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  • 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
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  • 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
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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
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  • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
• • 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
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  • 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
  • A61K47/6863—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 stomach or intestines cancer cell
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
  • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
  • C07D263/04—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
  • C07D263/06—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by oxygen atoms, attached to ring carbon atoms
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  • 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
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  • 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
  • C07D487/14—Ortho-condensed systems
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  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
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  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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  • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
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  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
  • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala

Definitions

• the present invention relates to novel cytotoxic compounds and cytotoxic conjugates comprising these cytotoxic compounds and cell-binding agents. More specifically, this invention relates to novel benzodiazepine compounds (e.g., indolinobenzodiazepines or oxazolidinobenzodiazepines), derivatives thereof, intermediates thereof, conjugates thereof, and pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as antiproliferative agents.
• novel benzodiazepine compounds e.g., indolinobenzodiazepines or oxazolidinobenzodiazepines
• Benzodiazepine derivatives are useful compounds for treating various disorders, and include medicaments such as, antiepileptics (imidazo [2,l-b][l,3,5]benzothiadiazepines, U.S. Pat. No. 4,444,688; U.S. Pat. No. 4,062,852), antibacterials (pyrimido[l,2- c][l,3,5]benzothiadiazepines, GB 1476684), diuretics and hypotensives (pyrrolo(l,2- b)[l,2,5]benzothiadiazepine 5,5 dioxide, U.S. Pat. No. 3,506,646), hypolipidemics (WO 03091232), anti-depressants (U.S.
• medicaments such as, antiepileptics (imidazo [2,l-b][l,3,5]benzothiadiazepines, U.S. Pat. No. 4,444,688; U.S. Pat. No. 4,062,852),
• PBDs are described in US Publication Number 20070072846.
• the PBDs differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. Their ability to form an adduct in the minor groove enables them to interfere with DNA processing, hence their potential for use as antiproliferative agents.
• One object of the invention is to provide novel benzodiazepines of formula (I) and (II), in which the diazepine ring (B) is fused with a heterocyclic ring (CD), wherein the heterocyclic ring is bicyclic, wherein: the double line — between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H, or an amine protecting moiety that converts the compound into a prodrug that can be transformed into the free amine in vitro or in vivo;
• Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR', a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R", amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO3, a bisulfite -OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and are selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 20 carbon
• 6 to 18 carbon atoms 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P and R 1 O optionally is SRo or CORo, wherein Ro is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 ) n , wherein n is an integer from 1 to 2000, , a 5- or 6-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein at least one of the rings is aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P and R 11 can also be OR 14 , wherein R 14 is H or has the same definition as R, optionally, R"
• R 5 is selected from OR 15 , CRR'OH, SH, CRR'SH, NHR 15 or CRR'NHR 15 , wherein R 15 has the same definition as R., R and R' have the same definition as given above; optionally, R 5 is a linking group that enables linkage to a cell binding agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent;;
• R 6 is OR, SR, NRR', wherein R and R' have the same definition as given above, or optionally R 6 is a linking group;
• Z is selected from (CH 2 ) n , wherein n is 1, 2 or 3, CR 15 R 16 , NR 17 , O or S, wherein R 15 , R 16 and R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 )Ii, wherein n is an integer from 1 to 2000; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers of these compounds. provided that the compound has no more than one linking group that enables linkage to a cell binding agent via a covalent bond.
• a second object of the invention is to provide novel benzodiazepines of formula (III), in which the diazepine ring (B) is fused with a heterocyclic ring (C), wherein the heterocyclic ring is monocyclic,
• the double line ⁇ between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine protecting moiety that converts the compound into a prodrug;
• Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR', a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R", amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO3, a bisulfite - OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon
• 5 to 18 membered fused ring system wherein at least one of the rings is aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P and R 11 can also be OR 14 , wherein R 14 is H or has the same definition as R, optionally R" is OH;
• R 5 is selected from OR 15 , CRR'OH, SH, CRR'SH, NHR 15 or CRR 5 NHR 15 , wherein R 15 has the same definition as R. or is a linking group that enables linkage to a cell binding agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo- imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent;;
• R 6 is OR, SR or NRR', wherein R and R' have the same definition as given above, optionally R 6 is a linking group;
• X' is CH 2 , NR, CO, BH, SO or SO 2 ;
• Y' is O, CH 2 , NR or S
• Z' is CH 2 or (CH 2 ) n , wherein n is 2, 3 or 4; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these compounds; provided that the compound has no more than one linking group that enables linkage to a cell binding agent via a covalent bond.
• a third object of the invention is to provide cytotoxic dimers (IV), (V) and (VI)
• the dimer compounds optionally bear a linking group that allows for linkage to cell binding agents, wherein: the double line — between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine protecting moiety that converts the compound into a prodrug;
• Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR', a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R", amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO3, a bisulfite - OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and are selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon
• Z is selected from (CH 2 ) n , wherein n is 1, 2 or 3, CR 1 SR 16 , NR 17 , O or S, wherein R 15 , R 16 and
• R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 ) n , wherein n is an integer from 1 to 2000;
• R 6 is OR, SR or NRR', wherein R and R' have the same definition as given above, optionally R 6 is a linking group;
• X' is selected from CH 2 , NR, CO, BH, SO or SO 2 wherein R has the same definition as given above;
• Y' is O, CH 2 , NR or S, wherein R has the same definition as given above;
• Z' is CH 2 or (CH 2 ) n , wherein n is 2, 3 or 4, provided that X', Y' and Z' are not all CH 2 at the same time;
• a and A' are the same or different and are selected from O, -CRR'O, S, -CRR' S, -NR 15 or
• D and D' are same or different and independently selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, optionally substituted with any one of halogen,
• L is an optional phenyl group or 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P that is optionally substituted, wherein the substituent is a linking group that enables linkage to a cell binding agent via a covalent bond, or is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted with any one of halogen, OR 7 , NR 8 RcI, NO 2 , NRCOR', SR 1 O, a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by SO 2 NRR', cyano, an azido, -COR 11 , OCOR 11 or OCONR 11 R 12 , wherein R 7 , R 8 , R
• a fourth object of the invention is to provide conjugates of cell binding agents with the novel benzodiazepine compounds or derivatives thereof of the present invention. These conjugates are useful as therapeutic agents, which are delivered specifically to target cells and are cytotoxic.
• the present invention includes a composition (e.g., a pharmaceutical composition) comprising novel benzodiazepine compounds, derivatives thereof, or conjugates thereof, (and/or solvates, hydrates and/or salts thereof) and a carrier (a pharmaceutically acceptable carrier).
• the present invention also includes a composition (e.g., a pharmaceutical composition) comprising novel benzodiazepine compounds, derivatives thereof, or conjugates thereof, (and/or solvates, hydrates and/or salts thereof) and a carrier (a pharmaceutically acceptable carrier), further comprising a second therapeutic agent.
• a composition e.g., a pharmaceutical composition
• a carrier a pharmaceutically acceptable carrier
• the present compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human).
• the present compositions are also useful for treating depression, anxiety, stress, phobias, panic, dysphoria, psychiatric disorders, pain, and inflammatory diseases in a mammal (e.g., human).
• the present invention includes a method of inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human) comprising administering to said mammal a therapeutically effective amount of novel benzodiazepine compounds, derivatives thereof, or conjugates thereof, (and/or solvates and salts thereof) or a composition thereof, alone or in combination with a second therapeutic agent.
• a mammal e.g., human
• the present invention includes a method of synthesizing and using novel benzodiazepine compounds, derivatives thereof, and conjugates thereof for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, organisms, or associated pathological conditions.
• novel benzodiazepine compounds, derivatives thereof, and conjugates thereof, and compositions comprising them are useful for treating or lessening the severity of disorders, such as, characterized by abnormal growth of cells (e.g., cancer).
