pyrrolobenzodiazepine compound and conjugate and uses thereof

专利摘要:
PIRROLOBENZODIAZEPINES. Pyrrolobenzodiazepine dimers having a C2-C3 double bond and an aryl group at the C2 position in one monomer unit, and a C2-C3 double bond and a conjugated bond or triple bond in the C2 position or an alkyl group in the C2 position in the other unit monomer, and conjugates of these compounds.
公开号:BR112012026410B1
申请号:R112012026410-9
申请日:2011-04-15
公开日:2020-12-01
发明作者:Phillip Wilson Howard；Luke Masterson；Arnaud Tiberghien；Scott Jeffrey；Patrick Burke；Peter Senter
申请人:Seattle Genetics, Inc；Medimmune Limited；
IPC主号:

专利说明:

The present invention relates to pyrrolobenzodiazepines (PBDs), in particular to pyrrolobenzodiazepine dimers having a C2-C3 double bond and an aryl group at the C2 position in a monomer unit, and a C2-C3 single bond and still a double or triple bond conjugated at the C2 position or an alkyl group at the C2 position on the other monomer unit. Fundamentals of the Invention
[002] Some pyrrolobenzodiazepines (PBDs) have the ability to recognize and bind to specific DNA sequences; the preferred sequence is PuGPu. The first anti-tumor PBD antibiotic, anthramycin, was discovered in 1965 (Leimgruber, and col., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, and col., J. Am Chem, Soc., 87, 5791-5793 (1965)). Since then, several naturally occurring PBDs have been described, and more than 10 synthetic routes have been developed for a variety of analogs (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abeimicin (Hochlowski, and col., J. Antibiotics, 40, 145-148 (1987)), quicamycin (Konishi, and col., J. Antibiotics, 37, 200-206 (1984)) , DC-81 (Japanese Patent 58-180 487; Thurston, and col., Chem. Brit., 26, 767-772 (1990); Bose, and col., Tetrahedron, 48, 751-758 (1992)) , mazetramycin (Kuminoto, and col., J. Antibiotics, 33, 665-667 (1980)), neotramicins A and B (Takeuchi, and col., J. Antibiotics, 29, 93-96 (1976) ), porotramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), protracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomycin (DC-102) (Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, and col ., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, and col., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, and col., J. Antibiotics, 25, 437-444 (1972)). PBDs are of a general structure:
[003] They differ in the number, type and position of the substituents, both in their aromatic A rings and in the pyrrole C rings, and in the degree of saturation of ring C. In ring B there is an imine (N = C), or an carbinolamine (NH-CH (OH)), or a methyl ether carbinolamine (NH-CH (OMe)) at position N10-C11, which is the electrolytic center responsible for alkylating DNA. All known natural products have a configuration (S) in the chiral position C11a, which proves them with a rotation from left to right when viewed from ring C to ring A. This gives them the appropriate three-dimensional shape for isohelicity with the secondary groove of form B DNA, resulting in a small fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pages 3-11 (1975); Hurley and Needham- VanDevanter, Acc Chem, Res., 19, 230-237 (1986)). Their ability to form an adduct in the secondary groove enables them to interfere with DNA processing, for this reason their use as antitumor agents.
[004] It was previously reported that the biological activity of these molecules can be enhanced by joining two units of PBD together through their hydroxyl functions in C8 / C 'by means of a flexible alkylene linker (Bose, DS, and col., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, DE, et al., J. Org. Chem., 61, 8141-8147 (1996)). PBD dimers are believed to form selective DNA damage for the sequence, such as crosslinking between palindromic 5'-Pu-GATC-Py-3 'strands (Smellie, M., and col., Biochemistry, 42, 8232 -8239 (2003); Martin, C., and col., Biochemistry, 44, 4135-4147), which is believed to be primarily responsible for its biological activity. An example of a PBD dimer, SG2000 (SJG-136):
recently completed Phase I clinical trials in the field of oncology and is about to enter Phase II (Gregson, S., and col., J. Med. Chem., 44, 737-748 (2001); Alley, MC, and col., Cancer Research, 64, 6700-6706 (2004); Hartley, JA, and col., Cancer Research, 64, 6693-6699 (2004)).
[005] More recently, the present inventors previously disclosed in WO 2005/085251, the dimeric PBD compounds having C2 aryl substituents, such as SG2202 (ZC-207):
and in WO2006 / 111759, bisulfites of such PBD compounds, for example, SG2285 (ZC-423):

These compounds have been shown to be highly useful cytotoxic agents (Howard, P.W., and col., Bioorg. Med. Chem. (2009), doi: 10,1016 / j.bmcl.2009,09,012).
[007] Due to the way in which these highly potent compounds act in the crosslinking of DNA, these molecules have been prepared symmetrically. This provides for direct synthesis, by constructing the PBD portions simultaneously having already formed the bond of the dimer, or by reacting the PBD portions already constructed with the dimer-binding group.
[008] The International Co-pending Application PCT / GB2009 / 002498, filed on October 16, 2009 discloses an asymmetric dimeric PBD compound containing aryl groups at the C2 position of each monomer, where one of these groups contains a replacement designed for providing an anchor for connecting the compound to another portion. Disclosure of the Invention
[009] The present inventors have developed additional asymmetric dimeric PBD compounds containing an aryl group at the C2 position of a monomer, said aryl group containing a substitute designed to provide an anchor for binding the compound to another portion, and also an unsaturated bond the C2-C3 double bond or an alkyl group is conjugated to the other monomer unit.
[0010] The present invention comprises a compound of formula I:
where: R2 and formula II:
where A is an aryl group of C5-7, X and selected from the group comprising: OH, SH, CO2H, COH, N = C = O, NHNH2, CONHNH2,

, NHRN, where RN is selected from the group comprising H and C1-4 alkyl, and (OC2H4) mOCH3, and: (i) Q1 is a single bond, and Q2 is selected from a single bond and - Z- (CH2) n-, where Z is selected from a single bond, O, S and NH and n is 1 to 3; or (ii) Q1 is -CH = CH-, and Q2 is a single bond; R12 is selected from: (ii) aliphatic saturated C1-5 alkyl; (iiib) saturated C3-6 cycloalkyl; (iic)
, where each of R21, R22 and R23 is independently selected from H, saturated C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the group R12 is not more than 5; (iid)
wherein one from R25a and R25b and H and the other is selected from: phenyl, whose phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (iie)
where R24 is selected from: H; cloudy C1-3 alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, whose phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR ', nitro, Me3Sn and halo; where R and R 'are independently selected from optionally substituted C1-12 alkyl groups, C3-20 heterocyclyl and C5-20 aryl; R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR ', nitro, Me3Sn and halo; or: (a) R10 and H, and R11 and OH, ORA, where RA is C1-4 alkyl; (b) R10 and R11 form a double nitrogen-carbon bond between the nitrogen and carbon atoms to which they are attached; or (c) R10 and H and R11 and SOzM, where z and 2 or 3 and M are a pharmaceutically acceptable monovalent cation; R "is a C3-12 alkylene group, the chain of which may be interrupted by one or more heteroatoms, for example, O, S, NRN2 (where RN2 and H or C1-4 alkyl), and / or aromatic rings, for example example, benzene or pyridine; Y and Y 'are selected from O, S, or NH; R6', R7 ', R9 are selected from the same groups as R6, R7 and R9, respectively, and R10' and R11 'are the same as R10 and R11, where if R11 and R11' are SOzM, M can represent a pharmaceutically acceptable divalent cation.
[0011] A second aspect of the present invention provides for the use of a compound of the first aspect of the invention in the manufacture of a medicament for the treatment of a proliferative disease. The second aspect also provides a compound of the first aspect of the invention for use in the treatment of a proliferative disease.
[0012] Someone of ordinary skill in the technique is readily able to determine whether a candidate conjugate treats or not a proliferative condition for any particular type of cell. For example, assays that can be conveniently used to assess the activity offered by a particular compound are described in the examples below.
