)prop~2~yn~1-5/1)-1—(3—(2,5~dioxoe2,5—dihydro-1H— pyrroi—1—yi)propanamido)-3, 6,9, 12—tetraoxapentadecan~15—amide (1 d) o O H 7 \> H i \> OYVOWO~0¢ONNVN OYVOV‘ONOWONNHKVN HN O HN O O mm; 100 .BOCOTHP —> H o o N H jH- o o N\ j OMe MeO:C/),N H OMe MeO: : (fil’h O a O 0 0 A seiution o195:5 v/v -th (1 mt.) was added to a sampte of the BoclTHP—protected compound a (67’ mg, 45.5 prooi) at 0 “C (iceiacetone). After stirring at i) “C for 1.5 hours“ the reaction was deemed oompiete as judged by LCIMS, desired product peak at retention time 2.1.17 min (1553+) mfz 1073 (tit/1+ HT; ~"% reiative intensity). The reaction mixture was kept coid and added drop wise to a chiiied saturated aqueous soiution of Nat-1803 {St} mL). The mixture was extracted with DCiVi (3 x 15 mL) and the combined organie iayers washed with brine (40 mt), dried (tit/195364); fittered and evaporated in vacuo to provide the crude product Purification by tiash chromatography ent eiution: 103% CHCEg to 96:4 viv CHOig/i‘v’ieOt-i) gave 1%} as an orange foam (12 mg, 24% yieid).
Exampie 3 (a) (11S,11a$,11’8,11a’Sj—di—tert—butyi 8,8’—(({5-{4—(3—{(ter1— butoxycarbonyt}amine)propyttpiperaginn1 "ya" 1 , 3131":enyiene)bis(methy!ene))bis(oxy))bis(7’" xy—E—methyiene~5~exo~1 1{fietrahydre~2H~pyran—2—yi)exy)~2, 3, 1 1, 1 1a~terrahydre~1H~ og’é’, 1-C][1,djbertzediazepine113(5Hj-earbexyiate) (1 1) (VN‘BOC THPQ $00 '.3OCOTHP wow mom NWQCEOMe mom/N O O 0 2r; 0 H A eampie of the hie—ether 26 (250 mg, 0.24 mmet), NaQiBu (57’ mg, 0.59 mmei), RuPhee (11 mg, 23.7 dmei) and RuPhosPd (19 mg, 23,? turret) were added to an ovenndried seaiahie tube (which was aiiowed to coat in a desicator), The mixture was degased and flushed with argon 3 times before the addition of dry THF (5 mt.) and then d to stir under an inert atmosphere for ~10 minutes untii the red eeiour had dieeharged. A eeiution of 3—(oiperazinnt"yi)prepan—t—amine (58 mg, 9.26 mmoi) in dry THF (1 mi.) was added and the e again degased and flushed with argon 3 times, The reaction mixture was heated at 80 °C in a preheated oii bath for 2.5 hours at which point anaiyaie by LCD/MS reveaied a 3— component mixture: desired product at retention time 3.35 min (53+) 13712 1264 (tit/1+ it} + 7 ~60% reiative ity), majer side product at retention time 3.95 min (de—ehierinated anaiogue 0126;) and shouider 4,13 min (trace starting materiai). After being aiiewed to coat to room temperature the reaction mixture was ioned between water (20 mL) and EtQAC (20 mL). The adueeue phage wee extracted with EtQAe (3 X112") mt.) and the combined organic iayers were washed with water (30 mt) brine (5t) mL), dried (M9504), fiitered and evaporated in vacue to provide the crude product. Purification by flash chromatography (gradient eiution: 100% DCin to 95:5 v/v DCMiit/ieOi-i) gave the piperazine 11 as a foam (152 mg, 25% yieid). ’i 07 (b) (1 7613, 1 1a 'S)~8, 8’~(((5~(4~(3eminopropyijpiperazim1~yii~ 1, 3~ phenyiene)bishnethyienefibisq’oxyfibis(7»rnethoxy—2—methyiena2, 3—dihydro—1H—pynroiog’2, 1— c][1Aibenzodiezepin-5(1 1aH)-one,i (12) ago; Eek afiber: moi A eoiution 0195:51v TFA/HgQ (2 mi.) was added toadeampie oi the Bodizliiipwproteeted compound 11 (142 mg, 0.11 mmoi) at t} ”C (toe/acetone). After stirring at 0 °C for 1 hour, the reaction was deemed oornpiete as judged by LCIMS, desired produo‘t peak at retention time 2.23 min iES+> mi}: 778 (iii/1+ H201") ~5% reiaiive intensity). The reaction e was kept ooid and added ise to a ehiiied saturated aqueous ori oi 03 (50 mt). The mixture was extracted with DCM (3 x 20 mi.) and the combined c iayere washed with brine (15 mt). dried (it/@804), fii‘tered and evaporated in vacuo to provide the crude product 12 as a waxy eoiid (22.6 mg). Note that during the NaH003 iieation step the d product precipitated out of soiution as a waxy soiid which was. oniy partiaiiy eoiubie in DOM.
Additiehai product was obtained by dissoiving DCiVi—iheoiubie soiids in DMF toiiowed by evaporation in vacuo. The resuiting oiiy residue was triturated with diethyiether to provide a soiid which was dried in vacuo to provide additionai crude 12 (54.4 mg, totai amount = 7’7 mg. 85% yieid) which was carried through to the next step without further purification or anaiyeie. (o) N~(3~(4~(3, 5~bis({((Si—7—methony—methy!ene—5—oxo~2, 3, 5. 1 1a—tetrahydro~1H—pyrroio[2, 1 — CH1, 4jbenzodiazepin831/1)oxy)methyijphenyiipiperazin—1—yl)propyi)1“(3(2.5011on2,5- dihydrd1H—pyrro:—1—_vi,ipropanemido)-3, 6, 9, 12—tetraoxapentadecen-15—amide (13).
O\/\o/\/ \/\o’\/ W QO N N I/\/N O O N N O E] —’ U H ’N O\/©\/O N‘ H H ’N O\/©\/O N‘ H o o o 12 13 o MAL~dPEG®4—acid (42 mg, 0.10 rnmoi) was added to a stirred on of EDCi (20 mg, 0.10 n‘imoi) and the crude primary amine 12 (7? mg, 0.10 mmoi) in drgvi DCiv’i (4 mL) at room temperature The reaetion e was d under an argon atmosphere for 3 heure at which point anaiyeie by LCIMS showed compiete consumption of starting materiai, a aubstantiai amount of deeired t at retention time 2.42 min (ES+) miz i176 + HZQF', ~5% reiative intensity“) and excess MALadPEGCRDet-acid at ion time 2.05 min (weak eignai on diode array hut detectabie eh ES+iES—). The reaction mixture wee diiuted with DEM (30 mi.) and washed with H30 (15 mL), brine (20 mt), dried (MgSQ4). fiitered and evaporated in vacuo to provide the crude product. Purification by tiaeh chromatography (gradient eiution: 190% CHCi3 to 93:? v/v CHCh/MeOi-i) gave the maieimide 13 as a team (46 mg, 55%). Note that trace amounts of excess MAL—dPEG®4~acid couid not he removed using flash chromatography.
Exampie d phenyiene)oisr‘methyienejjbisr’exviibisr’flmethoiymethyiene-5—exe—1(a) (113,1 1a3,11'3,1ta'3)*di~tert-t3rtyi 3,8(((5 utoxycerbonyiipiperazin— 1—3/1)—13 1—{(tetrahydr‘o-2H— pyi‘an—2—yijoxy)n2, 3 1 1, 1 1antetrahydrom ‘1anyrroiof'2, 1nc][1,4jbenzoo’iazepine—10(5Hjn carboxylate) (14) | N ea: ewe: at: an 2—t3icyciohexyioheeehine—Z‘,6‘—diieeoropoxyhiphenyi (18 mg, 38 omoi, [)2 ed), chiorotilidicyciehexyipheephinen2 ’,8'ndiisooropoxyn1,t'nhiphenyi){2n(2'nainino~1,‘i’~ hiphenyifloaiiadiumai) (t8 mg, 22 pmoi, 0.12 ea), caesium ate (0.35 9,1 .t mmoi, 5.0 ed) and iodo derivative (2a) (0.3%? g, 0.2? mn‘ioi, 1.9 ed) were piaced in a microwave viai which was. evacuated and flushed with Argon (x 3). Anhydrous THF (5 mt.) was added feiiowed by tertwbutyi pieerazinewt—carbexyiate (70 mg, 0.37 mmei. 1.1 eq) and the resuitant mixture was heated at 85043 for 4h then overnight at room temperature. The reaction mixture was d with saturated sodium hydrogen carbonate and extracted with ethyiacetate (3 x ttit) mL). The combined ethyiacetate extracts were weehed with brine (100 mt), dried (M9804) and evaporated under reduced pressure. The product id was purified by fiash coiumn chromatography [CHCiglivieOH 0/o to 1.5%in O.5%. increments} (0.1M g, 51%) Anaiytieai Data: RT 412 min; MS (ES‘) {Ii/2 (reiative intensity) tZO? (iii/i + ti") 30). (b) (1 1523, 1 1a’S)—8,8’—(((5—(eieerazin—1—3/0—1,3—5)henyiene)bis(methyiene)}bng’dxytibi'Sg’fl methaxyn2nmethyiene—2, Bdrm/Ciro"1anyrrdiog’2, 7 "CH7, 4jbenzediazepin~5(7 1 aid/inane) (‘3 5) Eco H [N] [N] 59d M deg; figfio/ £23; A eeid (ice bath) soiution at 95% tritiuordaeetie acid (4 mi.) was added to eempeund (14) (0.2 g“ 0.165 mmei, 1 eq.) which had been coated in an ice hath. The soiution was stirred at 0°C for 30 min when reaction was shown to he comptete by LCMS. The reaction mixture was added drop—wise to a mixture of ice and saturated sodium bicarbonate on te neutraiise the ereaeetid acid. The mixture was extracted with DCivi (4 x 75 mi.) and the combined extracts were washed with water (100 mi.) saturated brine (100 mL), dried (MQSCM) and evaporated under reduced pressure to give the t 15 as a yeiiow seiid which was used without further purification (0.116 g, o) Anaiytieai Data: RT 233 min; M8 (5.8") m/z ive intensity) 703 (iii/i + ti“, 100).
(C) N-( 15—(4-(3, 5-bis(((($ * Zilmethexy-2—methyienesdsoxofl 3, 5, 1 tastetrahydrd— 1H— pyrroidféi ‘i—ejf“! , 4ibenzodiazepin~8-yijexytmethyi)piien_yi)piperazin— 1 -yi)— ”idem—3, 5, 9, 72— tetradxapentadecytimtEi—(Z, 5~didanZ 5—dihydrom 7 H—pyi‘i‘fli— 1ny!)prepanamide (‘3 it”) 0 0 N 0 a twww [N] {:1 itV WO O ° [N] fifi:(N O O H, _) o o N~ H, H 0’ \0 1s :71)“;“k H affix/If,N 17 “lira 0 o o o N—(B—Bimethyiaminepropyi)mw’wethyiearhediimide hydreehieride (35 mg, 0.18 mmei, "i.“t ed) was added to a seiutien of compound (15) (1 t6 mg, 0.165 mmei, it) ed) and ’i~(2,5—dioxe— 2,5~dihydrdt H~pyrrei~t~yi)-3—exe—7,t6,13,t6—tetradxa~4-azanenadeean—tEadie acid (1%) (69 mg, 0.165 mmdi, 1.0 eq) in anhydreus DCiVi (5 mt.) under Argon. The resuitant soiution was stirred at room temperature for 2h. The eh mixture was diiuted with DCi‘t/i (5t) mL), washed with water (100 mt)a saturated sodium hydrogen carbonate eh (iQO rnL), water (tut) n‘iL), brine (100 mL), dried (M9894) and ated under reduced pressure.