• compounds and conjugates of this invention include, but are not limited to, treating osteoporosis, depression, anxiety, stress, phobias, panic, dysphoria, psychiatric disorders, and pain or as antiepileptics, antibacterials, diuretics and hypotensives, hypolipidemics, and anti-depressants.
• FIGS. 1-10 show the schemes for the synthesis of indolinobenzodiazepine and oxazolidinobenzodiazepine monomers, the representative linkers and the dimers in the present invention.
• FIG. 11 shows the scheme for the synthesis of the representative B-ring modified indolinobenzodiazepine monomer.
• FIG. 12 shows the scheme for the synthesis of the representative isoindolinobenzodiazepine monomer.
• FIG. 13 shows the scheme for the synthesis of the representative dimer with the linker directly attached on the indolinobenzodiazepine moiety in the present invention.
• FIGS. 14 and 15 show the schemes for the synthesis of the representative dimers containing (PEG) n moieties on the linkers.
• FIG. 16 shows the schemes for the synthesis of the representative mixed imine-amine and imine-amide indolinobenzodiazepine dimers.
• FIG. 17 shows the scheme for the synthesis of the representative IBD-poly(N- methylpyrrole-imidazole) conjugates .
• FIGS. 18-19 show the synthetic scheme for the preparation of polypyrrolo and polyp yrrolo-imidazo Io derivatives of the monomers.
• FIG 20 shows a scheme for the synthesis of piperidinylbenzodiazepines bearing a hydrazone linker.
• FIGS. 21-26 show the dose dependent in vitro antiproliferative activity of muB38.1 -IGN-
• FIG. 27 shows in vivo efficacy of huN901-IGN-07 conjugate in mice bearing Molp-8 tumors.
• FIGS. 28-30 show data that demonstrate that IGN-Ol, IGN-02, and IGN-09 bind and covalently adduct to double stranded DNA containing guanine residues on opposite strands.
• FIG. 31 contains TABLE 1, which shows the IC50 values for in vitro antiproliferative activity of indolinobenzodiazepine dimers and oxazolidinobenzodiazepine dimer on several cancer cell lines.
• FIG. 32 contains TABLE 2, which shows the comparison of the IC50 values for in vitro antiproliferative activity of indolinobenzodiazepine dimers with and without linkers.
• FIGS. 33-36, 39, 42, 43, 44, 48, 49 and 50 show synthetic schemes for the preparation of compounds of the present invention.
• FIGS. 37, 38, 40 and 41, 45, 46, and 47 show synthetic schemes for the preparation of linkable compounds of the present invention.
• FIG. 51 shows the in vitro cytotoxicity of compounds of the present invention.
• FIGS. 52, 54, 56, 57 and 58 show the in vitro cytotoxicity and specificity of chB38.1 conjugates.
• FIGS. 53 and 55 show the in vitro cytotoxicity and specificity of huMy9-6 conjugates.
• FIG. 59 shows the in vivo anti-tumor activity of chB38.1 conjugate
• Linear or branched alkyl refers to a saturated linear or branched-chain monovalent hydrocarbon radical of one to twenty carbon atoms.
• alkyl include, but are not limited to, methyl , ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2 -methyl- 1 -propyl, — CH 2 CH(CH 3 ) 2 ), 2-butyl , 2-methyl-2 -propyl, 1-pentyl , 2-pentyl 3-pentyl, 2-methyl-2 -butyl, 3- methyl-2-butyl , 3 -methyl- 1 -butyl, 2-methyl-l -butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2- pentyl,, 3-methyl-2-pentyl, 4-methyl-2-pentyl,, 3-methyl-3-pentyl,, 2-methyl-3-pentyl, 2,3
• Linear or branched alkynyl refers to a linear or branched monovalent hydrocarbon radical of two to twenty carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, triple bond. Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1- pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like.
• cyclic alkyl refers to a monovalent non-aromatic, saturated or partially unsaturated ring having 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring.
• Bicyclic carbocycles having 7 to 12 atoms can be arranged, for example, as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10 ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or as bridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.
• monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-I- enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-I-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.
• Aryl means a monovalent aromatic hydrocarbon radical of 6-18 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar”. Aryl includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring.
• Typical aryl groups include, but are not limited to, radicals derived from benzene (phenyl), substituted benzenes, naphthalene, anthracene, indenyl, indanyl, 1 ,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.
• heterocycle refers to a saturated or a partially unsaturated (i.e., having one or more double and/or triple bonds within the ring) carbocyclic radical of 3 to 18 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms being C, where one or more ring atoms is optionally substituted independently with one or more substituents described below.
• a heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P,_and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.
• Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W. A.
• Heterocyclyl also includes radicals where heterocycle radicals are fused with a saturated, partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring.
• heterocyclic rings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,_dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2- pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithi
• heteroaryl refers to a monovalent aromatic radical of 5- or 6-membered rings, and includes fused ring systems (at least one of which is aromatic) of 5-18 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, furazanyl,
• heterocycle or heteroaryl groups may be carbon (carbon-linked) or nitrogen
• carbon bonded heterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3,
• nitrogen bonded heterocycles or heteroaryls are bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3 -imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3- pyrazoline, piperidine, piperazine, indole, indoline, lH-indazole, position 2 of a isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or O-carboline.
• heteroatoms present in heteroaryl or heterocyclcyl include the oxidized forms such as
• halo or halogen refers to F, Cl, Br or I.
• compound or "cytotoxic compound” or “cytotoxic agent” as used herein is intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof that has been incorporated by reference.
• the term also includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs, and prodrug salts of a compound of all the formulae disclosed in the present invention.
• the term also includes any solvates, hydrates, and polymorphs of any of the foregoing.
• linkable to a cell binding agent referes to the novel benzodiazepine compounds (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine), derivates thereof or dimers thereof comprising at least one linking group or a precursor thereof suitable to bond these compounds, derivatives thereof or dimers thereof to a cell binding agent.
• precursor'Of a given group refers to any group which may lead to that group by any deprotection, a chemical modification, or a coupling reaction.
• the term "linked to a cell binding agent” refers to a conjugate molecule comprising at least one the novel benzodiazepine compounds (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine), derivates thereof or dimers thereof bound to a cell binding agent via a suitable linking group or a precursor thereof.
• novel benzodiazepine compounds e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine
• stereoisomer refers to compounds which have identical chemical constitution and connectivity, but different orientations of their atoms in space that cannot be interconverted by rotation about single bonds.
• Diastereomer refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as crystallization, electrophoresis and chromatography.
• Enantiomers refer to two stereoisomers of a compound which are non- superimposable mirror images of one another.
• the compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention.
• Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s).
• d and I or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory.
• a compound prefixed with (+) or d is dextrorotatory.
• these stereoisomers are identical except that they are mirror images of one another.
• a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
• racemic mixture and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
• tautomer or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier.
• proton tautomers also known as prototropic tautomers
• Valence tautomers include interconversions by reorganization of some of the bonding electrons.
• a substituent is "substitutable” if it comprises at least one carbon, sulfur, oxygen or nitrogen atom that is bonded to one or more hydrogen atoms. Thus, for example, hydrogen, halogen, and cyano do not fall within this definition.
• a substituent is described as being “substituted,” a non-hydrogen substituent is in the place of a hydrogen substituent on a carbon, oxygen, sulfur or nitrogen of the substituent.
• a substituted alkyl substituent is an alkyl substituent wherein at least one non- hydrogen substituent is in the place of a hydrogen substituent on the alkyl substituent.
• monofluoroalkyl is alkyl substituted with a fluoro substituent
• difluoroalkyl is alkyl substituted with two fluoro substituents. It should be recognized that if there is more than one substitution on a substituent, each non-hydrogen substituent may be identical or different (unless otherwise stated).
• substituent may be either (1) not substituted, or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogens on the carbon (to the extent there are any) may separately and/or together be replaced with an independently selected optional substituent. If a nitrogen of a substituent is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogens on the nitrogen (to the extent there are any) may each be replaced with an independently selected optional substituent.