[0013] A third aspect of the present invention comprises a compound of formula II:
where: R2 and formula II:
where A is an aryl group of C5-7, X and selected from the group comprising: OH, SH, CO2H, COH, N = C = O, NHNH2, CONHNH2, -,

,, NHRN, where RN is selected from the group comprising H and C1-4 alkyl, and: (i) Q1 is a single bond, and Q2 is selected from a single bond and -Z- (CH2) n-, where Z is selected from a simple bond, O, S and NH and n from 1 to 3; or (ii) Q1 and -CH = CH-, and Q2 is a single bond; R12 is selected from: (iia) aliphatic saturated C1-5 alkyl; (iiib) saturated C3-6 cycloalkyl; (iic)
, where each of R21, R22 and R23 and independently selected from H, saturated C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of atoms of carbon in group R12 is not more than 5; (iid)
wherein one of R25a and R25b is H and the other is selected from: phenyl, whose phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (iie)
, where R24 is selected from: H; saturated C1-3 alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, whose phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR ', nitro, Me3Sn and halo; where R and R 'are independently selected from optionally substituted C1-12 alkyl groups, C3-20 heterocyclyl and C5-20 aryl; R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR ', nitro, Me3Sn and halo; or: (a) R10 is a carbamate nitrogen protecting group, and R11 and O-ProtO, where ProtO is an oxygen protecting group; (b) R10 and a hemiaminal nitrogen protecting group and R11 and an oxo group; R is a C3-12 alkylene group, the chain of which can be interrupted by one or more heteroatoms, for example, O, S, NRN2 (where RN2 and H or C1-4 alkyl), and / or aromatic rings, for example , benzene or pyridine; Y and Y 'are selected from O, S, or NH; R6 ', R7', R9 'are selected from the same groups as R6, R7 and R9, respectively, and R10' and R11 'are the same as R10 and R11.
[0014] A fourth aspect of the present invention comprises a method of preparing a compound of formula I from a compound of formula II by deprotecting the imine bond.
[0015] The asymmetric dimeric PBD compounds of the present invention are prepared by different strategies than those previously employed in the preparation of symmetric dimeric PBD compounds. In particular, the present inventors have developed a method that involves adding each C2 substitute to a symmetric PBD-sized nucleus in separate steps of the method. Accordingly, a fifth aspect of the present invention provides a method of preparing a compound of the first or third aspect of the invention, comprising at least one of the steps of the method specified below.
[0016] In a sixth aspect, the present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein the PBD is a dimer of formula I (above).
[0017] In some modalities, the Conjugates have the following formula III: L - (LU-D) p (III) in which L is a Ligand unit (ie, a targeting agent), LU and a Ligand unit and D is a Pharmaco unit comprising a number of PBDs. The subscript p is an integer from 1 to 20. Consequently, the Conjugates comprise the Ligand unit covalently linked to at least one Pharmaco unit by a Ligand unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cellular component (a Cell Binding Agent) or other target molecules of interest. Consequently, the present invention also provides methods for treating, for example, various cancers and autoimmune disease. These methods include the use of the Conjugates in which the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antibody binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.
[0018] The dimer of PBD D and formula I, except that X is selected from the group comprising: O, S, C (= O), C =, NH (C = O), NHNH, CONHNH,
NRN, where RN is selected from the group comprising H and C1-4 alkyl. Brief Description of the Figure
[0019] Figure 1 shows the effect of a conjugated steel of the invention on a tumor. Definitions Pharmaceutically acceptable cations
[0020] Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated into this document by reference.
[0021] The pharmaceutically acceptable cation can be inorganic or organic.
[0022] Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not imitated to, alkali metal tones such as Na + and K +. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations, such as Ca2 + and Mg2 +. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium fonts (i.e., NH4 +) and substituted ammonium tones (e.g., NH3R +, NH2R2 +, NHR3 +, NR4 +). Examples of some suitable substituted ammonium shades are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such like lysine and arginine. An example of a common quaternary ammonium fon is N (CH3) 4+. Substitutes
[0023] The term "optionally substituted", as used in this document, refers to a main group that can be unsubstituted or that can be substituted.
[0024] Unless otherwise stated, the term "substituted", as used in this document, refers to a major group containing one or more substitutes. The term "substitute" is used in this document in the conventional sense and refers to a chemical moiety that is covalently linked to, or if appropriate, fused to, a major group. A wide variety of substitutes are well known, and the methods for their formation and introduction into a variety of major groups are also well known. Examples of substitutes are described in more detail below.
[0025] C1-12 alkyl: The term "C1-12 alkyl", as used in this document, refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which can be aliphatic or alicyclic, and which can be saturated or unsaturated (for example, partially unsaturated, totally unsaturated). Thus, the term "alkyl" includes the subclasses alkenyl, alkynyl, cycloalkyl, etc., discussed below.
[0026] Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl ( C7).
[0027] Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) ( C5), n-hexyl (C6) and n-heptyl (C7).
[0028] Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl ( C5).
[0029] C2-12 alkenyl: The term "C2-12 alkenyl", as used herein, refers to an alkyl group having one or more carbon-carbon double bonds.
[0030] Examples of unsaturated alkenyl groups include, but are not limited to, ethylene (vinyl, -CH = CH2), 1-propenyl (-CH = CH-CH3), 2-propenyl (ally, -CH-CH = CH2 ), isopropenyl (1-methylvinyl, -C (CH3) = CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).
[0031] C2-12 alkynyl: The term "C2-12 alkynyl", as used herein, refers to an alkyl group having one or more carbon-carbon triple bonds.
[0032] Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C = CH) and 2-propynyl (propargyl, -CH2-C = CH).
[0033] C3-12 cycloalkyl: The term "C3-12 cycloalkyl", as used in this document, refers to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which has 3 to 7 carbon atoms, including 3 to 7 atoms in the ring .
[0034] Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cyclo-eptane (C7) ), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcycliclutene (C6) C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and saturated political hydrocarbon compounds: norcaran (C7), norpinane (C7), norbornane (C7).
[0035] C3-20 heterocyclyl: The term "C3-20 heterocyclyl", as used herein, refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, this moiety which has 3 to 20 atoms in the ring, of which 1 to 10 are ring heteroatoms. Preferably, each ring has 3 to 7 atoms in the ring, of which 1 to 4 are ring heteroatom.
[0036] In this context, the suffixes (for example, C3-20, C3-7, C5-6, etc.) signify the number of atoms in the ring, or the range of number of atoms in the ring, whether they are atoms carbon, whether heteroatom. For example, the term "C5-6 heterocyclyl", as used herein, refers to a heterocyclyl group having 5 or 6 atoms in the ring.
Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from: N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (for example, 3-pyrroline, 2 , 5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7); O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxol (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepine ( C7); S1: thyrane (C3), tiethane (C4), thiolane (tetrahydrothiophene) (C5), thiano (tetrahydrothiopyran) (C6), tiepane (C7); O2: dioxolane (C5), dioxane (C6), and dioxepan (C7); O3: trioxane (C6); N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydroppyrazole) (C5), piperazine (C6); N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6); N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6); N2O1: oxadiazine (C6); O1S1: oxathiol (C5) and oxatian (thioxane) (C6); and, N1O1S1: oxatiazine (C6).
Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in the cyclic form, for example, furanoses (C5), such as arabinofuranose, lixofuranose, ribofuranose, and xylofuranose, and pyraneses (C6), such as allopyranosis, altropiranosis, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
[0039] C5-20 aryl: The term "C5-20 aryl" as used in this document refers to a monovalent moiety obtained by removing a hydrogen atom from an atom of the aromatic ring of an aromatic compound , this portion has 3 to 20 atoms in the ring. Preferably, each ring has 5 to 7 atoms in the ring.
[0040] In this context, the suffixes (for example, C3-20, C5-7, C5-6, etc.) mean the number of atoms in the ring, or the range of number of atoms in the ring, whether they are atoms carbon, whether heteroatom. For example, the term "C5-6 aryl", as used in this document, refers to an aryl group having 5 or 6 atoms in the ring.
[0041] The ring atoms can all be carbon atoms, as in "carboaryl groups".
[0042] Examples of carbohydrate groups include, but are not limited to, those derived from benzene (ie, phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphtacene (C18), and pyrene (C16).
[0043] Examples of aryl groups comprising fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H- indene) (C9), indene (C9), isoindene (C9), tetralin (1,2,3,4-tetrahydronaphthalene (C10), acenaphene (C12), fluorene (C13), phenalene (C13), acephenenthrene (C15) , and aceanthrene (C16).
[0044] Alternatively, ring atoms can include one or more heteroatom, as in "heteroaryl groups". Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from: N1: pyrrole (azole) (C5), pyridine (azine) (C6); O1: furan (oxol) (C5); S1: thiophene (thiol) (C5); N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6); N2O1: oxadiazole (furazan) (C5); N3O1: oxatriazole (C5); N1S1: thiazole (C5), isothiazole (C5); N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) ( for example, cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); N3: triazole (C5), triazine (C6); and, N4: tetrazole (C5).