Purification by ‘iiash eoiumn chromatography {CHCig/i‘v’ieGH 9% to 5% in 1% increments} gave the product “i? as a yeiiow giass (0.658 g, 32%) Anaiytieai Data: {oi‘gg z {+6281 (c 2 0.25, ; RT 2.85 min; MS (ES?) m/z (reiative intensity) 1101 (iii/i + 1}“, 40) Exampie 5 (a) tart—buty.’ (4242, on2, 5ndihydro~ 1 H—pyrroin 1’ nyi)n3 ?’—oxo— 3, 6, 9, 7' 2, 15, 18, 2 7', 24, 27, 30, Sit—Lindacaoxa—Sdazadoa‘etracontyi)oxycarbamare (1d) IowoA/Owowowowowowowowowt.H2 , H 13 IOWONOwONOwO~O$oNOwOwowNH N \ (I) 0W BOC’ 0 S—it/iaieimidohexanoie acid (64 mg 0.30 mrnot) was added to a stirred soiutien of EDCi (64 mg, 033 mmoi) and the primary amine 18 (200 mg, 030 mmoi) in dry DCit/i (6 mi.) at room temperature. The reaction mixture was stirred under an argon atmosphere for 16 hours at which point anatyste by LClivtS showed a substantiai amount of desired t at retention time 1.38 min {(ES+) m/z 854 + it?” ~30% reiative intensity)“ 8?? (tit/H Nay.a ~100% reiative inteneityfi anied by ted 18 at retention time 1.0? min, note that both starting rnateriai and product had weak UV absorption (214 and 254 nm) and were best detected on 58+ TiC. Additionai 6—maieimidohexanoie acid (32 mg, 0.15 minot) and EDCt (32 mg, 0.1? mmei) were added to the stirred mixture untii starting materiai was eompieteiy consumed (aejudged by LCIMSL The reaction mixture was diiuted with DCi‘t/i (100 mt.) and washed with its?) (3 x 30 mL), brine (40 rot.) dried (M9304), tiitered and evaporated in vacuo to provide the crude product. Purification hy‘ flash chromatography (gradient eiutien in 1% ineremente: 100% DOM to 96:4 w’v DCM/MeQH) gave the amide “it? as an oii (214 mg, 83% yieid). (b) N~(35~(arriinooxy)—3. 6, 9, 12. 15, 18, 21, 24, 27, 30, 33—uridecaoxapentatriacorityijhdfl 5—dioxo~ 2, dedihydro— 1 H—pyrrofl1»y{)hexanamide (2d) IOWONOWONOWONOwowowO~O\/\NH 800’ O OJO\/\o’\/O\/\o/\/O\/\o’\/O\/‘o/\/O\/\o’\/O\/\NHOWN ‘ H’ O 'H 2% A sciutioh o195:5 v/v TFAli-igO (2 mt.) was added to a samoie of the BociTHP—oroteoted compound 1% (214 mg, 0.25 mmoi) at 0 °C (ioeiaoetooe). After stirring at 0 °C for1 hour the reaction was deemed compiete as judged by LCiiViS. desired t peak at retention time 1.06 min {(ES") rrtiz 754 + H}".. ~100% reiative intensityi}, note that both starting materiai and product had weak UV absorption (214 and 254 nm) and were best detected on ES+ Th3.
The on e was kept eoid and added drop wise to a d saturated aqueous soiiition of NaHCCig (100 mL). The mixture was extracted with DCin (3 x 30 mL) and the combined organic iayers washed with brine (50 mt.). dried $th04), tiitered and evaporated in vacuo to provide the oxyamihe 21.} as an oii (11.11 mg, 85% yieid) which was carried through to the hext step without further purification. (c) (1 1S,11233,11’3“,11a’S}~di—tert—buiyi ((5~formyi—1,3— phenyiehe)bis(rnethyiene))t>is(oxy)}bis(?—me1hoxy~2~methyiene-S—oxo—1 1*((tetrahydro-2H— pyran—2—yi)oxy)—2, 3, 1 1 , 1 1a—tetrahydro-1H—pyrroio[2, 1—c}[1,djbenzodiazepine— 10(5111— carboxyi‘ate) ('21) T 21 3,5~bis<brorriorrrethyi)benzaidehyde (260 mg, 0.90 mmoi) [Enrique Di'ez—Barra et at J. Org.
Chem. 2061, 65, Seed—5670} was added to a stirred soiution of Boo/'THRprctected PBD g uhit "i (828 mg. 1.79 mmoi). TBAE (33 mg. 89.? pmoi) and K2003 (247 mg, 1.79 mmoi) in dry DMF (12 rrrL). The reaction mixture was heated to {:30 °C and stirred under an argon atmosphere for 2.5 hours at which point artaiysis by LC/EVES reveaied h‘tiai product formation at retention time 1.92 min {(ESW mi}: 1051 (iii/1+ H12 ~fi5% reiative intensity) 1073 (iii/1+ Na} ~25‘”o reiative intensity)}. The reaction mixture was aiiowed to cost to room temperature and the DMF was removed by evaporation in vacuo. The resetting residue was partitioned between water (50 mt.) and EtOAe (50 mi.) and the aqueous phase was extracted with EtOAC (3 x 15 inL). The combined o iayers were washed with water (2 x 20 int), brine (30 int), dried (M9804), tittered and evaporated in vacuo to e the crude product. cation by flash chromatography (gradient eiution in ‘i % increments: 50:50 Viv EtQAC/hexane to 80:20 v/v EtQAo/hexahe) gave the biswether 21 as a white foam (717 mg, 76% yieid). Note that 21 was isoiated as a mixture of diasteroisomers arising from THP protecting group. (d) (1 1 S, 1 1aS, 1 1 ’3, 1 1 a’Sj-di—terrebutyi 8, 8’—(((5-((syn/anti}—45*(2, 5—dioxo-2, 5~dihydro—1Hs ~1-}!!1-40—oxo—3fifl, 1 2, 1 5, 18,21, 24, 2?,30, 33, Ltd—dodecaoxa—Z’,fig—diazapentatetraconthen —1111)" 1 1 n . (gt—phenyiene)bis{methyiene))bis{o>ry),ibis(7—methoxy—Zmethyi‘enem5—oxon1 ({tetrahydro—2H—pyran~2~yi)oxy)—2, 3, 1 1 , 1 ‘rahydro—1H—pyrroio[2, 1 —ej[1,djbenzodiazepina 1 01’5H2*carboxyiare) (22) O H Refine: IOWONOWONOWONOWONOWO/Vowh‘H N \i O O Bfifiafi 22 PTSA (4.1 mg, 21.4 nmoi) was added to a stirred soiution of the de 21 (224 mg, 0.21 mrnoi) and the oxy—amihe 21} (161 mg 0.21 mrnoi) in dry DOM (3 mi.) at 0 “C (ice/acetone).
The reaction mixture was aiiowed to stir at O ”C under an argon here and stirring for 3 hours ahaiysis by LCliviS retreated oompiete consumption of ne 211 (retention time 1.08 min). presence of desired product {retention time 1.85 min (EST) m/z 178? (iii/1+ HY” ~25% reiative intensity) 1810 (iii/1+ Nat” ~QG% reiative intensity» and onreaeted aidehyde 21 (retention time 1.91 rnin). in order to avoid ed THP oieavage (observed in eariier test reactions), the reaction was quenched at this point aithough aidehyde had not been compieteiy consumed: The mixture was diiiited with DCit/i (51) int.) and washed with i‘taHCCe (3 x 15 mt), brine (30 mid; dried 804), 'iiitered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient eiution in t% increments: 100% DCi‘v’i to 96:4 Viv DCi‘vi/‘MeQi-i) gave the ti oximes 22 as a white foam (215 mg, 56% yieid). Unreacted aidehyde 21 (83 mg) was recovered during fiash chromatography.
Note that 22 was iseiated as a mixture of diasteroisomers arising from THP protecting group. (e) N—(i'syri/ariti}~1~(3, ((g’$)—7—methoxy~2~metityierie—d—oxo—Z, 3, 5, 1 ”i a—tetrahydro 1H~ pyrroiog’é’, 1-e][1, djbenzodiazepins8~yi)oxy)methyi}phenyi)~3, 6, 9, 12, 15, 18, 21, 24,27, 30. 33,36- dodecaoxanienazaoctatriacon tm 1—enn38—yi)—6—{2, 5ndioxow2, 5~dihydro— 7 H~pyrroim 1 - yi)hexanamide (23) IOwO/VowoNOwONOwo/Vowo/VOV\NH N \ C.’ o H /N O THPO’ .300 HP Hit. N O O N 22 wOMe MeomN H O O o o o o o o I \/\o/\/ \/\o/\/ \/\o/\/ \/\o/\/ \/\o/\/ \/\NH N \ C.’ o 0&0/N O «N N\ 23 H H o o A soiution 0195:?) w’v TFA/HZO (1 mi.) was added to a sampie oi the BoelTHP—proteoted compound 22 (204 mg, 0.11 mmoi) at G ”C (ice/acetone). After stirring at 0 °C for minutes, the reaction was deemed te as judged by LCliviS, desired product peak at retention time 1.42 min {(ES") mi’z 1383 i- iti"., <5% ve intensity)}. The reaction mixture was kept eoid and added drop wise to a ehiiied saturated aqueous soiution of NaHCQg (50 min). The e was extracted with DCiVi (3 X 15 mi.) and the combined organic iayers washed with brine {Cit} mL), dried (MgSQ4), tiitered and evaporated in vacuo to provide the crude product, Purification by tiash chromatography (gradient eiution in 1% increments: 100% CHCig to 96:4 Viv CHCigih'teQH) gave the eeted syn/anti oximes 23 as a yeiiow thin fiim (85 mg, 54% yieid). Aneiysis by reversewphese nitra~higheperiormanoe iiduid chromatography (see Generai information section for conditions) reveaied predominantiy two peaks at 16.15 min (syn isomer. minor component) and 16.42 min (anti , major component). (f) nti)— 1 ~13, 5~bis((((8)— oxy—E—methyiene~5~oxo~2, 3, 5, 1 1a~tetrahydro~ 1H— eflg, 4jbenzodiezepin~8-yi}oxy)me1hyi)phenyi)—3, 6, 9, 12, 15, 18, 2 1, 24, 2?, 30, 33, 36* docieceexe—Q—azaoctetriecont— 1—en-38~y{i-5—(2, 5~dioxo—2, 5—dihydro- 1H—pyrroi— 1— yi)hexanamide (24) IO\/\0/\/o\/\o/\/o\/\o’\/o\/\o/\/o\/\o~0%NH H N O ’N Che N‘ H” H N OMe MeO N o 0 Compound 23 was subjected to puritioation toy preparative HPLC (see generai information section for conditione). The peak eEuting at a retention time of 16.42 min was ieoieted and iyopniiised to provide the enti—oxin‘ie 24 (9.9 mg): "H NEVER (406 MHZ, CDCig) 5 8.12 (s, ‘i it), 7.66 (d, 2H, J = 4.4 Hz), 7.524.591 (m, 2H), 7.534751 (m, 3H), 5.82 (3, 2H), 6.58 (8, 2H), .16 (or s, 2H), 5.25-5.14 (m, 8H), 433(1, 2H, d 2 4.8 Hz), 4.28 (tar s, 4H), 3.96 (s, 6H), 3.904384 (m, 2H), 3.82~3.78 (m, 2H), 3.8?~3.49 (m, 43H), 3.4?)“342 (m, 2H), 3.15~3.08 (m,2H),2.98n2.90(m,21-i), 2.15 (t, 2H, J = 7.5 Hz), 1.70—1.54 (m, 4H), .24 (m, 2H). (g) Nm(g’eyri)n1—(3, 5nbis{(((3)~ 7~methoxy—2—methyiene~5moxon2 3, 5, 1 1amtetrai1ydro—1H~ pyrrolofl, ,4ft)enzodiazepin~8~yijoxygin2ethyi)pher:yi)~3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36— dodeceoxae2*azaeetatriacont— 1—ent38~yi,i~5-(2,5~dioxo—2, 5—dihydro-1H—pyrroi— 1— Whexenemide (25) H \”‘0J/CK/‘QNOWQNOWQ/V'DV‘QNDWQNOWN H0%)); ofi/o o fipwe,N a; H H ,, MeOIZrh o 0 Compound 23 was subjected to puritioation toy preparative HPLC (see generai information section for conditions). The peak eEuting at a retention time ot16.15 min was ieoiated and iyophiiised to provide the syn—mime 25 (5.2 mg): 1i-E NEVER (400 MHZ, CDCEg) 5 ?.§3m7.91 (m, 2H), 7.65 (d, 2H, J = 4.4 Hz), ?.57—?.55 (m, 1H), "1’52 (3, 2H), 7.32 (3, 1H), 5.82 (s, 2H), 6.68 (s, 2H), 5.18 (or 5.25M5.14(m,8H),4.35(t,21-i,.i = 5.1 Hz), 4.28 (or s, 4H), 3.95 (3, 5H). 3.QOK---3.84(m,21i),3.81 (t, 2H, J = 5.1 Hz), 3.68---3.48 (m, 43H), 3.45----3.42 (m, 2H), 3.154357 (m, 2H), 2.97~2.90 (m, 2H), 2.16 (t, 2H, J z 7.6 Hz), 1.704.514 (m, 4H), 1.364.241 (m, 2H).