• One exemplary substituent may be depicted as --NR 1 R," wherein R' and R" together with the nitrogen atom to which they are attached, may form a heterocyclic ring.
• the heterocyclic ring formed from R and R" together with the nitrogen atom to which they are attached may be partially or fully saturated.
• the heterocyclic ring consists of 3 to 7 atoms.
• the heterocyclic ring is selected from the group consisting of pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl and thiazolyl.
• substituents are collectively described as being optionally substituted by one or more of a list of substituents, the group may include: (1) unsubstitutable substituents, (2) substitutable substituents that are not substituted by the optional substituents, and/or (3) substitutable substituents that are substituted by one or more of the optional substituents.
• substituent may be either (1) not substituted; or (2) substituted by up to that particular number of non-hydrogen substituents or by up to the maximum number of substitutable positions on the substituent, whichever is less.
• any heteroaryl with less than 3 substitutable positions would be optionally substituted by up to only as many non-hydrogen substituents as the heteroaryl has substitutable positions.
• prodrug refers to a precursor or derivative form of a compound of the invention that is capable of being enzymatically or hydro lyrically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985).
• the prodrugs of this invention include, but are not limited to, ester-containing prodrugs, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, .beta.-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
• prodrug examples include, but are not limited to, compounds of the invention and chemotherapeutic agents such as described above.
• prodrug is also meant to include a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms.
• prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulae disclosed herein that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
• Other examples of prodrugs include derivatives of compounds of any one of the formulae disclosed herein that comprise —NO, -NO 2 , --ONO, or -ONO 2 moieties.
• Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs”.
• biohydrolyzable amides include, but are not limited to, lower alkyl amides, .alpha.-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
• biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters.
• biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines.
• Particularly favored prodrugs and prodrug salts are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal.
• salts refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention.
• Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., l,l'-methylene-
• a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion.
• the counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
• a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.
• the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesul
• an inorganic acid such as hydrochloric
• the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.
• an inorganic or organic base such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.
• suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
• amino acids such as glycine and arginine
• ammonia such as glycine and arginine
• primary, secondary, and tertiary amines such as piperidine, morpholine and piperazine
• inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
• solvate means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2- propanol, or the like, bound by non-covalent intermolecular forces.
• Solvates or hydrates of the compounds are readily prepared by addition of at least one molar equivalent of a hydroxylic solvent such as methanol, ethanol, 1-propanol, 2-propanol or water to the compound to result in solvation or hydration of the imine moiety.
• abnormal cell growth and “proliferative disorder” are used interchangeably in this application.
• abnormal cell growth refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition).
• tumor cells tumor cells
• tumors tumor cells
• any tumors that proliferate by receptor tyrosine kinases any tumors that proliferate by aberrant serine/threonine kinase activation
• benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation occurs.
• cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
• a “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
• squamous cell cancer e.g., epithelial squamous cell cancer
• lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, acute leukemia, as well as head/brain and neck cancer.
• NSCLC non-small cell lung cancer
• adenocarcinoma of the lung and squamous carcinoma of the lung cancer of the peritoneum, hepatocellular cancer, gas
• a “therapeutic agent” encompasses both a biological agent such as an antibody, a peptide, a protein, an enzyme or a chemotherapeutic agent.
• a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
• chemotherapeutic agents include Erlotinib (TARCEV A®, Genentech/OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SUl 1248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs), and Gefitinib (IRESSA®, AstraZeneca), AG1478,
• dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
• chemotherapeutic agent also included in the definition of "chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMA
• anti-angiogenic agents include MMP-2 (matrix -metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, COX-II (cyclooxygenase II) inhibitors, and VEGF receptor tyrosine kinase inhibitors.
• VEGF receptor tyrosine kinase inhibitors include 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(l- methylpiperidin-4-ylmethoxy)qu- inazoline (ZD6474; Example 2 within WO 01/32651), 4-(4- fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin- 1 -ylpropoxy)- -quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO 98/35985) and SUl 1248 (sunitinib; WO 01/60814), and compounds such as those disclosed in PCT Publication Nos.
• chemotherapeutic agents that can be used in combination with the present compounds include inhibitors of PI3K (phosphoinositide-3 kinase), such as those reported in Yaguchi et al (2006) Jour, of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. No. 7,173,029; U.S. Pat. No. 7,037,915; U.S. Pat. No. 6,608,056; U.S. Pat. No. 6,608,053; U.S. Pat. No. 6,838,457; U.S. Pat. No.
• PI3K phosphoinositide-3 kinase
• PBK inhibitors include SF-1126 (PBK inhibitor, Semafore Pharmaceuticals), BEZ-235 (PBK inhibitor, Novartis), XL- 147 (PBK inhibitor, Exelixis, Inc.).
• a “metabolite” is a product produced through metabolism in the body of a specified compound, a derivative thereof, or a conjugate thereof, or salt thereof. Metabolites of a compound, a derivative thereof, or a conjugate thereof, may be identified using routine techniques known in the art and their activities determined using tests such as those described herein. Such products may result for example from the oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterif ⁇ cation, deesterif ⁇ cation, enzymatic cleavage, and the like, of the administered compound.
• the invention includes metabolites of compounds, a derivative thereof, or a conjugate thereof, of the invention, including compounds, a derivative thereof, or a conjugate thereof, produced by a process comprising contacting a compound, a derivative thereof, or a conjugate thereof, of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof.
• composition indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
• protecting group refers to a substituent that is commonly employed to block or protect a particular functionality while reacting other functional groups on the compound, a derivative thereof, or a conjugate thereof.
• an "amino- protecting group” or an “amino-protecting moiety” is a substituent attached to an amino group that blocks or protects the amino functionality in the compound.
• Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9- fluorenylmethylenoxycarbonyl (Fmoc).
• hydroxy-protecting group refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable protecting groups include acetyl and silyl. A “carboxy-protecting group” refers to a substituent of the carboxy group that blocks or protects the carboxy functionality.
• Common carboxy-protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2- (trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2- (diphenylphosphino)-ethyl, nitroethyl and the like.
• Common thiol-protecting groups include those that convert the thiol into a thioester, such as acetyl, benzoyl or trifluoroacetyl, into a thioether, such as benzyl, t-butyl, triphenylmethyl, 9-fluorenylmetyl, methoxymethyl, 2- tetrahydropyranyl or silyl, into a disulfide, such as methyl, benzyl, t-butyl, pyridyl, nitropyridyl, phenyl, nitrophenyl or dinitrophenyl, into a thiocarbonate, such as t-butoxycarbonyl, into a thiocarbamate, such as N-ethyl.
• a thioester such as acetyl, benzoyl or trifluoroacetyl
• a thioether such as benzyl, t-but
• the double line ⁇ between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H, or an amine protecting moiety that converts the compound into a prodrug that can be transformed into the free amine in vitro or in vivo;
• Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR', a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R", amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO 3 , a bisulfite -OSO 3 , a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and are selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from
• 6 to 18 carbon atoms 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P and R 1 O optionally is SRo or CORo, wherein Ro is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 ) n , wherein n is an integer from 1 to 2000, , a 5- or 6-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein at least one of the rings is aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P and R 11 can also be OR 14 , wherein R 14 is H or has the same definition as R, optionally, R"
• R 5 is selected from OR 15 , CRR'OH, SH, CRR'SH, NHR 15 or CRR'NHR 15 , wherein R 15 has the same definition as R., R and R' have the same definition as given above; optionally, R 5 is a linking group that enables linkage to a cell binding agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent;;
• R 6 is OR, SR, NRR', wherein R and R' have the same definition as given above, or optionally R 6 is a linking group;
• Z is selected from (CH 2 ) n , wherein n is 1, 2 or 3, CR 15 R 16 , NR 17 , O or S, wherein R 15 , R 16 and R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 ) n , wherein n is an integer from 1 to 2000; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers of these compounds. provided that the compound has no more than one linking group that enables linkage to a cell binding agent via a covalent bond.