[0045] Examples of heteroaryl comprising fused rings include, but are not limited to: C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1 ), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxol (O2), benzofurazane (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S); C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxane (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4); C11 (with 2 fused rings) derived from benzodiazepine (N2); C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); e, C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxycine (O1S1), phenazine (N2), phenoxazine (N1O1), thiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
[0046] The groups mentioned above, either alone or as part of another substitute, may themselves be optionally substituted with one or more groups selected from themselves and from the additional substitutes listed below.
[0047] Halo: -F, -Cl, -Br, and -I.
[0048] Hydroxy: -OH.
[0049] Ether: -OR, where R is an ether substitute, for example, a C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyl group), or a C5-20 aryl group (also referred to as a C520 aryloxy group), preferably a C1-7 alkyl group.
[0050] Alkoxy: -OR, where R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O (nPr) (n-propoxy), -O (iPr) (isopropoxy), -O (nBu) (n-butoxy), -O (sBu) (sec-butoxy), -O (iBu) (isobutoxy), and -O (tBu) (tert-butoxy).
[0051] Acetal: -CH (OR1) (OR2), where R1 and R2 are independently acetal substitutes, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5 aryl group -20, preferably a C1-7 alkyl group, or, in the case of a "cyclic" acetal group, R1 and R2, considered together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached. are united, form a heterocyclic ring having 4 to 8 atoms in the ring. Examples of acetal groups include, but are not limited to, -CH (OMe) 2, - CH (OEt) 2, and -CH (OMe) (OEt).
[0052] Hemiacetal: -CH (OH) (OR1), where R1 is a hemiacetal substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group , preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -CH (OH) (OMe) and -CH (OH) (OEt).
[0053] Ketal: -CR (OR1) (OR2), where R1 and R2 are as defined for acetals, and R is a ketal substitute other than hydrogen, for example, a C1-7 alkyl group, a heterocyclyl group of C3-20, or aryl group of C5-20, preferably a C1-7 alkyl group. Examples of ketal groups include, but are not limited to, -C (Me) (OMe) 2, -C (Me) (OEt) 2, -C (Me) (OMe) (OEt), -C (Et) ( OMe) 2, - C (Et) (OEt) 2, and -C (Et) (OMe) (OEt).
[0054] Hemicetal: -CR (OH) (OR1), where R1 is as defined for hemietals, and R is a hemicetal substitute other than hydrogen, for example, a C1-7 alkyl group, a heterocyclyl group of C3-20, or aryl group of C5-20, preferably a C1-7 alkyl group. Examples of hemicetal groups include, but may not be limited to, -C (Me) (OH) (OMe), -C (Et) (OH) (OMe), -C (Me) (OH) (OEt), and -C (Et) (OH) (OEt). Oxo (keto, -one): = O. Tiona (thiocetone): = S.
[0055] Imino (imine): = NR, where R is an imino substitute, for example, hydrogen, C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ester groups include, but are not limited to, = NH, = NMe, = NEt, and = NPh.
[0056] Formyl (carbaldehyde, carboxaldehyde): -C (= O) H.
[0057] Acyl (keto): -C (= O) R, where R is an acyl substitute, for example, a C1-7 alkyl group (also referred to as C1-7 alkylacyl or C1-7 alkanophile ), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, -C (= O) CH3 (acetyl), -C (= O) CH2CH3 (propionyl), -C (= O) C (CH3) 3 (t-butyryl) , and -C (= O) Ph (benzoyl, phenone).
[0058] Carboxy (carboxylic acid): -C (= O) OH.
[0059] Tiocarboxy (thiocarboxylic acid): -C (= S) SH.
[0060] Thiolocarboxy (thiolocarboxylic acid): -C (= O) SH.
[0061] Thionocarboxy (thionocarboxylic acid): -C (= S) OH.
[0062] Imidonic acid: -C (= NH) OH.
[0063] Hydroxamic acid: -C (= NOH) OH.
[0064] Ester (carboxylate, carboxylic acid ester, oxycarbonyl); -C (= O) OR, where R is a ester substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a group C1-7 alkyl. Examples of ester groups include, but are not limited to, -C (= O) OCH3, -C (= O) OCH2CH3, -C (= O) OC (CH3) 3, and -C (= O) OPh.
[0065] Acyloxy (inverted ester): -OC (= O) R, where R is an acyloxy substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a group C5-20 aryl, preferably a C1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC (= O) CH3 (acetoxy), -OC (= O) CH2CH3, -OC (= O) C (CH3) 3, -OC (= O) Ph , and -OC (= O) CH2Ph.
[0066] Oxycarboyloxy: -OC (= O) OR, where R is an ester substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, -OC (= O) OCH3, -OC (= O) OCH2CH3, -OC (= O) OC (CH3) 3, and -OC (= O) OPh.
[0067] Amino: -NR1R2, wherein R1 and R2 are independently substituted for amino, for example hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or C1-7 dialkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a "cyclic" amino group, R1 and R2, considered together with the atom of nitrogen to which they are attached, form a heterocyclic ring having 4 to 8 atoms in the ring. The amino groups can be primary (-NH2), secondary (- NHR1), or tertiary (-NHR1R2), and in cationic form, they can be quaternary (- + NR1R2R3). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, -NHC (CH3) 2, -N (CH3) 2, -N (CH2CH3) 2, and -NHPh. Examples of amino caloric groups include, but are not limited to, aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholino, and thiomorpholino.
[0068] Starch (carbamofla, carbamyl, aminocarbonyl, carboxamide); - C (= O) NR1R2, where R1 and R2 are independently amino substitutes, as defined for amino groups. Examples of starch groups include, but are not limited to, -C (= O) NH2, -C (= O) NHCH3, -C (= O) N (CH3) 2, -C (= O) NHCH2CH3, and -C (= O) N (CH2CH3) 2, as well as starch groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure, as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
[0069] Thioamido (thiocarbamyl): -C (= S) NR1R2, where R1 and R2 are independently amino substitutes, as defined for amino groups. Examples of starch groups include, but are not limited to, -C (= S) NH2, -C (= S) NHCH3, -C (= S) N (CH3) 2, and -C (= S) NHCH2CH3.
[0070] Acylamido (acylamino): -NR1C (= O) R2, where R1 is an amide substitute, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or an aryl group of C5-20, preferably hydrogen or a C1-7 alkyl group, and R2 is an acyl substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5- 20, preferably hydrogen or a C1-7 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC (= O) CH3, -NHC (= O) CH2CH3, and -NHC (= O) Ph. R1 and R2 can together form a cyclic structure, as in, for example, succinimidyl, macheimidyl, and phthalimidyl:

[0071] Aminocarbonyloxy: -OC (= O) NR1R2, where R1 and R2 are independently amino substitutes, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, -OC (= O) NH2, - OC (= O) NHMe, -OC (= O) NMe2, and -OC (= O) NEt2.
[0072] Ure ^ do:: -N (R1) CONR2R3, where R2 and R3 are independently amino substitutes, as defined for amino groups, and R1 is a urea substitute, for example, hydrogen, a group C1-7 alkyl, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of urea groups include, but are not limited to, - NHCONH2, -NHCONHMe, -NHCONHEt, -NHCONMe2, -NHCONEt2, -NMeCONH2, - NMeCONHMe, -NMeCONHEt, -NMeCONMe2, and -NMeCONEt2.
[0073] Guanidino: -NH-C (= NH) NH2.
[0074] Tetrazolyl: an aromatic ring of five elements having four nitrogen atoms and one carbon atom,

[0075] Imino: = NR, where R is an imino substitute, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group. Examples of imino groups include, but are not limited to, = NH, = NMe, e = NEt.
[0076] Amidine (amidino): -C (= NR) NR2, where each R is an amidine substitute, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a group C5-20 aryl, preferably H or a C1-7 alkyl group. Examples of amidine groups include, but are not limited to, -C (= NH) NH2, -C (= NH) NMe2, and -C (= NMe) NMe2.
[0077] Nitro: -NO2.
[0078] Nitrous: -NO.
[0079] Azido: -N3.
[0080] Cyan (nitrile, carbonitrile): -CN.
[0081] Isocian: -NC.
[0082] Cyanate: -OCN.
[0083] Isocyanate: -NCO.
[0084] Thiocyanate (thiocyanate): -SCN.
[0085] Isothiocyanate (isothiocyanate): -NCS.
[0086] Sulfidrila (thiol, mercapto): -SH.
[0087] Thioether (sulfide): -SR, where R is a thioether substitute, for example, a C1-7 alkyl group (also referred to as a C1-7 alkylthio group), a C3-20 heterocyclyl group , or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of C1-7 alkylthio groups include, but are not limited to, -SCH3 and -SCH2CH3.