Exampie 6 (a) Dietertetmtyi 8,8'—(((5—(1—emin0~15-oxo-3, 6, 9, 12~tetraoxe~1 6—azanonedeo18~yri~1Q-yi}-1,3~ phenyienejbisr’memyiene,i)bisg’oxy))(1 18, 1 1 e8, 1 1 ’8, 1 1e ’Sj—bisi’7—metr‘10xy-2—methyiene—fi— exam 1 1—{('tetrahydro—EHmpyranQ—yt)oxyiméf, 3, 1 1, 1 1amtetrahydro—1H~pyrroio[2, 1~C][1, 4jbenzo diazepine—10(5H)—oarbexyiate} (25.?) H OWOWONOWONN‘BOC OWOWONOWONBKBOC —> HN + \I‘I 27 | OYVOWO/VOWONN‘BQC THPO SOC E; :M\/©\/2:Me: o”Biggiefifth; JfiiIo BOCOTHP THPO’ OWOW ~O\/\ 0 O , || THPO IO BOCOTHF’ Eflimm “”2125?th (i) tert—Buty! (15—oxo~3, 6, 9, 12—tetraexa~it’d—azanonadeo—18—yn—1—yi)oarbamete (2 7) EDCi (263 mg, 1.37 rnmoi) was added to a stirred soiutiort ot reboomN—arnidodPEG®4—aeid (2&3) (500 mg, 1.37 mmoi, Strateoh Scientific Limited) and gyiamine (88 iiL, 7% mg, 1.37 mmoi) in dry DOM (10 mi.) at room ature. The reaction mixture wee d under an argon atmosphere for 18 hours at which point eneiysis by LC/MS showed a eubstantiei amount of desired product at retention time 1 26 minutes (ES+) miz 403 (iii/1+ Ht ~50% reiative intensity} 425 + Na} ~10G% reiative ity), note that both starting meteriai and product had week UV absorption (214 and 254 nm) and were best detected on ES+ TEC. The reaction mixture was diiuted with DCiv’i (100 mi.) and washed with H20 (30 mt), brine (40 mL). dried (iVigSm) tittered and evaporated in vacuo to provide the crude product.
Purification by tiesh chromatography ent eiution in 1% inorerrtents:10@% DCiVi to 98:2 WV DCM/‘iVieQi-i) gave the amide 2? as an oii (3912 mg, 719’o yieid). (it) Di—tert—butyi 8,8’4115—12,2ndi’methyi—4,2G—dioxon3,8, 1 1, 14, 1 7—pentaoxan5, 21—diazatetracesn 23~yrt~24~yi)— 1 , 3~phenyiene)bisg’methylene)')bisr’oxy))(1 18, 1 138,1 1 3,1 1e ’S)—bis(?~methoxy—2— rrrethyiene—5~oxo~t1«{(teErahydro—2H~pyran~2—yiioxy)~2,3,17',17a~tetrahydro—1H~pyrroio[2,7~ c][1, 4jhenzoo'iazepine—10(5H)~carboxyiate) (28) A oatatytic amount of Pd(PPhg)4 (23.0 mg, 19.5 umoi) was added to a mixture of the iodoaryi compound 2a (1.02 g, 0.89 mmoi), Boc—acetyiene 2? (393 mg, 0,98 mmoi), Cui (7.4 mg, 39.1 umoi), diethyiamine (2.02 mL, 143 g, 19.5 rnmoi) and oven—dried 4A moiecuiar sieve peiiets in dry DMF (9 mi.) in an oven—dried seaiahie vessei. The mixture was degased and tiushed with argon 3 times then heated in a microwave at ttii) °C for 26 minutes at which point anaiysis by Lil/MS reveaied substantiai product formation at retention time 1.8g minutes (ES+) miz i446 (iii/H Nat”, ~‘i00% ve intensity, 1424 ([M+ HT, 45% reiative ity). The reaction mixture was aiiowed to coat to room ature and was then eci through a sinter to remove the sieves (washed with EMF). The tiitrate was evaporated in vacuo and the resuiting residue dieeotved in BCM (190 mL) and washed with H20 (20 mt), brine (30 mt), dried (it/igSOt), red and evaporated in vacuo to give the crude product. Purification by fiash chromatography“ (gradient eiution in 1% increments: 100% DCM to 97:3 viv DCMlMeOi-i) provided the aikyhe 28 as a yeiiow foam (882 mg, 70% (iii) Di~tert~butyi 8, 8’~g’({5—(1—amino~15—oxo—3, d, 9, 72~tetraoxa—16~azanonadec~18~yrr~19—yi)— 1,3*phenyienefihis(nethyieneflbifloxyfi(1 13, 1 1aS, 1 1 ’3, 1 1a’S}-bis(?—methoxyrrtethyiene— —oxo~’i 1—({tetrahydro-QH-pyrart—B—yiion-Q, 3, t ”i, 1 1a~tetrahydro— ‘iH-pyrroion, 1-ci[1,4ihenzo diazepinen10(5Hj—carboxyiate) (253) TBDMSQT‘E (1,42 mL, 1.64 9, 6,2 rnmoi) was added to a stirred soiution of the tri—Boc protected compound 2% (882 mg, (WE mmoi) and tidine (oiee mL, 883 mg, 8.25. mmoi) in dry [30M (15 mi.) at room temperature. The reaction mixture was aiiowed to stir under an argon atmosphere for 16 hours during which time is by S reveaied formation of the T83 carhamate at retention time 2,09 minutes (ES+) mi}: 1504 ({va Naf', 400% reiative intensity). The reaction mixture was diiuted with DCivi (60 mL) and washed with saturated Nine: (2 x 20 rnL), H20 (20 mt), brine (30 mL), dried (M93304), tittered and evaporated in vacuo to give the crude TBS oarhamate. The product was re—dissoived in THF (15 mi.) and treated with a on of TBAF (744 pi. ot a tKGit/i soiution in THF, 0744 mmoi) at room temperature. The reaction mixture was aiiowed to stir for “i hour at room temperature at which point anaiysis by LCIMS reveated ntiai product formation at retention time 145 minutes (ES+) miz "i324 (EM-r Hi", ~60% reiative intensity) aiong with product corresponding to t NtOBocl’i THP eieaved at ion time 129 minutes (ES+) mlz ’i‘i2’i ({M+ HY, ~‘iu% reiative intensity), 1138 (iii/1+ HgOF', ~26% reiative intensity) and product corresponding to 2 N10 Soc/'2 THP oteaved at retention time 1.12 minutes (ES+) 1 i7 miz 919 (MM HF, ~2,5% reiative intensity), 937’ ({va HZOE'“, ~39?) reiative intensity). 955 (W+ 2H2Cif'. ~5% reiative intensity). The THF was removed by evaporation in vaouo and the resetting reeidue re—diseoived in DCiv‘i (60 mi.) and weehed with Saturated Ni-hCi (2 x 20 mL), H20 (20 ed), brine (30 mL). dried (MgSOQ, fiitered and evaporated in vacuo to give the key amine 23 as a h foam. (is) (R)—2-r’pyridih—2—yldisuifanyi)propyi {7 9—(3, 5—bis(‘(r’{8)— 7—methoxy—Q-methyiene—S—exe— 2. 3 5, "i trahydre— 1Hmpyrroiof2, 1~oj[‘i, 4jbenzooiiezepin—8ny!)oxyfiimethyiMhem/w "i Emoxo— 3. 6, 9, raoxa—16~azanonedec~18~yn~1—yiicarbamate (33) OYVOWQNOWONNHZ .\ D‘SNOH . C , II _) / 0 $0c i?Lif;ouie.. . 31 THPO H‘- N wordsO 51‘.erquO H ."i 1::- _ H H E I I N\ S‘SNOYNwONOwO/VOWN / O 0 || THPO EEOC weenie —> ’1 N O C! N H, NflOh/ie mom/NH O O _ H H E I N\ 3‘ s/\/(kn/Nw0/\/'z-K/‘ONO\/\n/N. .