• R 1 , R 2 , R3, R 4 are each H; optionally, independently, any one OfR 1 , R 2 , R3 and R 4 can be a linking group that enables linkage to a cell binding agent via a covalent bond;
• R 5 is selected from OR 15 , CRR'OH, SH, CRR'SH, NHR 15 or CRR 5 NHR 15 , wherein R 15 is H or has the same definition as given above for R, or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent, R and R' have the same definition as given above;
• R 6 is OCH 3 ;
• Z is selected from (CH 2 ) n , wherein n is 1 or 2, NH, NCH 3 or S; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these compounds.
• compounds of formula (I) and (II) are compounds of formulae (VII), (VIII) or (IX):
• the double line ⁇ between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine protecting moiety that converts the compound into a prodrug;
• Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR', a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R", amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO3, a bisulfite - OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon
• R 5 is selected from OR 15 , CRR'OH, SH, CRR'SH, NHR 15 or CRR 5 NHR 15 , wherein R 15 is H or has the same definition as R. or is a linking group that enables linkage to a cell binding agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo- imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent;is selected from a poly-pyrrolo, poly-indolyl, poly- imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent, optionally, R 5 is a linking group that enables linkage to
• R 6 is OR, SR or NRR', wherein R and R' have the same definition as given above, optionally R 6 is a linking group;
• X' is CH 2 , NR, CO, BH, SO or SO 2 ;
• Y' is O, CH 2 , NR or S
• Z' is CH 2 or (CH 2 ) n , wherein n is 2, 3 or 4; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these compounds; provided that the compound has no more than one linking group that enables linkage to a cell binding agent via a covalent bond.
• R 5 is selected from OR 15 , CRR'OH, SH, CRR'SH, NHR 1 S or CRR 5 NHR 15 , wherein R 15 is H or has the same definition as given above for R, or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrolloindolyl or polyimidazoloindolyl unit optionally bearing a linking group that enables linkage to a cell binding agent;
• R 6 is OCH 3 ;
• Y' is O, CH 2 , NR or S
• Z' is (CH 2 ) n , wherein n is 1 or 2, provided that X', Y' and Z' are not all CH 2 at the same time; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these compounds.
• compound of formula III is represented by a compound of formula (X) or (XI),
• the double line — between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine protecting moiety that converts the compound into a prodrug;
• Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR', a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R", amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO 3 , a bisulfite - OSO 3 , a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and are selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from
• NRCOR' NRCOR', SR 1 O, a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -
• OCOR 11 or OCONR 11 R 12 wherein R 7 , Rs, R9, Rio, Rn and R 12 are as defined above, optionally, any one OfR 1 , R 2 , R3, R 4 , R 1 ', R 2 ', R3', or R 4 ' is a linking group that enables linkage to a cell binding agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly- imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent,
• Z is selected from (CH 2 ) n , wherein n is 1, 2 or 3, CR 1 SR 16 , NR 17 , O or S, wherein R 1 S, R 16 and
• R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 ) n , wherein n is an integer from 1 to 2000;
• R 6 is OR, SR or NRR', wherein R and R' have the same definition as given above, optionally R 6 is a linking group;
• X' is selected from CH 2 , NR, CO, BH, SO or SO 2 wherein R has the same definition as given above;
• Y' is O, CH 2 , NR or S, wherein R has the same definition as given above;
• Z' is CH 2 or (CH 2 ) n , wherein n is 2, 3 or 4, provided that X', Y' and Z' are not all CH 2 at the same time;
• a and A' are the same or different and are selected from O, -CRR'O, S, -CRR' S, -NR 15 or CRR'NHRis, wherein R and R' have the same definition as given above and wherein R 1S has the same definition as R .
• D and D' are same or different and independently selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, optionally substituted with any one of halogen, OR 7 , NR 8 R 9 , NO 2 , NRCOR', SR 10 , a sulfoxide represented by SOR', a sulfone represented by - SO 2 R', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by SO 2 NRR', cyano, an azido, -COR 11 , OCOR 11 or OCONR 11 R 12 , wherein the definitions of R 7 , R 8 , R 9 , Rio, Rn and R 12 are as defined above, or a polyethylene glycol unit (-OCH 2 CH 2 ) n , wherein n is an integer from 1 to 2000;
• L is an optional phenyl group or 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P that is optionally substituted, wherein the substituent is a linking group that enables linkage to a cell binding agent via a covalent bond, or is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted with any one of halogen, OR 7 , NR 8 R 9 , NO 2 , NRCOR', SR 1 O, a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by SO 2 NRR', cyano, an azido, -COR 11 , OCOR 11 or OCONR 11 R 12 , wherein the definitions of R 7 , R
• Y is selected from -OR, NR'R", a sulfite -SO3, or a bisulfite -OSO3, wherein R is selected from
• H linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 )Ii, wherein n is an integer from 1 to 2000, aryl having from
• R 1 , R 2 , R3, R 4 , R 1 '. R 2 '. R3' and R 4 ' are each independently selected from H, NO 2 or a linking group that enables linkage to a cell binding agent via a covalent bond;
• R 6 is OR 1 S, wherein R 1 S has the same definition as R;
• Z is selected from (CH 2 ) n , wherein n is 1, 2 or 3, CR 1 SR 16 , NR 17 , O or S, wherein R 1 S, R 16 and
• R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2 CH 2 ) n , wherein n is an integer from 1 to 2000;
• Y' is O, NR, or S, wherein R is defined as above;
• Z' is CH 2 or (CH 2 ) 2 ; A and A' are each O;
• D and D' are same or different and independently selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;
• L is an optional phenyl group or a heterocycle ring having from 3 to 10 carbon atoms that is optionally substituted, wherein the substituent is a linking group that enables linkage to a cell binding agent via a covalent bond, or is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted with any one of halogen, OR 7 , NR 8 R 9 , NO 2 , NRCOR', SR 10 ,a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by SO 2 NRR', cyano, an azido, , - COR 11 , OCOR 11 or OCONR 11 R 12 , a polyethylene glycol unit (-OCH 2 CH 2 )n, wherein n is
• the compound of formula (IV), (V) or (VI) is represented by compounds of formulae (XII) and (XIII):
• Y is selected from OH, an ether represented by -OR, NR'R", a sulfite -SO 3 , or a bisulfite -OSO 3 , wherein R, R' and R" are selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; one ofR 2 , R 3 , R 2 ' and R 3 ' is a linking group that enables linkage to a cell binding agent via a covalent bond and the others are H, NRCOR' or NO 2 ;
• R 6 is OR, wherein R has the same definition as above;
• Z is CH 2 or NR, wherein R has the same definition as above;
• A is O or NR 15 ;
• L is (CH 2 ) I1n , wherein nn is 0 or an integer between 1 and 5, or a substituted or unsubstituted alkyl or alkenyl having from 2 to 4 carbon atoms, wherein the substituent is selected from halogen, OR 7 , NR 8 R 9 , NO 2 , NRCOR', SR 10 ,a sulfoxide represented by SOR', a sulfone represented by -SO 2 R', a sulfite -SO 3 , a bisulfite -OSO 3 , a sulfonamide represented by
• L itself is a linking group that enables linkage to a cell binding agent via a covalent bond
• one of L', L" or L'" is a linking group that enables linkage to a cell binding agent, while the others are H
• L' is the linking group
• G is CH or N or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these compounds.