[0088] Disulfide: -SS-R, where R is a disulfide substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group (also referred to herein as C1-7 alkyl disulfide). Examples of C17 alkyl disulfide groups include, but are not limited to, -SSCH3 and -SSCH2CH3.
[0089] Sulfine (sulfinyl, sulfoxide): -S (= O) R, where R is a sulfin substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or an aryl group of C5-20, preferably a C1-7 alkyl group. Examples of sulfin groups include, but are not limited to, -S (= O) CH3 and -S (= O) CH2CH3.
[0090] Sulfone (sulfonyl): -S (= O) 2R, where R is a sulfone substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or an aryl group of C5-20, preferably a C1-7 alkyl group. including, for example, a fluorinated or perfluorinated C1-7 alkyl group. Examples of sulfone groups include, but are not limited to, -S (= O) 2CH3 (methanesulfonyl, mesyl), -S (= O) 2CF3 (triflyl), -S (= O) 2CH2CH3 (esila), - S (= O) 2C4F9 (nonaflyl), -S (= O) 2CH2CF3 (tresyl), -S (= O) 2CH2CH2NH2 (tauryl), -S (= O) 2Ph (phenylsulfonyl, besila), 4-methylphenylsulfonyl (tosyl ), 4-chlorophenylsulfonyl (closila), 4-bromophenylsulfonyl (brosyl),
[0091] 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (naphthyl), and 5-dimethylamino-naphthalen-1-yl sulfonate (dansyl).
[0092] Sulfinic acid (sulfine): -S (= O) OH, -SO2H.
[0093] Sulphonic acid (sulfo): -S (= O) 2OH, -SO3H.
[0094] Sulfinate (sulfamic acid ester): -S (= O) OR; where R is a sulfinate substitute, for example, a C1-7 alkyl group, a C320 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, -S (= O) OCH3 (methoxysulfinyl; methyl sulfinate) and -S (= O) OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).
[0095] Sulphonate (sulfonic acid ester): -S (= O) 2OR, where R is a sulfonate substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, -S (= O) 2OCH3 (methoxysulfonyl; methyl sulfonate) and -S (= O) 2OCH2CH3 (ethoxysulfonyl; ethyl sulfonate).
[0096] Sulfinyloxy: -OS (= O) R, where R is a sulfinyloxy substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, -OS (= O) CH3 and -OS (= O) CH2CH3.
[0097] Sulfonyloxy: -OS (= O) 2R, where R is a substituted sulfonyloxy, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulphonyloxy groups include, but are not limited to, -OS (= O) 2CH3 (mesylate) and -OS (= O) 2CH2CH3 (esylate).
[0098] Sulfate: -OS (= O) 2OR; where R is a sulfate substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C520 aryl group, preferably a C1-7 alkyl group. Examples of sulfate groups include, but are not limited to, -OS (= O) 2OCH3 and -SO (= O) 2OCH2CH3.
[0099] Sulfamila (sulfamofla; surfonic acid amide; sulfinamide): -S (= O) NR1R2, where R1 and R2 are independently amino substitutes, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, -S (= O) NH2, -S (= O) NH (CH3), -S (= O) N (CH3) 2, -S (= O) NH (CH2CH3), -S (= O) N (CH2CH3) 2, and -S (= O) NHPh.
[00100] Sulfonamido (sulfinamofla; sulfonic acid amide; sulfonamide): -S (= O) 2NR1R2, where R1 and R2 are independently amino substitutes, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, -S (= O) 2NH2, -S (= O) 2NH (CH3), -S (= O) 2N (CH3) 2, -S (= O ) 2NH (CH2CH3), -S (= O) 2N (CH2CH3) 2, and -S (= O) 2NHPh.
[00101] Sulfamino: -NR1S (= O) 2OH, where R1 is an amino substitute, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS (= O) 2OH and -N (CH3) S (= O) 2OH.
[00102] Sulfonamino: -NR1S (= O) 2R, where R1 is an amino substitute, as defined for the amino groups, and R is a sulfonamino substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, -NHS (= O) 2CH3 and -N (CH3) S (= O) 2C6H5.
[00103] Sulfinamino: -NR1S (= O) R, where R1 is an amino substitute, as defined for amino groups, and R is a sulfinamino substitute, for example, a C1-7 alkyl group, a group C3-20 heterocyclyl, or a C520 aryl group, preferably a C1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, -NHS (= O) CH3 and -N (CH3) S (= O) C6H5.
[00104] Phosphine (phosphine): -PR2, where R is a phosphine substitute, for example, -H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5 aryl group -20, preferably -H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphine groups include, but are not limited to, -PH2, -P (CH3) 2, -P (CH2CH3) 2, -P (t-Bu) 2, and -P (Ph) 2.
[00105] Phospho: -P (= O) 2.
[00106] Phosphinyl (phosphine oxide): -P (= O) R2, where R is a phosphinyl substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or an aryl group of C5-20, preferably a C1-7 alkyl group or a C5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, -P (= O) (CH3) 2, -P (= O) (CH2CH3) 2, -P (= O) (t-Bu) 2, and -P (= O) (Ph) 2.
[00107] Phosphonic acid (phosphono): -P (= O) (OH) 2.
[00108] Phosphonate (phosphon ester): -P (= O) (OR) 2, where R is a phosphonate substitute, for example, -H, a C1-7 alkyl group, a C3- heterocyclyl group 20, or a C5-20 aryl group, preferably -H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphonate groups include, but are not limited to, -P (= O) (OCH3) 2, -P (= O) (OCH2CH3) 2, -P (= O) (Ot-Bu) 2, and -P (= O) (OPh) 2.
[00109] Phosphoric acid (phosphonooxy): -OP (= O) (OH) 2.
[00110] Phosphate (phosphonooxy ester): -OP (= O) (OR) 2, where R is a phosphate substitute, for example, -H, a C1-7 alkyl group, a heterocyclyl group of C3-20, or a C5-20 aryl group, preferably -H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphate groups include, but are not limited to, -OP (= O) (OCH3) 2, -OP (= O) (OCH2CH3) 2, -OP (= O) (Ot-Bu) 2, and -OP (= O) (OPh) 2.
[00111] Phosphorous acid: -OP (OH) 2.
[00112] Phosphite: -OP (OR) 2, where R is a phosphite substitute, for example, -H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5- aryl group 20, preferably -H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphite groups include, but are not limited to, -OP (OCH3) 2, -OP (OCH2CH3) 2, -OP (O-t-Bu) 2, and -OP (OPh) 2.
[00113] Phosphoramidite: -OP (OR1) -NR22, where R1 and R2 are substitutes for phosphoramidite, for example, -H, a C1-7 alkyl group (optionally substituted), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably - H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphamidamide groups include, but are not limited to, -OP (OCH2CH3) -N (CH3) 2, -OP (OCH2CH3) -N (i-Pr) 2, and -OP (OCH2CH2CN) -N (i -Pr) 2.
[00114] Phosphoramidate: -OP (= O) (OR1) -NR22, where R1 and R2 are substituted by phosphoramidate, for example, -H, a C1-7 alkyl group (optionally substituted), a heterocyclyl group of C3-20, or a C5-20 aryl group, preferably -H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP (= O) (OCH2CH3) - N (CH3) 2, -OP (= O) (OCH2CH3) -N (i-Pr) 2, and -OP ( = O) (OCH2CH2CN) -N (i-Pr) 2. Alkylene
[00115] C3-12 alkylene: The term "C3-12 alkylene", as used herein, refers to a bidentate moiety obtained by removing two hydrogen atoms, both from the same carbon atom, or one from each of two different carbon atoms, a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which can be aliphatic or alicyclic, and which can be saturated, partially unsaturated, or totally unsaturated. Thus, the term "alkylene" includes the subclasses alkenylene, alkylene, cycloalkylene, etc., discussed below.
[00116] Examples of linear saturated C3-12 alkylene groups include, but are not limited to, - (CH2) n-, where ne is an integer from 3 to 12, for example -CH2CH2CH2- (propylene), - CH2CH2CH2CH2- (butylene), -CH2CH2CH2CH2CH2- (pentylene) and -CH2CH2CH2CH2CH2CH2CH2- (heptylene).
[00117] Examples of branched saturated C3-12 alkylene groups include, but are not limited to, -CH (CH3) CH2-, -CH (CH3) CH2CH2-, -CH (CH3) CH2CH2CH2-, -CH2CH ( CH3) CH2-, -CH2CH (CH3) CH2CH2-, -CH (CH2CH3) -, -CH (CH2CH3) CH2-, and -CH2CH (CH2CH3) CH2-.