/ " OH N N H, O O N~‘(‘z : \ H OMB MeOIZ'rN O O (i) (Ft)(pyridih—2—yidisuifah_vi)propyi oarboneehioridate (31) Triphoegene (9.323 mg, St .5 pmoi) was added to a stirred eoiution of (R)—2~(pyridih—2— yidisuifahyiipropan—i—oi (3%) (18 mg, 0.09 mmoi) and pyridine (6.7 pL, 6.6 mg, 0.98 mmoi) in dry DCM (“i ihL). The on mixture was aiiowed to etir under an argon here for 39 minutes after which time the eeiveht was removed by evaporation in vacuo to provide the crude ehiorot‘ormate 3’3 as a white foam. Note: The product was carried through to the next step without purification or ahaiyeie. (ii) Di-tert-butyi 8, ’5er’(R)—5, 2 1—dioxo(pyridin~2~yidisuifenW4, 9, 12, 15, 18—pentaoxa~ 6,22-diazapentacos—Q4~ynyti-1,3~ phenyiene)bis(methyiene))hisr’oxy»(1 1S,11aS,1 1’S,11a’S)"bier/7m?ethoxy—Qnmethyienend— oxo~1 1 firetrahydro~2H~pyra{HE—y!)oxy)~2, 3, 1 1, 1 1a~tetrahydro—1H~pyrroio[2, 1~e][1, 4] henzodiazepine—10r’5i-{hcarboxyiate} (32) A soiution (3131 (~23 mg, ~01}?! mmot) in dry DCivi (1 mi.) was; added drop—wise to a stirred on of amine 29 (~116 mg, ~0.0Q mmoi) and pyridine (7.8 uL, 7.7 mg, 0.1 rnmot) in dry DCM (1 mt.) at room temperature. The on mixture was aiiowed to stir under an argon atmosphere for 3 hours at which point ahaiysie by LCD/MS (Kinetex® ooiumn) reveaied eubatantiai product formation at retention time 2.02 minutes (58+) 11712 1550 (tit/1+ HT; ~20% reiative intensity) atong with persistence of ted starting materiai 2% at retention time 1.51 minutes. The seivent was removed by evaporation in vacuo to provide the crude oaroamate 32 which was carried through to the next step without further purification or anatyeia. (iii) (R)n2n(pyddin—2—yidisuifany11propyi (18313,dubis((((8)~7~methony—methyieneémexew 2, 3, 5, 1 1a~terrahydro~1H~pynroio{2, 143,211,tithen.2:odiezepin~8—yi’)wry)methyi),ohenyi)~ 1 5~oxo~ 3, 6, Q 12—tetraoxa~16-azanonadec—18—yn—1eyi)carbamate (.33) A eoiution 0195::‘3 Viv TFAii-i20 (1 mi.) was added to a crude aampie of the Boo/Tint?— protected compound 32 (~136 mg. 88 umoi) at t} ”C (ice/acetone). After stirring at O ”C for 1 hour the on was deemed eornpiete as judged by LCli‘v’iS (Kinetexifi) coiumn), desired product peat: at retention time 1.42 minutes (ES+) mix 1146 (tit/1+ Hf, ~90% reiative inteneity). The reaction mixture was kept said and added drop—wise to a ehiiied saturated aqueous eoiutien of NaH003 (50 mt). The mixture was extracted with DOM (3 x 15 mi.) and the oomhined organic iayers washed with brine (20 inL), dried (it/19894), ed and evaporated in vaeuo to provide the crude t. Purification by ttaeh chromatography (gradient eiution: 100% CHCh to 95:5 Viv CHCi-gfi‘vieOi-t) gave 33 as a tiirn (10 mg, 7% yieid): LCiiViS (15mminute run). ion time 5.79 minutes (135+) miz 1146 (tit/1+ Ht“, ~8% reiative intensity); 1H NEVER (400 MHZ, (313613) 5 8.44 (d, 1H, J = 4.8 Hz), 7,75m7.58 (m, 2H), 7.66 (Ci. 2H, J = 4.4 Hz), 7.52 (s, 2H), 77.45—27.40 (m, 3H), 198—735 (m, 1H), 5 (in, 1H), $.79 (3,211), 5.434541 (m, 1H), 5.23%.09 (m, 8H), 4.304123 (in, 6H), 4.194110 (m, 4H), 3.9?“ 3.94tm,21~i). 3.96 (s, 6H), 3.9't---3.85(m,211), 3.75 , J = 5.8 Hz), 58 (m, 8H). 3.52 (t, 2H. J = 5.1 Hz), 3.34~3.30 (m, 12H), 3.23~3.08 (m, 3H), 2,96~2.90 (m,21~i), 2.52 (t, 2H, J = 5.7 Hz),1.31 (d, 3H, J = 17.13 Hz).
Exampie 3” OY\/O\/\0/\/O\/\0/\/NH2 O O NOH —> aNOH + SH \N 3 s’ 35 THPO 530C I|3OCOTHP N O O N .3“4 wOMeHI" MeomNH O 0 '1 'i N S N O O N \ \SM \/\O/\/ ’ \/\n/ / O O | | THPO‘ 530C I.3OCOTHP H" N O O N H N~‘(‘ : —> OMe MeO: : ’7’,N 36 O i‘ 'fi |N\ S‘sMNwONOwO/VOWN / O O H" ,N O O N\ H O O (a) (i)~4~{pyridinQ—yidisuifany!)panran0ic acid ('35) hioiTM—Z (176 mg, 0‘88 mmoi) was added to a stirred ori of (2)41“ mercaptepenianoie acid 34 (107 mg, 0.80 mmei, Aurora Fine Chemicais LLC) in EtOi—i (2 mi.) at more temperature. The reaction e was aiiewed to stir for 16 hours under an argon atmesphere at which point anaiysis by LCIMS reveaied substantiai product formation at retention time 1:32 minutes (ES+) ml}: 244 ([M+ Hit, ~95% retative ity)“ The eetveni was removed by“ evaporation in vaeue and the resetting residue purified by fiaeh Chromatography (gradient eiution: 90:13 WV hexane/‘EiOAe to 80:20 Viv hexane/EtOAc) tr.) give 35 as a White seiid (92 mgr 47% yietd) (b) (ij-Di—ieriebuiyi 8,8’—(((5-(5,21~dioxoe24~myridin“2*yidisuifanyit8, 1 “i, 14, 1?»?eiraexa-4, 21?- diazapeniacos—‘i—yn—‘i—yi)—1,3—phenylena}bis{maihyiene))bis{oxy})(1 1 S, 1 1e38, 1 7 ’8, “i 1a’8)- bis('7—merhoxy—2—mathyi‘anem5—exo—1 71’(terrahydro—Qanyran—Zyi)oxy)n2, 3 7 1, 7 1anz‘etrahydro— 1H~pyrreio[2, ”i ~cjig'1,4jberizediazepine—1 (3(5H)~carboxyiate) (35) EDGE (222 mg, 3.12 mmei) was added to a stirred eeiution of (:)~4-(pyridin~2~ yidisutfanyi)perttanote acid (35) (26 mg, {Mt} mmoi) and amine 2t} (~139 mg, 9.1 mmoi) in dry DCi‘v‘i (2 mt.) at room temperature. The reaction mixture was stirred under an argon atmosphere for 29 hours at which point anaiysis by LCIMS showed a substantiat amount of desired product (eoiit peak) at retention time 1.88 minutes (ES+) ml: t548 t- i-if', ~40% reiative ity) aiong with product corresponding to t NtOBoo/t THP cieaved at ion time tit“)? minutes (ES+) {rt/z 1348 ([M+ HT; ~20% reiative intensity), 1138 ({M+ Hth‘“. ~20% reiative intensity) and product corresponding to 2 N10 Boo/'2 THP oieaved at retention time 1.43 minutes (ES+ M+ not observed). The reaction mixture was diiuted with DCiVi (30 mi.) and washed with H20 (15 mt). brine (29 mL), dried (MgSQ4), tittered and evaporated in vacuo to provide the crude product 36 as a team. (0) (i)~N~(3~(3, (((8)—7—methoxy~2~methyiene—S—oxo—Q, 3, 5, 1 1a—tetrahydrc~1H-pyrrolo[2, 1- cjfl , 4]stnzodiezepin—B—yi)ext/finethyi)pitenyi)propyn~ 7 —yi,i{4—(pyridin yidisuifanyijpentanamidoj—B, d, 9, t Quietraoxapentadecan— “i Swamide (It?) A soiution of 95:5 va TFA/HgO (1 mL) was added to a crude sampie oi the BociTHPs protected compound 35 (~183 mg, {ltd mmoi) at Q “C (ice/acetone). After stirring at 0 “C for 1 hour the reaction was deemed cemeiete as judged by LC/i‘v’iS. desired product peak at ion time 1.44 minutes (ES+) miz H44 (iii/Pr Ht", ~3% reiative intensity). The reaction mixture was kept coid and added drop—Wise to a chiiied saturated aqueous sotution ct NaHCQg (60 mL). The mixture was extracted with DCiVi (3 X 30 mt.) and the combined organic iayers washed with brine (39 mL), dried (MgSQ4), tittered and evaporated in vacuo to e the crude product, Purification by tiash tography (gradient eiution: 100% CHCig to 923:4 viv iVieOi-i) gave 3'? as an orange team (69 mg, 57% yieid): LEE/MS (1%} minute run), retention time 5.72 minutes (ES+) mix 1144 (tit/H HT, ~3% reiative intensity); 1it NMR (400 MHZ, CDCig) a 8.42 (d, tit, J = 4.3 Hz), 539 (in, 2H), 7.66 (d, 2H, J = 4.3 Hz), 7.52 (8, 2H), 7.45-7.40 (m, 3H),, 7.09-7.04 (m, 1H), 6.98-6.94 (in, tit). 6.80 (3, 2H). 8.40%.35 (m, tit), 5.234339 (in, 8H). 4304.23 (m, 6H), 449411.10 (m, 4H), 3.923 (s, 8H), 3.9’i—335 (m, 2H), 3.?5 (t. 2H, J = 5.? Hz), add—3.58 (in, 8H), 3.51 (t, 2H, J = 5.6 Hz), 3.42— 3.39 (m, 2H), 3234.08 (in, 2H), 2964.90 (m, 3H), 2.52 (t, 2H, J I 5.6 Hz), 2.34 (t, 2H, J I 7.4 Hz), 1.94 (q, 2H, J = 24 Hz), 1.30 (d, 3H, J = 6.7 Hz).
Exempted OWQW /\/O\/\ NNHZo 0 i-i2N\/\lr0H , ‘i‘ §\’ + o N ')H _> o Y W‘ H o o 33 39 50p BOG THPO’ I | 0TH F' H ., N A .13. N H 0MP: 941501erQ 0 O 40 TH PC) | H F H \/UYNvYNwONO\/\O/\/OWNI I.