• the compound of formula (IV), (V) or (VI) is represented by compounds of formulae from formulae (XIV) and (XV):
• Y is selected from OH, an ether represented by -OR, a sulfite -SO 3 , or a bisulfite -OSO 3 , wherein R is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; nn is 0 or an integer from 1 to 5;
• One ofR 2 , R3, R 2 ' and R3' is a linking group that enables linkage to a cell binding agent via a covalent bond and the others are H, NRCOR', or NO 2 ;
• one of L', L" or L'" is a linking group that enables linkage to a cell binding
• G is CH or N or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these compounds.
• the cytotoxic compound comprises a linking moiety. While a linker that connects two moieties is bifunctional, one end of the linker moiety can be first reacted with the cytotoxic compound to provide the compound bearing a monofunctional linking group, which can then react with a cell binding agent. Alternatively, one end of the linker moiety can be first reacted with the cell binding agent to provide the cell binding agent bearing a monofunctional linking group, which can then react with a cytotoxic compound.
• a linker that connects two moieties is bifunctional
• one end of the linker moiety can be first reacted with the cytotoxic compound to provide the compound bearing a monofunctional linking group, which can then react with a cell binding agent.
• one end of the linker moiety can be first reacted with the cell binding agent to provide the cell binding agent bearing a monofunctional linking group, which can then react with a cytotoxic compound.
• the linking moiety contains a chemical bond that allows for the release of the cytotoxic moiety at a particular site.
• Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds and esterase labile bonds (see for example US Patents 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073).
• Preferred are disulfide bonds, thioether and peptidase labile bonds.
• Other linkers that can be used in the present invention include non- cleavable linkers, such as those described in are described in detail in U.S. publication number
• R 2 , R3, R 4 or R 5 preferred linkable groups are R 2 , R3, and R5, and the most preferred linkable group is R5.
• suitable substituents at R 1 , R 2 , R3, R 4 and R5 for compounds of formula (I), (II) and (III) include, but are not limited to:
• A" is an amino acid selected from glycine, alanine, leucine, valine, lysine, citrulline and glutamate or a polypeptide containing between 2 to 20 amino acid units;
• R20, R21, R22, R23, R24, R25, R26, R27 are the same or different and are H or a linear or branched alkyl having from 1 to 5 carbon atoms;
• R-28 is H or alkyl;
• R 2 9 and R30 are the same or different and are H or alkyl from 1 to 5 carbon atoms; optionally, one of R 4 0 and R 41 is a negatively or positively charged functional group " and the other is H or alkyl, alkenyl, alkynyl having 1 to 4 carbon atoms.
• the compounds of formula (IV), (V), (VI), (VII), (XII) and (XIII) can be linked through R 1 , R 2 , R 3 , R 4 , Ri', R2', R3', R4', L', L", L' ".
• preferred linkable groups are R 2 ', R3', R 4 ', L', L", L'" and most preferred linkable groups are R 2 ', R 3 ' and L'.
• Examples of linking groups for compounds of formula (IV), (V), (VI), (VII), (XII) and (XIII) include, but are not limited to:
• A" is an amino acid selected from glycine, alanine, leucine, valine, lysine, citrulline and glutamate or a polypeptide containing between 2 to 20 amino acid units;
• R 2 O, R 2 i, R 22 , R 2 3, R 24 , R25, R26, and R 2 7 are the same or different and are H or a linear or branched alkyl having from 1 to 5 carbon atoms;
• R 2 9 and R30 are the same or different and are H or alkyl from 1 to 5 carbon atoms;
• R 33 is H or linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 12 carbon atoms, a polyethylene glycol unit -(OCH 2 CH 2 ) n , or R 33 is -COR 34 , -CSR 34 , -SOR 34 , or -SO 2 R 34 , wherein
• R 34 is H or linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 20 carbon atoms or, a polyethylene glycol unit -(OCH 2 CH 2 ) n ; and one of R 4 o and R 41 is optionally a negatively or positively charged functional group and the other is H or alkyl, alkenyl, alkynyl having 1 to 4 carbon atoms.
• cytotoxic compounds e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine
• derivatives thereof, or dimers thereof bearing a linking moiety is described below in terms of an amide, thioether or disulfide bond containing linking moieties at the L' (in the compound of formula XIII) or R3 (in the compound of formula XII) positions
• linking moieties at other positions and with other chemical bonds, as described above can also be used with the present invention.
• sodium dithionite was used to conveniently convert to aldehyde 6 to the ring closed compound 7 after further treatment with methanol under acidic conditions.
• the benzyl protecting group was removed to furnish monomer 8.
• Dimers which possess linkers that can react with antibodies are prepared by converting the methyl esters to the corresponding reactive esters of a leaving group such as, but not limited to, N-hydroxysuccinimide esters, N-hydroxyphtalimide esters, N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters.
• a leaving group such as, but not limited to, N-hydroxysuccinimide esters, N-hydroxyphtalimide esters, N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters.
• Representative examples for the synthesis of the linkable dimers are shown in figures 8. Synthesis of dimers that bear a thiol or disulfide moiety to enable linkage to cell binding agents via reducible or nonreducible bonds is shown in Figures
• the B ring modified monomer 58 devoid of a carbonyl group is achieved from the benzyl acohol compound 52 by the steps shown in Figure 11.
• the isoindolino monomer 66 can be prepared from isoindole 59 as outlined in Figure 12.
• the linker can also be attached directly to the indolino moiety.
• Methyl indolino-2-carboxylate can be converted into the linkable dimer 82 via the synthetic steps shown in Figure 13.
• the synthesis of linkable dimers bearing a PEG moiety is shown in Figures 14 and 15.
• the invention provides a process for the preparation of the indolinobenzodiazepine (IBD) monomer of formula (I) ( Figure 1), the process comprising the steps of: a) coupling compound of formula (1) and compound of formula (2) to give compound of formula
• LG is a leaving group
• W is COOR or CH 2 OW", wherein R has the same definition as above and W" is a protecting group;
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , W, Z, X, Y and — have the same definition as described above.
• Another aspect of the invention provides a process for the preparation of compound of formula (II) comprising the steps of: a) coupling compound of formula (1) and compound of formula (5) to give compound of formula
• Another aspect of the invention provides a process for the preparation of compound of formula (III) comprising steps of: a) coupling compound of formula (1) and compound of formula (8) to give compound of formula
• Another aspect of the invention provides a process for the preparation of compound of formula (Another aspect of the invention provides a process for the preparation of compound of formula (IV) comprising the steps of: coupling compound of formula (11), compound of formula (H)' and compound of formula (12) to give compound of formula (IV),
• LG is a leaving group
• R 1 , R 2 , R 3 , R 4 , Ri', R2', R3', R4', Re, W, X, Y, Z, A, A', D, D', L and ⁇ have the same definition as above.
• Another aspect of the invention provides an alternative process for the preparation of compound of formula (IV) of the present invention comprising steps of: a) converting compound of formula (15) into aldehyde of formula (16); and b) converting compound of formula (16) into compound of formula (IV),
• W is COOR or CH 2 OW", wherein R has the same definition as above and W" is a protecting group; R 1 , R 2 , R 3 , R 4 , Ri', R 2 ', R 3 ', R 4 ', R 6 , W, X, Y, Z, A, A', D, D', L and — have the same definition as above.
• Another aspect of the invention provides a process for the preparation of compound of formula (V) comprising the step of coupling compound of formula (13), compound of formula (13)' and compound of formula (12) to give compound of formula (V),
• Another aspect of the invention provides an alternative process for the preparation of compound of formula (V) of the invention comprising the steps of: a) converting compound of formula (17) into aldehyde of formula (18); and b) converting compound of formula (18) into compound of formula (V),
• W is COOR or CH 2 OW", wherein R has the same definition as above and W" is a protecting group; R 1 , R 2 , R 3 , R 4 , Ri', R 2 ', R 3 ', R4', Re, W, X, Y, A, A', D, D', L and — have the same definition as above.