[00118] Examples of partially unsaturated linear C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, -CH = CH-CH2-, -CH2-CH = CH2-, -CH = CH-CH2-CH2-, -CH = CH-CH2-CH2-CH2-, -CH = CH-CH = CH-, -CH = CH-CH = CH-CH2-, -CH = CH-CH = CH-CH2-CH2-, -CH = CH-CH2-CH = CH-, -CH = CH-CH2-CH2-CH = CH-, and -CH2-C = C-CH2-.
[00119] Examples of branched partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and alkylene groups) include, but are not limited to, -C (CH3) = CH-, -C (CH3) = CH-CH2-, -CH = CH-CH (CH3) - and -CEC-CH (CH3) -.
Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (for example, cyclopent-1,3-ylene), and cyclohexylene (for example, example, cyclohex-1,4-ylene).
[00121] Examples of partially unsaturated alicyclic C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (eg, 4-cyclopenten-1,3-ylene), cyclohexenylene (eg example, 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cycloexadien-1,4-ylene).
[00122] Oxygen protecting group: the term "oxygen protecting group" refers to a portion that masks a hydroxy group, and these are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T.W. and Wuts, G.M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. Classes of particular interest include silicyl ethers (for example, TMS, TBDMS), substituted methyl ethers (for example, THP) and esters (for example, acetate).
[00123] Nitrogen protecting group of carbamate: the term "Nitrogen protecting group of carbamate" refers to a portion that masks nitrogen in the imine bond, and these are well known in the art. These groups have the following structure:
where R'10 and R as defined above. A large number of suitable groups are described on pages 503 to 549 by Greene, TW and Wuts, GM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated in this document. by reference.
[00124] Hemiaminal nitrogen protecting group: the term "hemiaminal nitrogen protecting group" refers to a group having the following structure:
where R'10 and R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups by Greene, TW and Wuts, GM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated in this document by reference. Conjugated
[00125] The present invention provides Conjugates comprising a PBD dimer connected to a Ligand unit through a Ligand unit. In one embodiment, the Binder unit includes an Elongation unit (A), a Specificity unit (L1), and a Spacer unit (L2). The Ligand unit is connected at one end to the Ligand unit and at the other end to the PBD dimer compound.
[00126] In one aspect, such Conjugate is shown below in the formula Illa: L- (A1a-L1s-L2y-D) p (IIIa) where: L is the Ligand unit; and -A1a-L1s-L2y- and the Binder Unit (LU), where: -A1- is a Lengthening unit, ae 1 or 2, L1 - and a Specificity unit, if an integer ranging from 1 at 12, - L2- and a Spacer unit, y 0, 1 or 2; - D is a PBD dimer; and p e from 1 to 20.
[00127] In another aspect, such Conjugate is shown below in the formula lllb: L1s | L - (A1a- L2y -D) p (lllb) Also illustrated as: L - (A1a- L2y (- L1s) -D) p (lb) where: L is the Ligand unit; e - A1a-L1s (L2y) - and the Binder unit (LU), in which: - A1- and a Stretching unit connected to a Stretching unit (L2), ae 1 or 2, L1 - and a Specificity linked to an Elongation unit (L2), if an integer ranging from 0 to 12, -L2- and a Spacer unit, y 0, 1 or 2; -D and a PBD dimer; and p e from 1 to 20. Preferences
[00128] The following preferences can apply to all aspects of the invention as described above, or can relate to a single aspect. Preferences can be combined together or in any combination.
[00129] In one embodiment, the Conjugate has the formula: L- (A1a-L1s-L2y-D) p where L, A1, a, L1, s, L2, D and p are as described above.
[00130] In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Pharmaco unit (D), CBA and the Cell Linking Agent, L1 and a Specificity unit, A1 and a Stretching unit connecting L1 to the Cellular Linking Agent, L2 and a Spreading unit , which is a covalent bond, a self-destructive group or together with -OC (= O) - forms a self-destructive group, and optional L2.
[00131] In another embodiment, the Ligand unit (L) is a Cellular Bonding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below: CBA - A1a- L1s - L2y - * where the asterisk indicates the point of attachment to the Pharmaco unit (D), CBA and the Cell Binding Agent, L1 and a Specificity unit, A1 and a Stretching unit connecting L1 to the Cell Linking Agent, L2 is a Paying Unit which is a covalent bond or a self-destructive group, eae 1 or 2, if 0, 1 or 2, eye 0 or 1 or 2.
[00132] In the modalities illustrated above, L1 can be a unit of Cleavable Specificity, and can be referred to as a “trigger” that, when cleaved, activates a self-destructive group (or self-destructive groups) L2, when a self-destructive group (s) is present. When the L1 Specificity unit is cleaved, or the bond (i.e., the covalent bond) between L1 and L2 is cleaved, the self-destructive group releases the Pharmaco unit (D).
[00133] In another embodiment, the Ligand unit (L) is a Cellular Linking Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula and illustrated below: L1s | CBA - A1a- L2y - * where the asterisk indicates the connection point to Farmaco (D), CBA and the Cellular Linking Agent, L1 and a Specificity unit connected to L2, A1 and a Stretching unit connecting L2 to the Agent Ligagao Celular, L2 is a self-destructive group, eae 1 or 2, if 1 or 2, eye 1 or 2.
[00134] In the various modalities discussed here, the nature of L1 and L2 can vary widely. These groups are chosen based on their characteristics, which can be dictated, in part, by the conditions at the location to which the Conjugate is delivered. Where the L1 Specificity unit is cleavable, the L1 structure and / or sequence is selected in such a way that it is cleaved by the action of enzymes present at the target site (for example, the target cell). L1 units that are cleavable by changes in pH (for example, base or unstable acid), temperature or after irradiation (for example, photolable) can also be used. L1 units that are cleavable under conditions of reduction or oxidation may also find use in Conjugates.
[00135] In some embodiments, L1 may comprise an amino acid or a sequence of amino acids. The amino acid sequence can be the target substrate for an enzyme.
[00136] In one embodiment, L1 is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L1 can be cleaved by a liposomal protease, such as a cathepsin.
[00137] In one embodiment, L2 is present and together with -C (= O) O- forms a self-destructive group or self-destructive groups. In some modalities, -C (= O) O- is also a self-destructive group,
[00138] In one embodiment, where L1 is cleavable by the action of an enzyme and L2 is present, the enzyme cleaves the link between L1 and L2, whereby the self-destructive groups release the unit of Farmaco.
[00139] L1 and L2, where present, can be connected by a link selected from: -C (= O) NH-, -C (= O) O-, -NHC (= O) -, - OC (= O) -, -OC (= O) O-, -NHC (= O) O-, -OC (= O) NH-, -NHC (= O) NH, and -O- (a glycoside link ^ hint).
An L1 amino group that connects to L2 can be the N-terminus of an amino acid or can be derived from an amino group of an amino acid side chain, for example, a lysine amino acid side chain.
A carboxyl group of L1 that connects to L2 can be the C-terminal of an amino acid or it can be derived from a carboxyl group of an amino acid side chain, for example, a side chain of glutamic acid amino acids .
[00142] A hydroxy group of L1 that connects to L2 can be derived from a hydroxy group of an amino acid side chain, for example, a serine amino acid side chain.
[00143] In one mode, -C (= O) O- and L2 together form the group:
where the asterisk indicates the point of attachment to the Pharmaco unit, the wavy line indicates the point of attachment to L1, Y and -N (H) -, -O-, -C (= O) N (H) - or - C (= O) O-, ene 0 to 3. The phenylene ring is optionally substituted with one, two or three substitutes as described in this document.
[00144] In one mode, Y and NH.
[00145] In one embodiment, n and 0 or 1. Preferably, n and 0.
[00146] Where Y and NH and n and 0, the self-destructive group is a p-aminobenzylcarbonyl ligand (PABC).
[00147] The self-destructive group will allow the release of the Pharmaco unit (ie, the asymmetric PBD) when a remote site in the ligand is activated, proceeding along the lines shown below (for n = 0):
where the asterisk indicates the connection to Farmaco, L * and the activated form of the remaining portion of the ligand and the released Pharmaco unit is not shown. These groups have the advantage of separating the Farmaco activation site.
[00148] In another modality, -C (= O) O- and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above. Each phenylene ring is optionally replaced with one, two or three substitutes as described in this document. In one embodiment, the phenylene ring having the substitute for Y is optionally substituted and the phenylene ring not having the substitute for Y is unsubstituted.