O O 41 j; :H ’N o 0 Pk H OM52 MeO: : ’w’h o o (a) 3~i’((pro,o~2~yn~1Mow)cart;oriyiiamirioipropanoic acid (39) A eoiution of propargyi chiorotormate (315 ui... 383 mg, 3.23 riimoi) in toiueoe (2 mL) was added drop—Wise to a stirred mixture of fi—aianihe (33) (250 mg, 2.8t mmoi) and Nat-i803 (678 mg, 8.1 mmoi) in H20 (7 mL) at room temperature. The reaction mixture was aiiewed to stir vigoroueiy for 16 hours after which time it was partitioned. The s Eeyer was diiuted with H20 (20 mL), washed with EEO (4 x 1!) mL), chiiied to 9—5 °C (ice/acetone) and acidified to pH 2 with concentrated HCi. The acidic eoiutiori was. ted with EtOAc (3 x mi.) and the combined organic iayei‘s washed with H20 (ti) mL), brine (20 mL), dried (M9804), fiitered and evaporated in vacuo to provide the crude product 33 as an oii. which was carried through to the hext step without r purification. (b) Di—tert~butyi 8, 8 ’—(((5n(5, 9, 25mtrioxom4, 13, “i 6, 19,22mpentaoxa—6, 1G,264riazanonacosa—128— diyn—QQ—yl)—1,3—pheriyieneibisr’met‘hyienei)bisiOxy»(”i 13, “i 1533‘, “i 1 ’8, 1 ”la s{?~methoxy—2~ methyiene—doxo»? trahydro—2H»pyran~2~yi)oxy)~2, 3, ‘i‘ “i, ‘i‘ ta~tetrahydro— “i H~pyrroio[2, “i“ c][1,4]benzodiazeoine-10(5Hi—carhcxyiete) (4t?) EDGE (24 mg, 0.13 rnmoi) was added to a stirred eoiution of 3—(((prop—2—yn—1— yioxy)oerbony‘i)emino)propenoie acid {39) (18 mg, 0.10 mmoi) and amine 2% (~130 mg, 0.10 mmoi) in dry [30M (3 mt.) at room ature. The reaction mixture wee stirred under an argon atmosphere for 1.5 hours at which point eneiysis by Lit/MS showed at substantiai amount of desired product at retention time 1.84 minutes (EEH) miz 1477 (tit/H H}"', «20% reiative intensity), 1499 (iii/2+ Na?’, ”22% reiative intensity) eiohg with product corresponding to 1 N1OBoo/1 THP oieeved et retention time 1.60 minutes (ES+) m/z 12% (Elwi- Hf, ~10% reiative intensity) and ted 5 at retention time 1.47 minutes 635+) miz 1324 (iii/it" Ht“, ~5% reietive intensity). The reaction mixture was diiuted with DCiVi (20 mL) and washed with H20 (2 x 10 mL), brine (20 mL), dried (ii/19304), tittered and evaporated in vaeuo to e the crude product 40 as e foam. (5) Prop—Qnyn— 1 my! (EBB—('3, 5~bis{(‘('(S)n7—methoxy—Qnmethyienend—oxo—E, 3, 5, ‘1 tantetrehydro— 1H" pyrroiog’é’, 1-C][1,diabenzodiazepinedit/dew)metityi}phertyi)~3, 1 9-dioxo* 7, ‘i 0, ‘i 3, 1' 6—tet‘raoxe- 4,20—diazatricos—22~yn~1~yiicarhemete (4 1') A eoiution of 95:5 v/v TFA/Hgfi (2 mL) was added to a crude sampie of the Boo/THP~ protected compound 4% (~155 mg, 0.10 rnmoi) at G “C oetohe). After stirring at 0 “C For 1.5 hours the reaction was deemed oompiete as judged by“ LC/‘MS, desired product peak at retention time 1.41 minutes (138+) miz 1073 ([M+ Hf, ~30% reiative intensity). The reeotion mixture was kept ooid end edded drop—wise to a ohiiied saturated aqueous soiution of NaHCQg (80 mL). The mixture was extracted with DCM (3 x 20 mi.) and the combined organic ieyere washed with brine (25 rnL), dried (MgSOt), iiitered and ated in vacuo to e the orude product. Purification by fiesh chromatography ent eiution: 100% CHCig to 95:5 WV CHCEgliMGOH) gave 41 as e yeiiow teem (51 mg. 45% yieid): Lil/”MS (15— minute run), retention time 5.71 minutes (ES+) mfz 1073 + Ht“, ~30% reiative ity); 1H NMR (400 MHZ, CDCig) 6 7.07 (d, 2H, J = 4.5 Hz), 7.52 (s, 2H), 7484.43 (rn, 3H), 7.07“ 7.02 (m AH), 6.80(s,21—i), d.64—6.57(m,1it), 5.78—5.72im ,1H), 5.21—5.09(m, 81-1), 4.64 (d. 2H, J = 2.2 Hz), 4.294125 (m .611), 3.96 (2;, 6H). 3.904385 (m, 2H), 3.76 (t, 2H, J = 5.9 Hz), 3.65----3.35 (m .1814), .07 (m, 2H), 2.93 (d, 2H, J z 16 Hz), 2.52 (t. 2H, J 2 5.9 Hz), 2.48m245 (m, 1H), 2.40 (t, 2H, J = 5.9 Hz).
Exempie 5 OYVOWONOWONNHZ (Ting HN + II N:/V\fl’/\HgLN 135,0,<1 Egg;800THPO BOCO 42 mick YNHZ OYVOWONOWO’VNI3%“?in THPO 53°C P H N wOMeo MeomrNHo O O o NH2 O 0 H E H OY\/O\/\o/\/O\/\o’\/N\nlo O O HN 0 H2313 1313235 (a) 131—15511bun/18,8”((15”(1(4((S)——2~((1’n812”(6(2,55dioxo~2, 5—d1hydron1H—pyrmi~1~ yijhexanamidc11~3me11211111111ana{riidOHS—ureidopentariamidmphenyi)319diex0~ 2, 7, 11:1, 13, 16-pentadxe—4, 20—diaza11100522117231!)-1,”?h.- 111101111151101131511215111yiene))e:s(dxy))(11S 115$ 11’S,11—a'S)13139-112911;oxy—2—me1hyiene5- oxon1 1—{(1e1rahydm—2anyran—2—yijoxy)n2, 3, 1 1 1 1anz‘etrahydm—1anyrreiof2, 1 _ 01111, 4jber2zediazepine~10(‘5H1—Carbexylate) (43) DiPEA (44 11L, 32 mg, 0.25. mmei) was added to a stirred on of key amine 23 (~155 mg, 0.11 ndthenitmpheny1 carbonate 42 (84 mg, (1.11 mmoi) in dry DMF (3 n1L1e1reom temperature. The reaction mixture was aiiewed 10 stir under an argon atmespnere for 3 days after which time anaiysis by LCIMS reveaied desired produd observed at retention time 1.80 s (ES+1 11712 1922 (1111+ H1; ~40% reietive intensity),1944 (1111+ Na1, ~26% reiative intensity) aidng with product corresponding to 1 111080911 THP d at retention time 1.61 minutes (ES+) {11/2 1720 (1111+ H1“, ~20% reietive intensity). The DMF was removed by atioh in vaetio and the resuitihg product 43 carried through to the next step without r purification or is. (b) 41’(S)m2m('(S)n2—(6—(2, Endioxoma 5—dihydron’i H—pyrroi~1 myi)hexanamido)~3— methylbutariamido)~5—ureidoper:tanamido)benzyi (19173, 5—his(g’(($)~?~methoxy~2~methylerie~ S—oxo—EE, 3, 5, 1 1a—tetrahyo’ro~1H-pyrroio[2, 1-e][1; 4jbenzediazepin—Seyimxy)methyijphenyi} 1 5- oxo—3, 6,. 9, 12~tetraexe—7 6-azanonadee~18-yn—1—yiiearbemare (add) A soiution of 95:5 v/v TFA/Hgfi (3 mL) was added to a crude sampie of the Boc/THP~ protected Compound 43 (~t?3 rng, Quit rhrnot) at 0 CC (ioeiaeetone), After stirring at 0 “C for t5 hours the reaction was deemed oompiete as judged by“ LC/‘MS, desired product peak at ion time 1.42 minutes (ES+) mi: 1518 (iii/H Hf, ~40% reiative intensity). The reaction mixture was kept eoid and added droenwise to a ohiiied saturated aqueous soiution ot Nat-i003 (iOO rrtL). The e was extracted with DOM (3 x 30 int.) and the ed organic. iay‘ers washed with brine (20 rrtL), dried (M9804), tiitered and evaporated in vacuo to provide the crude product. cation by tiash chromatography (gradient eiution: 103% CHCh to 80:20 Viv CHCh/i‘vieOH) gave crude product as a yeiiow team (72 mg, 42% crude yieid). The materiai was further purified by preparative HPLC to provide pure 44 as a thin tiirn (4.5 mg, 3% yieid): LCIiViS (t5eminute run), retention time 5.44 minutes (ES+) m/z 1518 + HP, ~3G% reiative intensity).
Exampie 1 Q (a) ‘i—(3—eminopropenamido}-N—(3—(3, 5-bis(({(8)eZmernoxy-Zmethyieneedoxosz 3, 5, 1 1e“ retranydro— 1 H—pyrroioffi‘, 7—c][1,4jbenzodiazepinfi-yiioxy)metrig/1);)henyi‘mromemynm 1 ~51!)- 3, 6,9, 12ntetraoxaoentadecanm75namide (4?) OwowowowowNHz H HN Ofi/\’O\/\O’\/o\/\o’\/N‘Fmoc BOC BOC THPO | | OTHP _> —> I BOC BOC H:' N O O N H THPO l l OTHP ' ii“ N o o N N OMe MeO N N OMe MeO N O O O O O O O H W / \/\O/\/ ‘Fmoc OY\/o\/\o/\/o\/\o/\/NH2 HN HN H ,N O O M H H ,N o o N\ H o o o o (i) t—butyl 8,8’~((r’5~(1~(9H~fiuoren—9—yl)—3,7,23—trioxo—2,ii1,”i4,1?,23~pentaoxe—4,8,24— triezeheptados~26-yn-27—yi)— ‘i,3-phenyiene)bis(methyleneiibis(oxyi)(1 18,11aSJ 13,? 13's,)“ bis(7-methoxy—2—methy!ene~5~oxo—1 1—((tetrehydro-QH—pyren—E—yi)ext/3L2,3, 1 7, 1 7a—tetrehydro~ 1H~pyrroio§2 ‘i~cj[1,4jbensodiazepine—7 0(5Hficarboxyiate) (45) EDCi (4Q mg, 0.25 mmoi) was added to a stirred soiution of Fmodfimaiemne (86 mg, 021 mmoi) and amine 23 (~279 mg, 3.21 mmoi) in dry DCM (5 mt.) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point is by LCr’EViS showed a substantiei amount of desired product at ion time 1,76 minutes (ES+) n’r/‘z 1617 ([va Hi“, ~10% reiative intensity), 163g ([Mt Nay, 430% reiative intensity) aiong with product corresponding to t N‘tQBoc/‘i THP oieaved at retention time 1.56 minutes 658+) miz 1415 ({M-r Hf; MOB/is reiative intensity). The on mixture was d with DCi‘t/i (39 mL) and washed with H20 (20 mt), brine (29 mL), dried (M9504), fittered and evaporated in vacuo to provide the crude product 45 as a foerni (ii) (QH—fiuoren~9~yl’)methyl (23—(3, 5—bis((r(S)»27~merhoxy—E—metnyienesdoxo-Z 3, 5, ‘i ta~ ydro-1H—pyrroio[2, 7~e}[1,djbenzodiazepin—8—yl)oxy)methyiipheny{)—3, redem— 7, 1d, 13.7dntetreoxa~4,20~diazatricos—22nynn1ny!)carbamete (add) A sotution of 95:5 v/v TFAli-th (4 mt.) was added to a crude sempie of the Boo/THP— protected compound 45 (~34’i mg, 0.21 mmot) at t) ”C (ice/acetone). After stirring at ti ”C: for 1 hour the reaction was deemed oompiete as judged try LCIMS, desired product peak at retention time 144 minutes (ES+) miz 1212 (iii/Pr HY, ~30% reiative intensity). The reaction mixture was kept eoid and added drop—wise to a ohiiied ted aqueous soiution ot Nat-i803 {St} mL). The mixture was extracted with [30M (3 x 20 mt.) and the combined organic iayers washed with NaHC03 (2 x 20 mL), brine (20 mt), dried (it/igSO4), fiitered and evaporated in vacuo to provide the crude product. cation by tiash chromatography (gradient eiution: 100% CHCig to 95:5 Viv CHOEg/i‘vieCti-i) gave pure product 45 as a yeiiow foam (179 mg, 70% yieid). (iii) 1—{S—aminopropanamido)—N—(3—(3,5—bis(((($)~?~rnethoxy—Z—methyiene~5~oxo~2,3,5, 1 1a~ tetrahydro~1H—pyrrolo[2, 1—c][1,4jbenzodiazepin—8M)oxygin:ethyi)phenyi)prop—2—yn— “it—y!)— 3, 6, Q 12—tetraoxapentadecan~iii-amide (4?) Bimethyiamine (735 uL of a 2.0M soiution in THF, 1.47 mmoi) was added to a stirred soiution ot the Finer; protected compound 46 (89 mg, 73.5 umoi) in THF (3 mL) at room temperature. After stirring for 3 hours at room temperature, anaiysis by LC/‘MS reveaied reaction oompietion with desired product at retention time 1.14 s (ES+) ml: 990 ({va HY; ~89?) ve intensity), 1008 (iii/iri- HQQY', ~’iO% reiative intensity), 1926 (iii/H 2H20}+', 45% reiative intensity) aiong with Frnoo oieavage duot at retention time i.88 minutes, The mixture was evaporated in vacuo and crude 4‘? was carried through to next step without r purification or anaiysis.