• Another aspect of the invention provides a process for the preparation of compound of formula (VI) of the invention comprising the step of coupling compound of formula (14), compound of formula (14)' and compound of formula (12) to give compound of formula (VI),
• LG is a leaving group
• R 6 , W, X, Y, X', Y', Z' A, A', D, D', L and — have the same definition as above.
• Another aspect of the invention provides a process for the preparation of compound of formula (VI) of the invention comprising the steps of: a) converting compound of formula (19) into aldehyde of formula (20); and b) converting compound of formula (20) into compound of formula (VI),
• the in vitro cytotoxicity of the cytotoxic compounds e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine
• derivatives thereof, dimers thereof or conjugates thereof of the present invention can be evaluated for their ability to suppress proliferation of various cancerous cell lines in vitro (Tables 1, 2 in FIGS. 31, 32.).
• cell lines such as the human breast carcinoma line SK-Br-3, or the human epidermoid carcinoma cell line KB, can be used for the assessment of cytotoxicity of these new compounds.
• Cells to be evaluated can be exposed to the compounds for 72 hours and the surviving fractions of cells measured in direct assays by known methods. IC50 values can then be calculated from the results of the assays.
• Cell-binding agents may be of any kind presently known, or that become known and includes peptides and non-peptides. Generally, these can be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell-binding molecule or substance.
• cell-binding agents that can be used include: polyclonal antibodies; monoclonal antibodies; fragments of antibodies such as Fab, Fab', and F(ab')2, Fv (Parham, J. Immunol. 131 :2895-2902 (1983); Spring et al. J. Immunol. 113:470-478 (1974); Nisonoff et al. Arch. Biochem. Biophys. 89:230-244 (I960)); interferons (e.g.
• lymphokines such as IL-2, IL-3, IL-4, IL-6; hormones such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte- stimulating hormone), steroid hormones, such as androgens and estrogens; growth factors and colony-stimulating factors such as EGF, TGF-alpha, FGF, VEGF, G- CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984)); transferrin (O'Keefe et al. J. Biol. Chem. 260:932-937 (1985)); and vitamins, such as folate.
• lymphokines such as IL-2, IL-3, IL-4, IL-6
• hormones such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte- stimulating hormone), steroid hormones, such as androgens and estrogens
• growth factors and colony-stimulating factors such as EGF,
• Monoclonal antibody techniques allow for the production of extremely specific cell- binding agents in the form of specific monoclonal antibodies.
• Particularly well known in the art are techniques for creating monoclonal antibodies produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins such as viral coat proteins.
• Sensitized human cells can also be used.
• Another method of creating monoclonal antibodies is the use of phage libraries of scFv (single chain variable region), specifically human scFv (see e.g., Griffiths et al., U.S. Patent Nos.
• the monoclonal antibody MY9 is a murine IgG 1 antibody that binds specifically to the CD33 Antigen ⁇ J.D. Griffin et al 8 Leukemia Res., 521 (1984) ⁇ and can be used if the target cells express CD33 as in the disease of acute myelogenous leukemia (AML).
• the monoclonal antibody anti-B4 is a murine IgG 1 , that binds to the CD 19 antigen on B cells ⁇ Nadler et al, 131 J. Immunol.
• HuB4 is a resurfaced antibody derived from the murine anti-B4 antibody (Roguska et al., 1994, Proc. Natl. Acad. ScL, 91, pg 969-973).
• HuN901 is a humanized antibody that binds to the CD56 antigen expressed on small cell lung cancer, multiple myeloma, ovarian cancer and other solid tumors including neuroendocrine cancers (Roguska et al., 1994, Proc. Natl. Acad.
• B38.1 is a chimeric antibody targeting EpCAM.
• Fully human antibodies such as panitumumab targeting the EGF receptor expressed on several solid tumors may also be used (Van Cutsem et al., J Clin Oncol. 2007;25(13): 1658-1664).
• the cell- binding agent that comprises the conjugates and the modified cell-binding agents of the present invention may be of any kind presently known, or that become known, and includes peptides and non-peptides.
• the cell-binding agent may be any compound that can bind a cell, either in a specific or non-specific manner.
• these can be antibodies (especially monoclonal antibodies and antibody fragments), interferons, lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell-binding molecule or substance.
• the cell-binding agent is an antibody, it binds to an antigen that is a polypeptide and may be a transmembrane molecule (e.g. receptor) or a ligand such as a growth factor.
• antigens include molecules such as renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha- 1 -antitrypsin; insulin A- chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor vmc, factor IX, tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t- PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (
• GM-CSF which binds to myeloid cells can be used as a cell-binding agent to diseased cells from acute myelogenous leukemia.
• IL-2 which binds to activated T-cells can be used for prevention of transplant graft rejection, for therapy and prevention of graft- versus-host disease, and for treatment of acute T-cell leukemia.
• MSH which binds to melanocytes, can be used for the treatment of melanoma.
• Folic acid can be used to target the folate receptor expressed on ovarian and other tumors.
• Epidermal growth factor can be used to target squamous cancers such as lung and head and neck.
• Somatostatin can be used to target neuroblastomas and other tumor types.
• the present invention also provides cytotoxic compound-cell-binding agent conjugates comprising a cell binding agent linked to one or more cytotoxic compounds via a variety of linkers, including, but not limited to, disulfide linkers, thioether linkers, amide bonded linkers, peptidase -labile linkers, acid-labile linkers, esterase-labile linkers.
• linkers including, but not limited to, disulfide linkers, thioether linkers, amide bonded linkers, peptidase -labile linkers, acid-labile linkers, esterase-labile linkers.
• Representational cytotoxic conjugates of the invention are antibody/cytotoxic compound, antibody fragment/cytotoxic compound, epidermal growth factor (EGF)/ cytotoxic compound, melanocyte stimulating hormone (MSH)/ cytotoxic compound, thyroid stimulating hormone (TSH)/ cytotoxic compound, somatostatin/cytotoxic compound, folate/cytotoxic compound, estrogen/cytotoxic compound, estrogen analogue/cytotoxic compound, androgen/cytotoxic compound, and androgen analogue/cytotoxic compound.
• the present invention provides an indolinobenzodiazepine dimer-cell-binding agent conjugate comprising the cytotoxic agent and the cell binding agent linked through a covalent bond.
• the linker can be cleaved at the site of the tumor/unwanted proliferating cells to deliver the cytotoxic agent to its target in a number of ways.
• the linker can be cleaved, for example, by low pH (hydrazone), reductive environment (disulfide), proteolysis (amide/peptide link), or through an enzymatic reaction (esterase/glycosidase).
• representatative cytotoxic conjugates of the invention are antibody/ indolinobenzodiazepine dimer, antibody fragment/indolinobenzodiazepine dimer, epidermal growth factor (EGF)/ indolinobenzodiazepine dimer, melanocyte stimulating hormone (MSH)/ indolinobenzodiazepine dimer, thyroid stimulating hormone (TSH)/ indolinobenzodiazepine dimer, somatostatin/ indolinobenzodiazepine dimer, folate/ indolinobenzodiazepine dimer, estrogen/ indolinobenzodiazepine dimer, estrogen analogue/ indolinobenzodiazepine dimer, prostate specific membrane antigen (PSMA) inhibitor/ indolinobenzodiazepine dimer, matriptase inhibitor/ indolinobenzodiazepine dimer, designed ankyrin repeat proteins (DARPins)/ indolinobenzodiazepine dimer, androgen/ indolino
• Disulfide containing cytotoxic conjugates can be made by reacting a thiol-containing cytotoxic agent such as 49 with an appropriately modified cell-binding agent. These conjugates may be purified to remove non-linked cytotoxic agent by using gel-filtration, ion exchange chromatography, ceramic hydroxyappetite (CHT) chromatography, hydrophobic interaction chromatography (CHT), tangential flow filtration (TFF), or by HPLC.