[00149] In another modality, -C (= O) O- and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above, E and O, S or NR, D and N, CH, or CR, and F and N, CH, or CR.
[00150] In one mode, D and N.
[00151] In one mode, D and CH.
[00152] In one mode, E and O or S.
[00153] In one mode, F and CH.
[00154] In a preferred embodiment, the covalent bond between L1 and L2 is an unstable cathepsin bond (for example, cleavable).
[00155] In one embodiment, L1 comprises a dipepUdeo. The amino acids in the dipeptide can be any combination of natural and unnatural amino acids. In some embodiments, dipepUdeo comprises natural amino acids. Where the binder is a
[00156] unstable cathepsin binder, dipepUdeide and the site of action for cathepsin-mediated cleavage. Dipepide, then, is a recognition site for cathepsin.
[00157] In one embodiment, the group -X1-X2- in dipepUdeo, -NH-X1-X2-CO-, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys- , -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-; where Cit and citrulline. In such a dipipe, -NH- is the amino group of Xi, and CO and the carbonyl group of X2.
[00158] Preferably, the group -X1-X2- in dipeptide, -NH-X1-X2-CO-, and selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, - Ala-Lys-, and -Val-Cit-.
[00159] More preferably, the group -X1-X2- in dipepUdeo, -NH-X1-X2-CO-, and -Phe-Lys-, Val-Cit or -Val-Ala-.
[00160] Other combinations of dipepUdeo of interest include: -Gly-Gly-, -Pro-Pro-, and -Val-Glu-.
[00161] Other combinations of dipepUdeo can be used, including those described by Dubowchik et al., Which are incorporated herein by reference.
[00162] In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group can be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipepUdeid sequence comprising a Lys residue protected by a Boc side chain and cleavable by cathepsin.
[00163] Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem Catalog. Additional protective group strategies are defined in Protective groups in Organic Synthesis, Greene and Wuts.
[00164] Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality: Arg: Z, Mtr, Tos; Asn: Trt, Xan; Asp: Bzl, t-Bu; Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt; Glu: Bzl, t-Bu; Gln: Trt, Xan; He: Boc, Dnp, Tos, Trt; Lys: Boc, Z-Cl, Fmoc, Z; Ser: Bzl, TBDMS, TBDPS; Thr: Bz; Trp: Boc; Tyr: Bzl, Z, Z-Br.
[00165] In one mode, -X2- and indirectly connected to the Farmaco unit. In such modality, the L2 Spagulator unit is present.
[00166] In one embodiment, dipepUdeo is used in combination with a self-destructive group (s) (the Spattering unit). The self-destructive group (s) can be connected to -X2-.
[00167] Where a self-destructive group is present, -X2- is directly connected to the self-destructive group. In one mode, -X2- is connected to group Y of the self-destructive group. Preferably the group -X2-CO- is connected to Y, where Y is NH. -X1- and connected directly to A1. In one mode, -X1- is connected directly to A1. Preferably, the NH-X1- group (the amino terminus of X1) is connected to A1. A1 can comprise the -CO- functionality to thereby form an amide bond with -X1-.
[00168] In one mode, L1 and L2 together with -OC (= O) - comprise the group -X1-X2-PABC-. The PABC group is directly connected to the Farmaco unit. In one example, the self-destructive group and the dipepUdeo together form the -Phe-Lys-PABC- group, which is illustrated below:
where the asterisk indicates the point of attachment to the Pharmaco unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
[00169] Alternatively, the self-destructive group and the dipepUdeo together form the group -Val-Ala-PABC-, which is illustrated below:
where the asterisk and the wavy line are as defined above.
[00170] In another modality, L1 and L2 together with -OC (= O) - represent:
where the asterisk indicates the point of attachment to the Pharmaco unit, the wavy line indicates the point of attachment to A1, Y and a covalent bond or a functional group, and E is a group that is susceptible to cleavage to thereby activate an auto group -destructive.
[00171] And it is selected such that the group is susceptible to cleavage, for example, by light or by the action of an enzyme. And it can be -NO2 or glucuronic acid (e.g., p-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a 0-glucuronidase. The group Y can be a covalent bond. The Y group can be a functional group selected from: -C (= O) - -NH- -O- -C (= O) NH-, -C (= O) O-, -NHC (= O) -, -OC (= O) -, -OC (= O) O-, -NHC (= O) O-, -OC (= O) NH-, -NHC (= O) NH-, -NHC (= O) NH, -C (= O) NHC (= O) -, SO2, and -S-.
[00172] The group Y is preferably -NH-, -CH2_, -O-, and -S-.
[00173] In some modalities, L1 and L2 together with -OC (= O) - represent:
where the asterisk indicates the point of attachment to the Pharmaco unit, the wavy line indicates the point of attachment to A, Y and a covalent bond or a functional group and E is glucuronic acid (for example, 0-glucuronic acid). Y is preferably a functional group selected from -NH-.
[00174] In some modalities, L1 and L2 together represent:
where the asterisk indicates the point of attachment to the remainder of L2 or the Pharmaco unit, the wavy line indicates the point of attachment to A1, Y and a covalent bond or a functional group and E is glucuronic acid (for example, 0-acid gluturonic). Y is preferably a functional group selected from -NH-, - CH2_, -O-, and -S-.
[00175] In some additional embodiments, Y and a functional group as defined above, the functional group is linked to the amino acid, and the amino acid is linked to the A1 Stretching Unit.
[00176] In some modalities, amino acid and 0-alanine. In such a modality, the amino acid is equivalently considered part of the Stretching unit.
[00177] The L1 Specificity unit and the Ligand unit are indirectly connected via the Stretching unit.
[00178] L1 and A1 can be connected by a connection selected from: -C (= O) NH-, -C (= O) O-, -NHC (= O) -, -OC (= O) - , -OC (= O) O-, -NHC (= O) O-, -OC (= O) NH-, and -NHC (= O) NH-.
[00179] In one modality, group A1 and:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n and 0 to 6. In one embodiment, n and 5.
[00180] In one modality, group A1 and:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n and 0 to 6. In one embodiment, n and 5.
[00181] In one modality, group A1 and:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 a 8, preferably 4 to 8, more preferably 4 or 8.
[00182] In one modality, group A1 and:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 a 8, preferably 4 to 8, more preferably 4 or 8.
[00183] In one modality, group A1 and:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n and 0 to 6. In one embodiment, n and 5.
[00184] In one modality, group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n and 0 to 6. In one embodiment, n and 5.
[00185] In one mode, group A1 and:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 a 8, preferably 4 to 8, more preferably 4 or 8.
[00186] In one modality, group A1 and:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 a 8, preferably 4 to 8, more preferably 4 or 8.
[00187] In one embodiment, the connection between the Ligand unit and A1 is through a thiol residue from the Ligand unit and a maleimide group of A1.
[00188] In one embodiment, the connection between the Ligand unit and A1 is:
where the asterisk indicates the point of attachment to the remaining portion of A1, L1, L2 or D, and the wavy line indicates the point of attachment to the remaining portion of the Ligand unit. In this embodiment, atom S is typically derived from the Ligand unit.
[00189] In each of the above modalities, an alternative functionality can be used in the seat of the group derived from maleimide shown below:
where the wavy line indicates the point of attachment to the Ligand unit as before, and the asterisk indicates the link to the remaining portion of group A1, or to L1, L2 or D.
[00190] In one embodiment, the group derived from maleimide and replaced with the group:
where the wavy line indicates the point of attachment to the Ligand unit, and the asterisk indicates the link to the remaining portion of group A1, or to L1, L2 or D.
[00191] In one embodiment, the group derived from maleimide is replaced with a group, which optionally together with the Ligand unit (for example, a Cell Binding Agent), is selected from: -C (= O) NH -, -C (= O) O-, -NHC (= O) -, -OC (= O) -, -OC (= O) O-, -NHC (= O) O-, -OC (= O ) NH-, -NHC (= O) NH-, -NHC (= O) NH, -C (= O) NHC (= O) -, -S-, -SS-, - CH2C (= O) - - C (= O) CH2-, = N-NH-, and - NH-N =.
[00192] In one embodiment, the group derived from maleimide and replaced with a group, which optionally together with the Ligand unit, is selected from:
where the wavy line indicates the point of attachment to the Ligand unit or the link to the remaining portion of the A1 group, and the asterisk indicates the other from the point of attachment to the Ligand unit or the link to the remaining portion of the A1 group.