WO 59981 ’t 27 (b) N~(3~(3, 5~bis(((g’$)— 7—methoxy~2~methyiene~5—0X0—2, 3, 5, 1 1a—tetrahydre~1H—pyrroiefl2, 1~ C][1, 4jbenzodiezepin-B—yi}ext/dinethyliiphenyl)pr0p~2~yn~1-yi,i~1-(3-(2~ bromoaeetamidejprepanamide)—3, 6,. 9, “i 2~tetreexapentadecen—15~amicie (’48) H H I I H2N\/\g/N\/\o/\/O\/\o/\/O\/\[OrN wOMe momH,N O O N\ H 0 O H 'i' 'i' BfitWVWVNWt” wOMe momH, IN 0 O N\ H 0 O Bremoacetic anhydride (23 mg, 88.2 tirnei) was added to a stirred eeiutien of the crude amine 4‘? (~73 mg, 73.5 untet) in DCivi (3 ntL). The reaction mixture was aiiowed to stir under an argon atmosphere at room ature ter 3 hours at which point anatyeie by LCIMS reveaied compietien of reaction with desired t observed at retention time 1.34 minutes (ES+) m/z ’i’i‘i2 flit/H Ht“, ~30% reiative intensity). The eeivent was removed by evaporation in vaeue to provide the crude nreduet. Purification by flash tegraphy (gradient eiutien: 100% CHCig to 93:? Viv CHCEgliMGOH) gave the nreduet as. a yettow team (38 mg, 46% crude yieid). The materiai was further purified by preparative HPLC te previde pure 48 as a thin fiim (5 mg, 8% yieid): LCih/iS innte run), retention time 4.96 minutes (ES+) mi: 1112 (Vt/H HY, ~tO% retative intensity); 1H NMR (400 MHZ, CDCEg) 5 7’6? (d, 2H, J I 4.4 Hz), 7.52 (s, 2H), 7’.46~7.43(m, 4H), 7.1040701: AH), 6.630(3, 2H), 52143.09 (in, 8H), 4.29-4.25 (in ,6H), 3.96 (3, 6H)“ 3.90-6.85 (m, 2H), 3.82 (at 2H), 3.?7 (t, 2H, J = 5.9 Hz), 3.65%.40 (m ,tBH), 3,16%33? (m, 2H), 2.94 (d, 2H, J = 16 Hz), 2.53 (t, 2H; J = 5.9 Hz), 2.43 (t, 2H, J = 5.9 Hz).
WO 59981 Exampie 11 m Aiternete eyntheeis et 15 OYVOwONOwO/VNHZ OWOWQNOWONIQWN;\ HN HN O 0 || || THPO SOC ::\§O§;—> mm 300 .BOCOTHP *9 \ OWOWONOWONNWN HN o o —’ || jam WZJCCDi (a) rtnButy.’ 8,81—m“51/1(2,5—o’ioxo2 5—~o'ihydro 1H—pyrroi~1~yi)~,3 19dioxo 7,10,13,15" tetreexa~4, 20~diazairicos—QZ—yn—Zdyit— 1, 3— phenyiene)bis(methyiene))bis(oxy))(1 15,11e5,1 18,1 1e’3}~bis(?—methoxymethyiene—5— axe-1 1 —((tetrehydre—2H-pyran~2~yijoxy)~2, 3, 1 1, 1 ta-tetrehydre— 1H-pyrroio[2, 1- cjft,4jhenzodiezepinen1G(5H)~cerboxyiate {5) EDGE (61 mg, 0.32 mmoi) was added to a stirred en oi N~rneieoyi—§5~eiartine (53 mg, 0.32 mmoi) and amine 2% (~418 mg, 0.32 mmoi) in dry DOM (6 mL) at room temperature.
The reaction e was stirred under an argon atmosphere for 3 hours at which point is by Lit/MS showed a substantiai amount of d product at ion time 1.80 minutes (ES+) miz 14M (tit/1+ Hi", «45% reiative intensity), 149? (EA/1+ Net", ~100% reietive intensity), atong with product corresponding to 1 NtOBeeI‘i THP cleaved at retention time ’t 56 minutee 1272i(tit/1+ H3", ~80°o reietive inteneitv) 1295 (tit/1+ Na} ~45/lo reietive intensity) and precinct corresponding to 2 N10 80012 THP eieeved at retention time 1.31 minutes (153+ M+ not observed), The reaction mixture was diiuted with DCi‘x/i (30 mi.) and washed with H20 (15 mL), brine (20 niL), dried ), fittered and evaporated in recur) to provide the crude product 9 as a foam. (b) N~(3m(3, 5—bis('(r’{S)n 7—methoxy~2~methyienen5noxon2, 3. 5, 1 1antetrehydrom ‘1anyrroiof'2, 1— cj[1, £1,159r220diazepin~8~yijexy)meihyi)pheny1)prop—2—yn— 1~W— 117312, 5—dioxo~2, 5~dihydro— 1H~ pyrroi~1~yi),orepenarnido)—3, 5,9, 12~tetreoxepentadecen~15*amio’e (15) A soihtien et 95:5 v/v TFA/itgo (5 mi.) was added to a crude sampie of the Bee/THPn protected compound 5 (~46?) mg, 0,32 mrnoi) at 0 "C (icelecetone). After stirring at 0 ”C for t hour the reaction was deemed cornoiete as judged by LCiiViS, desired product peak at retention time 1.32 s (ES+) miz ’iQTO ([M+ Hf, ~‘i00% reiative intensity). The reaction mixture was kept coid and added drop—wise to a chiiied saturated aqueous soiution of NaHC03 (120 mL). The mixture was extracted with DCiVi (3 x 40 mL) and the combined organic iayers washed with brine (50 mL), dried (MgSOQ. ed and ated in vacuo to provide the crude product. Purification by tiash chromatography (gradient eiution: 103% CHCig to 96:4 viv CHCisli‘t/ieQH) gave 1% as an orange foam (202 mg, 60% yieid): [dimg = +351O (c = 0.47, CHCig); LC/i‘v‘iS nute run), retention time 4.88 minutes (ES+) m/z tGTO ({th HT, ~tt‘id% reiative intensity); 1H NMR (490 hit-i2, CDC-E3) 5 7’66 (d, 2H, J = 4.4 Hz), 7’52 (s, 2H), 7.454.413 (m, 3H), 6.98%.94 (m, 1H), 6.80 (s, 2H), 6.661ss 2H), 6.55% 8.50 (rn, 1H), 5.22-5.07 (m, 8H). 4.33-4.22 (m. 8H), 3.96 (s. 6H). 3.Qt----3.85 (m, 2H). 3.82 (t, 2H, J a 7.2 Hz), 3.76 (t. 2H, J = 5.8 Hz), 43 (m, 16H), 3.18~3.08 (m, 2H), 2.94 (d, 2H.
J = 15.7 Hz), 2.54—2.44 (m, 4H).
Reductioniflxidation of Thioiti’iahs for Conjugation Fuii iength, cysteine engineered monociohai antibodies (ThioiViabs — dunutuia, et at, 2008b Nature Biotech, 925-932; Dorhan et at (2009) Biood 114(t3):2721~2729; US 752154’i; US 7723485; WOZOGQIOSEMQ, Shen et at (2012) Nature Biotech, 30(2):184-t‘3=t; dunutuia et at (2008) dour of immun. Methods 332:41n52)exoressed in CHO ceiis were reduced with about a 20~40 toid excess of TCEP (trisiZ~carboxyethyi)ohosohine hydroohioride or DTT othreitoi) in 50 miv’i Tris pH 17.5 with 2 mivi EDTA for 3 hrs at 37°C or ght at room temperaturetGetz et a! (1999) Anal. Biochem. Voi 273:73—80; Soitec Ventures, Beveriy, MA). The reduced Thioitiiab was diiuted and ioaded onto a HiTrap S eoiumn in ti) rnivi sodium acetate, pH 5. and eiuted with PBS containing 0.3M sodium chioride. Aiterhativeiy, the antibody was acidified by addition ct 1/2ch vciume of 16% acetic acid. diiuted with ti) mi‘vi succinate pH 5, ioaded onto the coiumn and then washed with ti) coiumn veiurnes of succinate butter. The coiumn was eiuted with 50 miVi Tris pH7.5, 2 rniVi EDTA.
The eiuted reduced Thioiv’iab was treated with 15 toid moiar excess of DHAA (dehydroascorhic acid) or 200 nit/t aqueous copper e (6:15.304). Oxidation of the interchain disuitide bonds was compiete in about three hours or more. t air oxidation was aiso effective. The re~oxidized dy was diaiyzed into 20 miVi sodium succinate bit . ’iSQ rniVi NaCi, 2 mid EDTA and stored frozen at ~20°C. ation of Thionh‘iahs with Compounds to orepare antibodyvdreg conjugates The dehiocked, reoxidized, thiosahtihodies (Thick/tab) were reacted with 88 foid moiar excess of nds 7', it}, 13, 17, 24, 25, 33, 37', 44, 48 (from a DMSO stock at a concentration of 20 miyi) in 50 miVi Tris, pi-i 8 untii the reaction was oompiete (1624 hours) as determined by LC—MS anaiysis of the reaction mixture The crude antihody~drug conjugates (ADC) were then appiied to a cation exchange coiumn after on with 20 miVi sodium succinate, pH 5. The ooiumn was washed with at ieast it) coiumn voiumes of 20 mi\/i sodium succinatea pH 5, and the dy was eiuted with PBS.
The antibody drug conjugates were tormuiated into 20 mi‘v’i Hisfacetate, pH 5, with 240 miVi sucrose using gei fiitration ceiurnns. The antibody—drug conjugates were characterized by UV spectroscopy to ine protein concentration, anaiyticai SEC (sizenexciusion chromatography) for ation anaiysis and LCmi‘t/iS before and after treatment with Lysine C ptidase.
Size exciusion chromatography was performed using a Shodex Kin/802.5 coiumn in 0.2M potassium phosphate pH 6.2 with 0:25 rnh'i potassium chioride and 15% iPA at a ‘iiow rate of 0.75 milmih. Aggregation state of the conjugate was determined by ation of eiuted peak area ahsorhance at 28!) nm.
LOMS anaiysis was performed using an Agiient QTOF 6520 ESi instrument. As an e, an antibody~drug conjugate generated using this chemistry was treated with 1:500 w/w Endoproteinase Lys C (Promega) in Tris, pH 7’5, for 38 min at 37°C. The resuiting cieavage fragments were ioaded onto a 1000A, 8 um PLRP~S coiumn heated to 80°C and eiuteci with a nt of 30% iii to 40% B in 5 minutes. iViohiie phase A was H20 with 0.05% TFA and mobiie phase B was itriie with 0.04% TFA. The fiow rate was 0.5miimim Protein eiution was monitored by UV absorbance ion at 289 nm prior to eiectrospray ionization and MS anaiysis. Chromatographic resoiution of the unconjugated Fc fragment, residuai unoonjugated Fab and drugged Pat; was usuaiiy achieved. The obtained mlz spectra were deconvoiuted using Mass i-iuhterTM software (Agiient Technoiogies) to caicuiate the mass of the antibody fragments.