• CHT ceramic hydroxyappetite
• CHT hydrophobic interaction chromatography
• THF tangential flow filtration
• a solution of an antibody in aqueous buffer may be incubated with a molar excess of an antibody modifying agent such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or with ⁇ /-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) to introduce dithiopyridyl groups.
• SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
• SPDB ⁇ /-succinimidyl-4-(2-pyridyldithio)butanoate
• the modified antibody is then reacted with the thiol-containing cytotoxic agent such as compound 49 to produce a disulf ⁇ de-linked antibody- indolinobenzodiazepine dimer conjugate.
• the cytotoxic- cell binding conjugate may then be purified using any of the above mentioned methods.
• the antibody may be incubated with a molar excess of an antibody modifying agent such as 2-iminothiolane, L-homo cysteine thiolactone (or derivatives), or N- Succinimidyl-S-acetylthioacetate (SATA) to introduce sulfhydryl groups.
• an antibody modifying agent such as 2-iminothiolane, L-homo cysteine thiolactone (or derivatives), or N- Succinimidyl-S-acetylthioacetate (SATA) to introduce sulfhydryl groups.
• the modified antibody is then reacted with the appropriate disulfide-containing cytotoxic agent, such as, compound 51 to produce a disulf ⁇ de-linked antibody-cytotoxic agent conjugate.
• the antibody- cytotoxic agent conjugate may then be purified by gel-filtration or other methods mentioned above.
• the number of cytotoxic molecules bound per antibody molecule can be determined spectrophotometrically by measuring the ratio of the absorbance at 280 nm and 330 nm. An average of 1-10 cytotoxic molecules/antibody molecule(s) can be linked by this method. The preferred average number of linked cytotoxic molecules per antibody molecule is 2-5, and the most preferred is 3-4.5.
• a solution of an antibody in aqueous buffer may be incubated with a molar excess of an antibody-modifying agent such as N-succinimidyl-4-(N-maleimidomethyl)- cyclohexane-1-carboxylate to introduce maleimido groups, or with JV-succinimidyl-4- (iodoacetyl)-aminobenzoate (SIAB) to introduce iodoacetyl groups.
• the modified antibody is then reacted with the thiol-containing cytotoxic agent to produce a thioether-linked antibody- cytotoxic conjugate.
• the antibody-cytotoxic conjugate may then be purified by gel-filtration or other methods mentioned above or by methods known to one of skill in the art.
• Cytotoxic agents containing linkers terminating in an N-Hydroxy succinimidyl (NHS) ester, such as compounds 43, 44, and 46, can be reacted with the antibody to produce direct amide linked conjugates such as huN901-IGN-03 and huN901-IGN-07.
• the antibody-cytotoxic agent conjugate may then be purified by gel-filtration or other methods mentioned above.
• the following cell-binding agent/cytotoxic agent conjugates can be prepared using the appropriate linkers.
• Dimer 1 and 2 with peptide cleavable linkers can be prepared from the corresponding NHS esters, Dimer 3 can be made by reacting the appropriate thiol-containing cytotoxic agent with SMCC modified cell binding agent, and acid-labile hydrazone Dimer 4 can be prepared through condensation of a cytotoxic agent containing an alkyl, aryl ketone with a hydrazide modified cell binding agent.
• Asymmetric indolinobenzodiazepine dimer conjugates such as Dimers 5-8 can also be prepared using similar methods to those described above.
• Conjugates of cell-binding agents with cytotoxic agents of the invention can be evaluated for their ability to suppress proliferation of various unwanted cell lines in vitro.
• cell lines such as the human colon carcinoma line COLO 205, the rhabdomyosarcoma cell line RH- 30, and the multiple myeloma cell line MOLP-8 can be used for the assessment of cytotoxicity of these conjugates.
• Cells to be evaluated can be exposed to the compounds for 1-5 days and the surviving fractions of cells measured in direct assays by known methods. IC50 values can then be calculated from the results of the assays.
• Fig. 21-26 Examples of in vitro potency and target specificity of antibody-cytotoxic agent conjugates of the present invention are shown in Fig. 21-26. All of the conjugates with cytotoxic agent/antibody ratios of 1-3 are extremely cytotoxic on the antigen positive cancer cells with an IC50 in the low picomolar range. Antigen negative cell lines remained viable when exposed to the same conjugates.
• the target specificity of conjugates of the indolinobenzodiazepine dimers are >1000 with the antibodies huN901 (anti-CD56) and muB38.1 (anti-EpCAM).
• the B38.1-IGN-3 conjugate killed antigen positive COLO 205 cells with an IC50 value of 1.86 pM, while the antigen negative Namalwa cell line was about 200-fold less sensitive with an IC50 value of 336.3 pM, demonstrating antigen specificity.
• the conjugate is also highly potent towards the multidrug resistant COLO 205 MDR cell line with an IC50 value of 16 pM.
• the huN901-IGN3 conjugate was highly potent, with an IC50 value of 15 pM for antigen positive RH30 cells (Fig. 22).
• huN901-IGN-07 Another huN901-IGN conjugate (huN901-IGN-07) also showed high potency towards antigen expressing RH-30 cells, with drug load dependent cytotoxicity and IC50 values of 16 pm, 3 pM and 2 pM respectively for conjugates bearing 1.2, 2.0 and 3.0 linked drugs per antibody molecule (Fig. 23). Similar results were obtained with huN901-IGN07 and huN901-IGN03 towards antigen-positive Molp-8 cells.
• Hu901-IGN07 gave IC 50 values of 5 pM, 3 pM and 2 pM respectively for IGN07 loads of 1.2, 2.0 and 3.0 (Fig. 24).
• the huN901-IGN07 and IGN03 conjugates were much less potent towards antigen negative Namalwa cells with IC50 values ranging from 1000 pM to >3000 pM (Fig. 25).
• the B38.1 -IGN 10 conjugate was also specifically potent killing antigen positive COLO 205 cells, with an IC50 of 17 pM, and less potent (170 pM) for antigen-negative Ramos cells (Fig . 26).
• the indolinobenzodiazepine dimers of the present invention bind and alkylate double- stranded DNA (dsDNA) containing guanine residues on opposite strands spaced 4 base pairs apart.
• Figures 28-30 present data from reverse-phase ion pair chromatography assays showing rate of IGN-01, IGN-02, and IGN-09 binding and crosslinking to dsDNA.
• the indolino group (IGN-01) is preferred to the oxazole group (IGN-02) for rapid DNA binding and interstrand crosslinking (ICL).
• ICL interstrand crosslinking
• IC50 values of various compounds of the present invention towards a panel of cell lines is listed in Fig. 31. Comparative in vitro potency of linkable and non-linkable compounds of the present invention are shown in Fig. 32. Incorporation of a linker does not significantly affect potency of the parent compounds.
• compositions and methods of use are provided.
• the present invention includes a composition (e.g., a pharmaceutical composition) comprising novel benzodiazepine compounds (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine), derivatives thereof, or conjugates thereof, (and/or solvates, hydrates and/or salts thereof) and a carrier (a pharmaceutically acceptable carrier).
• a composition e.g., a pharmaceutical composition
• novel benzodiazepine compounds e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine
• derivatives thereof e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine
• a carrier a pharmaceutically acceptable carrier
• the present invention also includes a composition (e.g., a pharmaceutical composition) comprising novel benzodiazepine compounds, derivatives thereof,, or conjugates thereof, (and/or solvates, hydrates and/or salts thereof) and
• the present compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human).
• the present compositions are also useful for treating depression, anxiety, stress, phobias, panic, dysphoria, psychiatric disorders, pain, and inflammatory diseases in a mammal (e.g., human).
• the present invention includes a method of inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human) comprising administering to said mammal a therapeutically effective amount of novel benzodiazepine compounds (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine), derivatives thereof, or conjugates thereof, (and/or solvates and salts thereof) or a composition thereof, alone or in combination with a second therapeutic agent.