[00193] Other groups suitable for connecting L1 to the Cell Binding Agent are described in WO 2005/082023.
[00194] In one embodiment, the Stretching unit A1 is present, the Specificity unit L1 is present and Spacer unit L2 is absent. In this way, L1 and the Pharmaco unit are directly connected via a link. Equally in this modality, L2 is a bond. - 1 and D can be connected by a link selected from: -C (= O) NH-, -C (= O) O-, -NHC (= O) -, -OC (= O) -, - OC (= O) O-, -NHC (= O) O-, -OC (= O) NH-, and -NHC (= O) NH-.
[00195] In one embodiment, L1 and D are preferably connected by a link selected from: -C (= O) NH-, and -NHC (= O) -.
[00196] In one embodiment, L1 comprises a dipipe and an end of the dipeptide and linked to D. As described above, the amino acids in the dipeptide can be any combination of natural and unnatural amino acids. In some embodiments, dipepUdeo comprises natural amino acids. Where the linker is an unstable cathepsin linker, dipepUdeide is the site of action for cathepsin-mediated cleavage. Dipepide, then, is a recognition site for cathepsin.
[00197] In one embodiment, the group -X1-X2- in dipepUdeo, -NH-X1-X2- CO-, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys- , -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-; where Cit and citrulline. In such a dipipe, -NH- is the amino group of X1, and CO and the carbonyl group of X2.
[00198] Preferably, the group -X1-X2- in dipeptide, -NH-X1-X2-CO-, and selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, - Ala-Lys-, and -Val-Cit-.
[00199] More preferably, the group -X1-X2- in dipepUdeo, -NH-X1-X2-CO-, and -Phe-Lys- or -Val-Ala-.
[00200] Other combinations of dipepUdeo of interest include: -Gly-Gly-, -Pro-Pro-, and -Val-Glu-.
[00201] Other combinations of dipepUdeo can be used, including those described above.
[00202] In one embodiment, L1-D is: - -NH-X1-X2-CO-NH- * where -NH-X1-X2-CO and dipepUdeo, -NH- and part of the Pharmaco unit, the asterisk indicates the point of attachment to the remainder of the Pharmaco unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
[00203] In one embodiment, dipepUdeo and valine-alanine and L1-D and:
where the asterisk, -NH- and the wavy line are as defined above.
[00204] In one embodiment, dipepUdeo and phenylalanine-lysine and L1-D and:
where the asterisk, -NH- and the wavy line are as defined above.
[00205] In one embodiment, dipepUdeo and valine-citrulline.
[00206] In one embodiment, groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00207] In one embodiment, groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00208] In one embodiment, groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, and 0 to 30. In a preferred embodiment, ne 1 and 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00209] In one embodiment, groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, and 0 to 30. In a preferred embodiment, ne 1 and 0 to 10, 1 to 7, preferably 3 to 7, more preferably 3 or 7.
[00210] In one embodiment, groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00211] In one embodiment, groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00212] In one embodiment, groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00213] In one mode, groups A1-L1 and:
where the asterisk indicates the point of attachment to L2 orD, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, and 0 to 30. In a preferred embodiment, ne 1 em and 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00214] In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S and a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the rest of the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00215] In one embodiment, the group L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S and a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00216] In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S and a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, ne 0 or 1, and 0 to 30. In a preferred embodiment , ne 1 eme 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00217] In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 to 7, preferably 4 to 8, more preferably 4 or 8.
[00218] In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00219] In one mode, the groups L-A1 -L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and 0 to 6. In one embodiment, ne 5.
[00220] In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, ne 0 or 1, em 0 to 30. In a preferred embodiment, ne 1 em and 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00221] In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, ne 0 or 1, em 0 to 30. In a preferred embodiment, ne 1 em and 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00222] In one embodiment, the Stretching unit is an acetamide unit, having the formula: / -CH2-CO-N- * where the asterisk indicates the point of attachment to the remainder of the Stretching unit, L1 or D, and the wavy line indicates the connection point to the connecting unit.
[00223] In other modalities, Ligand-Pharmaco Compounds are supplied for conjugation to a Ligand unit. In one embodiment, the Ligand-Pharmaco compounds are designed for connection to a Cellular Binding Agent.
[00224] In one embodiment, the Pharmaco-Ligand compound has the formula:
where the asterisk indicates the point of attachment to the Pharmaco unit, G1 and a Stretching group (A1) to form a connection to a Ligand unit, L1 and a Specificity unit, L2 (the Spacer unit) and a covalent bond or together with -OC (= O) - forms a self-destructive group (s). In another mode, the Pharmaco-Ligand compound has the formula: G1-L1-L2- * where the asterisk indicates the point of attachment to the Farmaco unit, G1 and a Stretching unit (A1) to form a connection to a Ligand unit, L1 and a Specificity unit, L2 (the Spacer unit) and a covalent bond or a self-destructive group (s). L1 and L2 are as defined above. References to the connection to A1 can be constructed here as referring to a connection to G1.
[00225] In one embodiment, where L1 comprises an amino acid, the side chain of that amino acid can be protected. Any suitable protecting group can be used. In one embodiment, the side chain protecting groups are removable with other protecting groups in the compound, where present. In other embodiments, the protecting groups may be orthogonal to other protecting groups in the molecule, where present.
[00226] Suitable protecting groups for amino acid side chains include those groups described in the Novabiochem Catalog 2006/2007. Protective groups for use in an unstable cathepsin binder are also discussed in Du-bowchik et al.
[00227] In certain embodiments of the invention, the L1 group includes an amino acid residue Lys. The side chain of this amino acid can be protected with a Boc or Alloc protective group. A Boc protecting group is more preferred.
[00228] Functional group G1 forms a connection group when reacting with the Ligand unit (for example, a Cell Binding Agent.
[00229] In one embodiment, the functional group G1 and or comprises an amino, carboxylic acid, hydroxy, thiol, or maleimide group for reaction with an appropriate group on the Ligand unit. In a preferred embodiment, G1 comprises a maleimide group.
[00230] In one embodiment, the G1 group and an alkyl maleimide group. This group is suitable for reaction with thiol groups, particularly cysteine thiol groups, present in the cell binding agent, for example present in an antibody.
[00231] In one modality, the G1 group and:
where the asterisk indicates the point of attachment to L1, L2 or D, and n and 0 to 6. In one embodiment, ne 5.
[00232] In one modality, the G1 group and:
where the asterisk indicates the point of attachment to L1, L2 or D, and n and 0 to 6. In one embodiment, ne 5.
[00233] In one modality, the G1 group and:
where the asterisk indicates the point of attachment to L1, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 to 2, preferably 4 to 8, and more preferably 4 or 8.
[00234] In one modality, the G1 group and:
where the asterisk indicates the point of attachment to L1, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 to 8, preferably 4 to 8, and more preferably 4 or 8.
[00235] In one modality, the G1 group and:
where the asterisk indicates the point of attachment to L1, L2 or D, and n and 0 to 6. In one embodiment, ne 5.
[00236] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n and 0 to 6. In one embodiment, ne 5.
[00237] In one modality, the G1 group and:
where the asterisk indicates the point of attachment to L1, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 to 2, preferably 4 to 8, and more preferably 4 or 8.
[00238] In one modality, the G1 group and:
where the asterisk indicates the point of attachment to L1, ne 0 or 1, eme 0 to 30. In a preferred embodiment, ne 1 eme 0 to 10, 1 to 8, preferably 4 to 8, and more preferably 4 or 8.
[00239] In each of the above modalities, an alternative functionality can be used in place of the maleimide group shown below:
where the asterisk indicates the link to the remaining portion of group G.
[00240] In one embodiment, the group derived from maleimide and replaced with the group:
where the asterisk indicates the link to the remaining portion of group G.
[00241] In one embodiment, the maleimide group is replaced with a group selected from: -C (= O) OH, -OH, -NH2, -SH, -C (= O) CH2X, where X and Cl, Br or I, -CHO, -NHNH2 -C = CH, and -N3 (azide).
[00242] In one embodiment, L1 is present, and G1 and -NH2, NH2N-NHMe, - COOH, -OH or -SH.
[00243] In one embodiment, where L1 is present, G1 and -NH2 or NH2N-NHMe. Either group can be the N-terminus of an L1 amino acid sequence.
[00244] In one embodiment, L1 is present and G1 and -NH2, and L1 is an amino acid sequence -X1-X2-, as defined above.
[00245] In one embodiment, L1 is present and G1 and COOH. This group can be the C-terminus of an L1 amino acid sequence.