ADC This-coniugates with Ti" DAR (drug to antibody ratio) LCit/ié; resuits Thio Ho Anti— r 2343982 LC HerZ arose 5168186 no T1110 Hu Anti— H612 4131558: H8 A1188 24034.96 LC CD222 97 HG 1OF4V'3 HG A1188 ADC: Thio~mn‘gugates with 11) Ab ADC DAR (drug to antibady r3116) LCMS s T1110 Hu Anti— 23449.18 L8 Heriz 413543 51697.51 1-18 H8 A1188 T1110 Hu Anti— He12 4131558: H8 A1188 T1110 hu anti- 24035.14 LC 8822 51744.95 HC 10154113 H8 A1188 This 1111 Anti— 23440.16 LC HerZ 41358 512397.07 H8 H8 A1188 T1110 Hu Anti— 51698.55 HG Her2 485—8 H8 A1188 This Hu Anti- 23932.47 LC 81333 51116.61 1-18 (Sh/115.33 HG A1188 T1110 Hu Anti— 23956.85 L8 LGR5 5143243 118 8E11.v2 H8 A1188 T1110 1-11.: Anti— Nap13b 101-11 .1 1.48 1-18 A1188 This hu anti“ CD22 ‘EGF4V3 HG A1188 T1110 Hu Anti— 297 2,0 48502 Fab HerZ 4135—8 HG A1186 .......................................................................................
ADC oniugates with 13 Ab DAR (drug 10 antibady ratio) LCMS resuits T1110 Hu Anti— HerE 41358 HC A1180 This Hu Anti- CD22 ‘EGF4V3 HG A1180 Thicr Hu Anti— 23934164 LC CD33 51219.40 HG (Sh/115.33 HG A1186 T1110 Hu Anti— 146970 ADC MUC16 3A5 HG A118C This Hu Anti— 14740? ADC CD33 GM1533 HG A1186; ADC Thiowceniugates with 1? LCMES resuits Thicr Hu Anti— 23440315 LC He12 45358: 51 72825 HO HO A1188 T1110 Hu Anti“ 51727.48 HC H6122 435—8 HG A118C This Hu Anti— 23440.15 LC CD22 51728.25 HG ‘EGF4V3 HG A1180 T1110 Hu Anti— 2403511 LC CD22 51717432 Hf; 10F4v3 HG A1186 T1110 Hu Anti— 23932.93 LC CD33 51148.14 HG GM1533 HG A1188 T1110 Hu Anti“ Herz 4135-8 HG A1186; This Hu Anti— HerE 41358 11C A1180 ADC m‘iiugates with 24 LCMS resuits T1110 Hu Anti— 23932.14 LC CD33 51428.37" HG GM1533 HG A118C T1110 Hu Anti“ 23479.80 LC MUC18 3A5 51845.58 HC HG A1186; ADC Thio~mn‘gugates with 25 AR (drug to antibady r3116) LCMS resuits T1110 Hu Anti— 23933 LC CD33 51431 HG (Sh/115.33 HG A1186 T1110 Hu Anti— MUC16 3A5 HG A1188 ABC Thiowceniugates with 33 DAR (drug to antibady ratio) LCMES resuits Thicr Hu Anti— 1 CD33 131,115.33 HG A1 1131: T1110 1-111 Anti— .
Napi3b 1131-1111413 1-10 A1186 ADC Thio—con‘gugates with 3? Ab - DAR (drug to aniibcrdy 13110) LCMS s Thio Hu Anti— CD33 (BR/£15.33 HC A1188 T1110 Hu Anti— NapiSb 1011311145 HG A1 1813 ADC Thioncon'gugates with 44 DAR (drug ta antibody ratio) LCMS resuits This Hu anti" . (31322 10121113 H13 A1186 This 1-111 anti" . 01333 15131513 HG A1 1131: ADC Thia~con§ugates with 48 ABC DAR (drug to 10111: ratio) LCMS resuits Thia Hu Anti“ . 147329 ADC Herz 4D5-8 HG A1 18C 1 35 Thin Hu Anti- CD22 ‘iGFliVS HG A1188 The foiiowing in vitro and in vivo assays are aisc described in Pniiiips et at (2008) Cancer Res. 68(22):9280—9290. in vitra ceii areiiferatien assay Efficacy of ADC were measured by a oeii preiiteration assay empioying the toiiowing protocei (CeiiTiter Gio Luminescent Ceii Viabiiity Assay, a Corp. Technicai Buiietin T8288; Mendoza et ai (2002) Cancer Res. 62:5485n5488). Ait oeii tines were obtained from American Type Cuiture Coiiectien: 1. An aiiduot ef 100 at of ceii cuiture containing about 104 ceiis (for exameie, KPL—4, a human breast cancer eeii tine, Kurehayashi et ai (1999) Brit. dour. Cancer 79(5sd):70?e717)9 or SKBR—B) in medium was deposited in each weii of a Std—wait, opaque—waned niate. 2 Controi weiis were prepared containing medium and without oeiis. 3. ADC was added to the experimentai weiis and ted for 3~5 days, 4 The states were eduiiibrated to room ature for approximateiy 30 minutes.
A voiume of CeiiTitenGio Reagent equai to the voiume of ceii cuiture medium present in each wait was added. 8. The contents were mixed for 2 minutes on an orbitai shaker to induce ceii iysis. ".7. The piate was incubated at room temperature for ti) minutes to stabiiize the iuminescence signai. 8. Luminescence was recorded and reported in graphs as RLU I reiative iuminescence units.
Certain ceiis are seeded at tflfiu—ZOOfii’weii or OQO/weii in a Qti—weii piste, SO utiweii.
After one or two days“ ADC are added in 50 at. voiumes to finai tration of 9000, 3000, 1000, 333, 111, 37, 12.4, 4.1, or 1.4 ng/mL, with "no ADC" eontrci weiis receiving medium aiene. Conditions are in ate or tripiicate After 3—5 days, 100 eii Ceii TiterGio ii is added (iuoiferasemhased assay; oroiiferation measured by ATP ieveis) and ceii counts are determined using a iurninemeter, Data are piotted as the mean at iuminescenee for each set at ates, with standard deviation error bars. The protecoi is a modification of the CeiiTiter Gio Luminescent Ceii ity Assay (Promega); ’i 36 i. Piate i000 ceiisi wait in 50 i ot FBSlgiutai‘nine media. Aiiow ceiis to attach overnight. 2. ADC is seriaiiy diiuted 1:3 in media beginning at at working concentration 18 uglmi (this resuits in a tinai concentration of 9 uglmi). 5t) uL of diiuted ADC is added to the 50 ui. of ceiis and media aiready in the weii, 3. incubate 72—96 hrs (the standard is 7’2 hours, but watch the Q ug/n‘it. concentration to stop assay when the ceiis are 85—95% confluent). 4. Add 100 uLiweii of a Ceii Titer Gie reagent, shake 3 min. and read on iuminometer Results Antibody~drug conjugates, trastuzumab—‘i (11d) trastuzumahn’it) {12th and trastuzumabd? (139) were tested against SK—BRmS, KPL—4, and i‘viCFm? (Levenson et ai (1%?) Cancer Res. 57(15):307‘i~3078) ceiis to measure in vitro oeii viabiiity' in five day studies. The i050 vaiue ) for tit) against SK—ER—B was 22.90. The i050 vaiue for 12% against 3 was i 1 pint. The i650 vaiue for 13% against SKnBR—Ci was 16.8. SK—BRwfi ceiis are HER2+ expressing, trastuzumah Sensitive 11%, “£23 and 13% were e‘itectiveiy inactive against MCF~7, which is a HERZ non—expressing human breast adenocarcinoma ceii iine. Thus, conjugates 11G, 12% and 13% demonstrate targetted ceii kiiiing potency.
Tumor growth inhibition, in viva eftioaey in high expressing HERE transgenio expiant mice Animate suitahie for transgenic experiments can he obtained from standard commerciai sources such as c (Germantown, NRC). iviany strains are suitabie, hut FVB femaie mice are preferred because of their higher susceptihiiity to tumor formation. FVB mates were used for mating and vasectornized GEM studs were used to stimuiate pseudopregnancy.
Vasectomized mice can he obtained from any oommerciai suopiier. Founders were bred with either FVB mice or with 129.43% x FVB n53 heterozygous mice. The mice with heterozygosity at 1053 aiieie were used to potentiaiiy increase tumor formation. However, this has proven unnecessary. Therefore, some Ft tumors are of mixed strain. Founder tumors are FVB oniy. Six rs were ed with some deveioning tumors without having iitters.
Animais having tumors rait ated from F05 mmtv transgenic mice) were d with a singie or oie dose by“ iv injection of ADC. Tumor voiume was assessed at various time points after injection.
Tumors arise readiiy in transgenic mice that express a mutationaiiy activated form ct neu, the rat nemeiog of HERE, but the HERE that is everexpressed in human breast cancers is not mutated and tumor formation is much iess robust in transgenic mice that overexpress nonmutated HERE (Webster at at “994) . Cancer Biol. 8).
To e tumor formation with nonmutated HERE, transgenic mice were produced using a HERE CDNA piasrnid in which an upstream ATG was deieted in order to prevent initiation of ation at such upstream ATG codons, which wouid otherwise reduce the frequency of transiaticn initiation from the downstream tic initiation oodon of HERE (tor exampie. see Chiid at a! (1999) J. Biol. Chem. EM: 24335—24341). Additionaiiy, a chimeric intron was added to the 5’ end, which shouid aiso enhance the ievei of expression as reported r (Neuberger and Wiiiiarns (t988) Nucleic Acids Res. 16:67‘i3; Buchman and Berg (t988) Moi. Ceii. Bioi. ; Brihster etai (t988) Proc. Nati. Acad. Sci. USA ). The ic intron was derived from a Promega vector. Poi—nee mammaiian expression vector the sec—1022). The CDNA 3‘—end is tianked by human growth hormone exons 4 and 5, and ooiyadenyiaticn sequences. Moreover, FVB mice were used because this strain is more susceptihie to tumor deveioprnent. The promoter from i‘x/iMTV—LTR was used to ensure tissue—specific HERE expression in the mammary giand. Animais were fed the AEN 76A diet in order to increase susceptihiiity to tumor formation (Rao et ai (1997’) Breast Cancer Res. and Treatment 45."?4Q~158).
F05 murine mammary tumor modei The F05 mode! is a transgenic mouse modei in which the human HERE gene, under transcriptionai reguiation ot the murine mammary tumor virus er (iViiViTV—HERE), is overexpressed in y epitheiium. The overexpression causes spontaneous pment of mammary tumors that overexpress the human HERE receptor. The mammary tumor of one of the founder animais (founder #5 {Feet} has been propagated in subsequent generations of FVB mice by seriai transpiantation of tumor fragments. Before being used for an in vivc efficacy study, the MMTV—HERE F05 transgenic mammary tumor was surgicaiiy transpianted into the No. 2!?) mammary fat pad of nuinu mice (from Charies River Laboratories) in fragments that measured approximateiy 2x2 mm. When tumors reached desired voiumes, the tumornhearing mice were randomized and given a singie dose by iv injection of the ADC.