• novel benzodiazepine compounds e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine
• derivatives thereof or conjugates thereof, (and/or solvates and salts thereof) or a composition thereof, alone or in combination with a second therapeutic agent.
• the present invention also provides methods of treatment comprising administering to a subject in need of treatment an effective amount of any of the conjugates described above.
• the present invention provides a method for inducing cell death in selected cell populations comprising contacting target cells or tissue containing target cells with an effective amount of a cytotoxic agent comprising any of the cytotoxic compound-cell-binding agents (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine dimer linked to a cell binding agent) of the present invention, a salt or solvate thereof.
• the target cells are cells to which the cell-binding agent can bind.
• Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those of ordinary skill in the art as the clinical situation warrants.
• suitable carriers, diluents and/or excipients include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, containing or not containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.
• the method for inducing cell death in selected cell populations can be practiced in vitro, in vivo, or ex vivo.
• Examples of in vitro uses include treatments of autologous bone marrow prior to their transplant into the same patient in order to kill diseased or malignant cells: treatments of bone marrow prior to their transplantation in order to kill competent T cells and prevent graft- versus- host-disease (GVHD); treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen.
• GVHD graft- versus- host-disease
• the conditions of non-clinical in vitro use are readily determined by one of ordinary skill in the art.
• Examples of clinical ex vivo use are to remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from autologous or allogenic bone marrow or tissue prior to transplant in order to prevent GVHD.
• Treatment can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the cytotoxic agent of the invention, concentrations range from about 10 ⁇ M to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and time of incubation, i.e., the dose, are readily determined by one of ordinary skill in the art.
• the bone marrow cells are washed with medium containing serum and returned to the patient intravenously according to known methods.
• the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.
• the cytotoxic agent of the invention will be supplied as a solution or a lyophilized powder that are tested for sterility and for endotoxin levels. Examples of suitable protocols of conjugate administration are as follows. Conjugates are given weekly for 4 weeks as an intravenous bolus each week.
• Bolus doses are given in 50 to 1000 ml of normal saline to which 5 to 10 ml of human serum albumin can be added. Dosages will be 10 ⁇ g to 2000 mg per administration, intravenously (range of 100 ng to 20 mg/kg per day). After four weeks of treatment, the patient can continue to receive treatment on a weekly basis. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc., can be determined by one of ordinary skill in the art as the clinical situation warrants.
• Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of inducing cell death in selected cell populations include malignancy of any type including, for example, cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphatic organs; autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft rejections, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft versus host disease; viral infections, such as CMV infection, HIV infection, AIDS, etc.; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and others as determined by one of ordinary skill in the art.
• PDR Physician's Desk Reference
• the PDR discloses dosages of the agents that have been used in treatment of various cancers.
• the dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
• the contents of the PDR are expressly incorporated herein in its entirety by reference.
• One of skill in the art can review the PDR, using one or more of the following parameters, to determine dosing regimen and dosages of the chemotherapeutic agents and conjugates that can be used in accordance with the teachings of this invention. These parameters include:
• cytotoxic agents [207]
• each of the cytotoxic agents described herein can be modified in such a manner that the resulting compound still retains the specificity and/or activity of the starting compound.
• the skilled artisan will also understand that many of these compounds can be used in place of the cytotoxic agents described herein.
• the cytotoxic agents of the present invention include analogues and derivatives of the compounds described herein.
• IBD Indolinobenzodiazepine (IBD) monomer 8:
• the mixture was stirred at RT for 0.5 h, diluted with ethanol/toluene (6 ml, 1 :1), evaporated and co-evaporated with ethanol/toluene (3 x 10 ml), and dried over a vacuum.
• the solid compound was used directly without further purification.
• huN901 antibody that binds to the CD56 antigen was selected for conjugation of IGN derivatives.
• a solution of huN901 antibody at a concentration of 3 mg/mL in an aqueous buffer containing 0.05 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) and 2 niM ethylenediaminetetra-acetic acid (EDTA), pH 8 was treated with a 20-fold molar excess of a solution of IGN-07 NHS ester in dimethylacetamide (DMA) such that the final concentration of DMA in the buffer was 10% v/v.
• HEPES N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid
• EDTA niM ethylenediaminetetra-acetic acid
• the reaction mixture was stirred at room temperature for 120 min and then loaded onto a Sephadex G25 gel filtration column (HiPrepTM 26/10 Desalting Column GE# 17-5087-01) that has been previously equilibrated into an aqueous buffer containing 0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5.
• the conjugated antibody- containing fractions are collected and pooled to yield product.
• the pooled sample was dialyzed overnight against the same elution buffer (0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5) to further purify the product.
• muB38.1 antibody that binds to the EpCAM antigen was selected for conjugation of IGN derivatives through a disulfide bond.
• a solution of muB38.1 antibody at a concentration of 2.5 mg/mL in an aqueous buffer containing phosphate buffered saline (PBS) pH 7.4 was treated with 120 molar excess of 1-homocysteine thiolactone for 12 hr at 37 0 C.
• the reaction mixture was loaded onto a Sephadex G25 gel filtration column (HiPrepTM 26/10 Desalting Column GE# 17- 5087-01) that was previously equilibrated in PBS pH 7.4.
• Fractions containing antibody are collected and pooled and assayed for reactive thiol content using the Ellman's assay.
• the modified antibody was then treated with a 4-fold molar excess of IGN-10 (in DMA) per free thiol and allowed to react at room temperature for 8 hr.
• the reaction mixture was loaded onto a Sephadex G25 gel filtration column (HiPrepTM 26/10 Desalting Column GE# 17-5087-01) that has been previously equilibrated into an aqueous buffer containing 0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5.
• the conjugated antibody-containing fractions are collected and pooled to yield product.
• the pooled sample was dialyzed overnight against the same elution buffer (0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5) to further purify the product. [295]
• DNA annealing single stranded DNA (Invitrogen) was annealed into dsDNA using a Peltier thermal cycler (PTC-200, MJ Research). 1 mM DNA in 100 mM TRIS, 1 mM EDTA pH 8 was heated to 80 0 C and then gradually cooled to 4 0 C over 90 min in 15 degree steps. The resulting dsDNA was kept at 4 0 C until used in the assay. IGN-01, IGN-02, and IGN-09 did not form covalent adducts with single stranded DNA (ssDNA) in control experiments.
• ssDNA single stranded DNA
• the organic layer was dried over anhydrous sodium sulfate, filtered, evaporated and high vacuumed to give the mesylate as colorless oil.
• the mesylate was transferred to a 10 mL round bottom flask with ethyl acetate, evaporated and high vacuumed.
• Compound 8 (221 mg, 0.75 mmol) was added followed by addition of anhydrous DMF (2 mL) and anhydrous potassium carbonate (207 mg, 1.5 mmol). The mixture was stirred at room temperature overnight. It was diluted with dichloromethane and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated.
• N-hydroxysuccinimide (NHS, 5.09 mg, 0.044 mmol) and N- (3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 8.48 mg, 0.044 mmol) was added subsequently.
• the mixture was stirred at room temperature overnight and diluted with dichloromethane, washed with brine and dried over anhydrous sodium sulfate. It was filtered, evaporated and the residue was purified by preparative reverse phase HPLC (C 18 column, eluted with acetonitrile/water). The product fractions were combined and extracted with dichloromethane.
• the white residue was placed on the high vacuum for a few hours. It was re-dissolved in methanol :dichloromethane [1 :1], filtered through celite, and stripped. The filter step was repeated until dilution in methanol appeared clear with no particles.
• the intermediate was placed on the high vacuum until completely dry then dissolved in anhydrous methanol (50ml). Acetyl chloride (1.9ml, 26.7 mmol) was added drop wise at room temperature, causing a yellow precipitate to form. It stirred at room temperature for 30 minutes and was quenched with saturated sodium bicarbonate. The mixture was diluted with dichloromethane and water (130mL/85mL) and extracted with dichloromethane.

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