Resuits Figure 1 shows a piet of the in vivo meah tumor vetume change overtime ih breast cancer“ medei MMTV—HERE F05 y att tumore thoeuiated ihte CRL hu/‘hu mice after singie iv dosing eh day 0 with: (‘1)Vehieie 29mm Histidirie e, pH 5.5, t sucrose, (2) XCD22—7 (115) at 6 mglkg, (3) trastuzumah—i’ (11%) at t mg/itgg (4) traetuzumabfi (11%) at 3 rhg/kg, and (5) trastuzumah-T (HQ) at 6 mg/kg. The iihes in the figure are indicated with the toitewihg symbeie: +Vehieie +ADC‘E tit) Témaibe? HG A1181}, ‘1 mgtitg +ADC‘E til} Titttflibr? HG 53.11313; 3 mgr‘kg —E[—AD{Zt‘ztt} —T HG At‘tfifi} E mgfitg --‘i’--AB{31 t5 CBERw? HG At 186, 8- smgfttg Figure 2 shows. a ptet of the in viva meah tumer vehime charige ever time in breast caheer—modet MMTV—HERQ F05 mammary aft tumors ihceuiated ihte CRL hulhu mice after singie iV dosing on day 0 with: (t) Vehicte 20 mi‘v’i Histidihe acetate, pit 55, 240 mM sucrose, (2) XC022»‘it) (125)at3 mg/‘kg, (3) trastuzumehfiitfi (1263) at (LS mgikg, (4) trastuzumah-ti} (1283) at t mgl’kg, and (5)trastuzumah~1i} (1283) at 3 mgl’kg. The tines in the figure are indicated with the tetiowihg symbeis: _)(_ Vehieie +aemae Tittah-vtfl Ht: Attafis. 8‘3 mgt’kg +Aemae Tmata‘lt) Ht: M1335 t rhgikg +Afi~tfit12fl Treat-430 Ht: At tea 3 sm-grttg "vuhtDCt 225 {$222 HG At tree? 3 magma Figure 3 shows. a ptet of the in viva meah tumer vehime charige ever time in breast cancer— medei MMTVIHEREZ Fe5 mammary aiiegraft tumors inccuiateci ihte CRL hu/hu mice after singie iv dosing on day 0 with: (1)Vehicie 20 min Hietidihe acetate, pit 5K5, 240 miVi sucrose, (2) t? (135) at 3 mglkg, (3) trastuzumahfii? (13%) at 03 mgikg (4) tr‘astuzumah~1? (t 3%) at t mgfkg, and (5) trastuzumah~1T (t 3%) at 3 mgfkg. The iihee in the figure are indicated with the teiiowihg eymbeis; +VSNE§E +ADC13£§ Tmaabfii‘? Hi: M "k BC,- {13 :mgfkg —Q— A1311: 3% 3f} Tmaba’fi éfix‘if: A}? '1? 8C, 1 mgf‘ékg —E|—m:-I=Cii '30 ‘1‘mab~“§? HE M TEE? 3 mgg’kg E? 333 {$322. HIE: A1 18C, 3 mgz’kg Abbreviations Ac acetyi Acm acetamidomethyi AEEOC aiiyioxycarbonyi BOG t—butyi dicarbonate t—Bu Eert~butyi Bzi benzyi, where BzinMe is methoxybenzyi and BzE—Me is methyibenzene Cbz er Z benzyiaxy—carbanyi, where Z—CE and 2—8:“ are chioro~ and bromobenzyiaxy carbonyi respactiveiy DEVEF N,N~dimethyiformamide an dinitmphenyi DTT dithiothreitoi Fmac 9H~fluGran—guyimathoxycarbenyi imp N40 Emma protecting group: 3—(2—methoxyethoxy)propanoate~VaE~AEamPAB MC—QSU idocaproyE—OHMSEJccinimEde Mac methoxyoarbonyi MP maiaimidopropanamide Mir 4mmethoxy~2,3a6—trimethtyibenzenesuEfonyi PAS parawaminobenzyioxycarbonyi PEG ethyieneoxy PNZ p—nitmbenzyi carbamate P390 2—(phenyisuifonyi)ethoxycarbonyi TBDMS tyidimethyisiiyi TBDPS ter’t—butyidiphenyiaiiyi T886 2—(trimethyisiiyi)athoxycarbonyi Toss tosy! 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Claims (20)

  1. Claims 1. A conjugate of formula (A): 20 19 10 21 R 9 R R R R 11 H N X' X N H T' Z T N 17 7 N 22 R R R 2 16 A 6 O R R O wherein: R36a R2 is R , where R36a and R36b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are ally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R36a and R36b 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’, NO2, Me3Sn and halo; R7 is independently selected from H, R, OH, OR, SH, SR, NH 2, NHR, NRR’, NO2, Me3Sn and halo; Y is selected from formulae A2, A3, A4, A5 and A6: (A2) (A3) (A4) CBA CBA n n (A5) (A6) L is a linker connected to a cell binding agent; CBA is the cell binding agent; n is an integer ed in the range of 0 to 48; RA4 is a C1-6 alkylene group; either (a) R10 is H, and R11 is OH, ORA, where RA is C1-4 alkyl; or (b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R10 is H and R11 is OSOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; R and R’ are each independently ed from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups, and optionally in relation to the group NRR’, R and R’ er with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring; wherein R16, R17, R19, R20, R21 and R22 are as defined for R6, R7, R9, R10, R11 and R2 tively; wherein Z is CH or N; wherein T and T’’ are independently selected from a single bond or a C1-9 alkylene, the C1-9 alkylene may be interrupted by one or more heteroatoms selected from O, S, N(H) and NMe, provided that the number of atoms in the shortest chain of atoms n X and X’ is 3 to 12 atoms; and X and X’ are independently selected from O, S and N(H).
  2. 2. The conjugate according to claim 1, wherein R9 is H, and R6 is H.
  3. 3. The conjugate according to either claim 1 or claim 2, wherein R7 is OR7A , where R7A is Me.
  4. 4. The conjugate according to any one of claims 1 to 3, wherein X is O and T is selected from a single bond, C1, and a C2 ne group.
  5. 5. The conjugate according to claim 4, wherein T is a C1 alkylene group.
  6. 6. The conjugate according to any one of claims 1 to 5, wherein: (a) R36a and R36b are both H; (b) R36a and R36b are both methyl; or (c) one of R36a and R36b is H, and the other is selected from methyl and ethyl.
  7. 7. The conjugate according to any one of claims 1 to 6, wherein R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
  8. 8. The conjugate according to any one of claims 1 to 7, wherein R16 , R17 , R19 , R20 , R21 , R22 , X’ and T’ are the same as R6, R7, R9, R10 , R11 , R2, X and T tively
  9. 9. The conjugate according to any one of claims 1 to 8, wherein L is of formula: (a) -LA-(CH - (L1) where m is from 0 to 6; (b) -LA-(CH 2)m -O- (L2) where m is from 0 to 6; (c) H -O-C (=O)-NH-(CH 2)q 2)p- (L3) where q is from 1 to 3, and p is from 1 to 3; or 1 H A X N L 2 m X O O (d) (L4) where m is from 0 to 6; and X1 and X2 are amino acid groups, selected from natural amino acids; wherein LA is selected from: (LA1-1) O (LA6) O CBA N (LA1-2) O (LA7) CBA CBA N (LA2) O (LA8-1) CBA O N N CBA N (LA3-1) (LA8-2) N CBA S N N (LA3-2) (LA9-1) N N N (LA4) CBA ) N H N N (LA5) O where Ar represents a C5-6 arylene group.
  10. 10. The conjugate according to claim 9, wherein the group -X1-X2- is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-.
  11. 11. The conjugate according to any one of claims 1 to 10, wherein the cell binding agent is an antibody or an active fragment thereof.
  12. 12. The conjugate of claim 11 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)-(38): (1) BMPR1B (bone morphogenetic protein receptor-type IB); (2) E16 (LAT1, SLC7A5); (3) STEAP1 (six transmembrane epithelial antigen of te); (4) 0772P (CA125, MUC16); (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, elin); (6) Napi3b (NAPI-3B, Napi2b, NPTIIb, SLC34A2, solute carrier family 34 m 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 embrane lial antigen of prostate 2, six transmembrane prostate protein); (12) TrpM4 50, FLJ20041, TRPM4, TRPM4B, transient or potential cation 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 or) or Hs 73792); (15) CD79b , CD79β, IGb (immunoglobulin-associated beta), B29); (16) FcRH2 , IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), , SPAP1C); (17) HER2; (18) NCA; (19) MDP; (20) IL20Rα; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3); (27) CD22 (B-cell receptor CD22-B isoform); (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha); (29) CXCR5 tt's lymphoma receptor 1); (30) HLA-DOB (Beta t of MHC class II molecule (Ia antigen)); (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2); (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family); (34) FcRH1 (Fc receptor-like protein 1); (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2); (36) TENB2 (putative transmembrane proteoglycan); (37) CD33 (CD33 molecule, SIGLEC-3, SIGLEC3, p67; CD33 n (gp67); gp67; myeloid cell surface antigen CD33; sialic acid binding Ig-like lectin 3; sialic acid-binding Iglike lectin); and (38) LGR5/GPR49.
  13. 13. The conjugate according to any one of claims 1 to 12, for use in therapy.
  14. 14. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 12, and a pharmaceutically acceptable diluent, carrier or excipient.
  15. 15. The conjugate according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 14, for use in the treatment of a proliferative disease in a
  16. 16. A compound of formula (B): R 19 9 10 R R R R 11 H N X' X N H T' Z T N 17 7 22 R R N R 2 B R 16 6 O R R O wherein: R2, R6, R7, R9, R10, R11, R22, R16, R17, R19, R20, R21, Z, T, T’, X and X’ are as defined in any one of claims 1 to 8; YL is selected from a group of formulae B2, B3, B4, B5 and B6: (B2) (B3) (B4) n n (B5) (B6) G is a reactive group for connecting to a cell binding agent wherein n and RA4 are as defined in claim 1.
  17. 17. The compound according to claim 16, wherein G is of formula: (a) GA-(CH2)m -(G1) where m is from 0 to 6; (b) GA-(CH2)m -O- (G2) Where m is from 0 to 6; (c) GA-(CH2)q -O-C(=O)-NH-(CH2)p- (G3) Where q is from 1 to 3, and p is from 1 to 3; and 1 H A X G 2 N m X O O (G4) where m is from 0 to 6; X1 and X2 are amino acid groups, ed from natural amino acids; n GA is selected from: (GA1-1) O (GA4) O Hal N Where Hal = I, Br, Cl (GA1-2) O (GA5) O N Hal where Hal = I, Br, Cl (GA2) O (GA6) O O O N O O O (GA3-1) (GA7) Br S S (NO2) where the NO2 group is optional (GA3-2) (GA8) S S (NO2) where the NO2 group is al (GA3-3) (GA9) N3 S S where the NO2 group is optional (GA3-4) S S where the NO2 group is optional where Ar represents a C5-6 arylene group.
  18. 18. A compound of a (C): 41 R 19 9 30 R R R R 31 H N X' X N T' Z T N 17 7 R R N R22 2 16 C 6 O R R O wherein: R2, R6, R7, R9, R22, R16, R17, R19, Z, T, T’, X and X’ are as defined in any one of claims 1 to 8; YC is selected from a group of formulae C2, C3, C4, C5 and C6: (C2) (C3) (C4) n n (C5) (C6) n n and RA4 are as defined in claim 1; either (a) R30 is H, and R31 is OH, ORA, where RA is C1-4 alkyl; or (b) R30 and R31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R30 is H and R31 is OSOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or (d) R30 is a nitrogen protecting group and R31 is OProtO, where ProtO is a y protecting group; and R40 and R41 are as defined for R30 and R31 respectively.
  19. 19. A compound of formula (D): R 19 9 30 R R R R 31 H N X' X N H T' Z T N 17 7 R R N R22 2 16 D 6 R O R R O wherein: R2, R6, R7, R9, R22, R16, R17, R19, Z, T, T’, X and X’ are as defined in any one of claims 1 to 8; R30, R31, R40 and R41 are as defined in claim 18; YD is selected from a group of formulae D2, D3, D4 and D6: (D2) (D3) (D4) (D6) wherein RA4 is as defined in claim 1.
  20. 20. The conjugate according to claim 1, ntially as herein described with reference to any one of the Examples and/or
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