[00246] In one embodiment, L1 is present and G1 and OH.
[00247] In one mode, L1 is present and G1 and SH.
[00248] The G1 group can be convertible from one functional group to another. In one embodiment, L1 is present and G1 and -NH2. This group is convertible to another G1 group comprising a maleimide group. For example, the -NH2 group can be reacted with an acid or an activated acid (for example, forms of N-succinimide) of these G1 groups comprising maleimide shown above.
[00249] The G1 group can therefore be converted to a functional group which is more suitable for reaction with the Ligand unit.
[00250] As noted above, in one embodiment, L1 is present and G1 is - NH2, NH2N-NHMe, -COOH, -OH or -SH. In an additional embodiment, these groups are supplied in a chemically protected form. The chemically protected form is, therefore, a precursor to the Ligand which is provided with a functional group.
[00251] In one embodiment, G1 and -NH2 in a chemically protected form. The group can be protected with a carbamate protecting group. The carbamate protecting group can be selected from the group consisting of:
[00252] Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.
[00253] Preferably, where G1 and -NH2, this is protected with an Alloc or Fmoc group.
[00254] In a modality, where G1 and -NH2, this is protected with a Fmoc group.
[00255] In one embodiment, the protective group is the same as the carbamate protecting group of the capping group.
[00256] In one embodiment, the protecting group is not the same as the carbamate protecting group of the capping group. In this modality, it is preferable that the protective group is removable under conditions that do not remove the carbamate protective group from the capping group.
[00257] The chemical protecting group can be removed to provide a functional group to form a connection to a Ligand unit. Optionally, this functional group can then be converted to another functional group as described above.
[00258] In one embodiment, the active group is an amine. This amine is preferably the N-terminal amine of a peptide, and it can be the N-terminal amine of the preferred dipopeUides of the invention.
[00259] The active group can be reacted to provide the functional group which is intended to form a connection to a Ligand unit.
[00260] In other modalities, the linker unit and a precursor to the linker unit having an active group. In this embodiment, the Ligand unit comprises the active group, which is protected by means of a protecting group. The protective group can be removed to provide the Binder unit having an active group.
[00261] Where the active group is an amine, the protecting group can be an amine protecting group, such as those described in Green and Wuts.
[00262] The protecting group is preferably orthogonal to other protecting groups, where present, in a ligand unit.
[00263] In one modality, the protective group is orthogonal to the capping group. In this way, the protective group of the active and removable group retaining the capping group. In other embodiments, the protective group and the capping group are removable under the same conditions as those used to remove the capping group.
[00264] In one embodiment, the Binder unit is:
where the asterisk indicates the connection point to the Pharmaco unit, and the wavy line indicates the connection point to the remaining portion of the Binding unit, as applicable or the connection point to G1. Preferably, the wavy line indicates the point of attachment to G1.
[00265] In one embodiment, the Binder unit and:
where the asterisk and the wavy line are as defined above.
[00266] Other functional groups suitable for use in forming a connection between L1 and the Cell Linking Agent are described in WO 2005/082023. Linking Unit
The ligand unit can be of any type, and includes a protein, polypeptide, peptide and a non-peptide agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These ligand units may include antibodies or an antibody fragment that contains at least one target molecule binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that may specifically bind to a target.
[00268] The terms "specifically bind" and "spherical binding" refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (for example, an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1x107 M-1, and binds to the predetermined molecule with an affinity that is at least twice as high as the binding affinity to a non-molecule. -specific (for example, BSA, casein) different from the predetermined molecule or a closely related molecule.
[00269] Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
[00270] In some embodiments, the Ligand unit is a Cellular Bonding Agent that binds to an extracellular target in a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptide agent. In some embodiments, the Cell Binding Agent can be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent can also be an antibody or antigen binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).
[00271] In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; fully human antibody; or a single chain antibody. One modality is the antibody and a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab ', F (ab') 2 and Fv fragments.
[00272] The antibody can be a diabody, a domain antibody (DAB) or a single chain antibody.
[00273] In one embodiment, the antibody is a monoclonal antibody.
[00274] Antibodies for use in the present invention include those antibodies described in WO 2005/082023, which is incorporated herein. Particularly preferred are those antibodies to tumor-associated amphigens. Examples of these amphigens known in the art include, but are not limited to, those tumor associated amphigens defined in WO 2005/082023. See, for example, pages 41-55.
[00275] In some modalities, Conjugates are designed to target tumor cells through their cell surface amphigens. Amphigens can be cell surface amphigens that are overexpressed or expressed in cell types or abnormal times. Preferably, the target amphigene is expressed only in proliferative cells (preferably tumor cells); however, this is rarely seen in practice. As a result, target amphigens are generally selected on the basis of differential expression between healthy and proliferative tissue.
[00276] Antibodies have been raised to target amphigens related to spermatoid tumors including:
[00277] Crypto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2 / Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate sperm membrane amphene), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2.
[00278] The Ligand unit and connected to the Ligand unit. In one embodiment, the Ligand unit is connected to A, where present, of the Ligand unit.
[00279] In one embodiment, the connection between the Ligand unit and the Ligand unit is through the thioether connection.
[00280] In one embodiment, the connection between the Ligand unit and the Ligand unit is through the disulfide bond.
[00281] In one embodiment, the connection between the Ligand unit and the Ligand unit is through an amide bond.
[00282] In one embodiment, the connection between the Ligand unit and the Ligand unit is via an ester link.
[00283] In one embodiment, the connection between the Ligand unit and the ligand is formed between a thiol group of a cysteine residue from the Ligand unit and a maleimide group of the Ligand unit.
[00284] The ligand unit system residues may be available to react with the functional unit of the Ligand unit to form a connection. In other embodiments, for example, where the ligand unit is an antibody, the thiol groups of the antibody can participate in the intermediate disulfide bonds. These interchain bonds can be converted to thiol free groups by, for example, treating the antibody with DTT prior to reaction with the functional group of the Ligand unit.
[00285] In some embodiments, the cysteine residue is one introduced into the light or heavy chain of an antibody. Positions for cysteine insertion by substitution in antibody light or heavy chains include those described in United States Application Published No. 2007-0092940 and International Patent Publication WO2008070593, which are incorporated into this document. Treatment Methods
[00286] The compounds of the present invention can be used in a method of therapy. A method of treatment is also provided, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of formula I. The term "therapeutically effective amount" and an amount sufficient to show benefit to a patient. patient. Such a benefit can be at least the improvement of at least one symptom. The actual amount administered, and the rate and course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, for example, decisions about dosage, is within the responsibility of general practitioners and other doctors.
[00287] A compound can be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (administration of active agents, including, for example, drugs; surgery; and radiation therapy.
[00288] Pharmaceutical compositions in accordance with the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, ie, a compound of formula I, an excipient, vehicle, stopper, tabilizer or other materials well known to those skilled in the art, pharmaceutically acceptable. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the vehicle or other material will depend on the route of administration, which can be oral, or by injection, for example, cutaneous, subcutaneous, or intravenous.
[00289] Pharmaceutical compositions for oral administration can be in the form of tablets, capsules, powders or liquids. A tablet can comprise a solid vehicle or an adjuvant. The liquid pharmaceutical compositions generally comprise a liquid vehicle, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol, can be included. A capsule can comprise a solid vehicle, such as gelatin.
[00290] For intravenous, cutaneous or subcutaneous injection, or injection at the site of the treatment, the active ingredient will be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has adequate pH, isotonicity and stability . Those of relevant skill in the art are quite capable of preparing suitable solutions using, for example, isotonic vehicles, such as the Sodium Chloride Injection, the Ringer Injection, the Lactated Ringer Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as required.
[00291] Compounds and Conjugates can be used to treat proliferative disease and autoimmune disease. The term "proliferative disease" means an unwanted or uncontrolled cell proliferation of excessive or abnormal cells that are undesired, such as hyperplasic or neoplasic growth, either in vitro or in vivo.
[00292] Examples of proliferative conditions include, but are not limited to, benign, premalignant and malignant cell proliferation, including, but not limited to, neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer , bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (for example, connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, hematological; malignant, such as leukemias and lymphomas, such as non-Hodgkin's lymphoma and subtypes, such as DLBCL, marginal zone, mantle and follicular zone, Hodgkin's lymphoma, AML and other cancers of B or T cell origin.
[00293] Examples of autoimmune diseases include the following: rheumatoid arthritis, autoimmune demyelinating diseases (for example, multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveorretinitis, systemic lupus erythematosus, severe mupuncture, mousse de graphemes, msten , glomerulonephritis, autoimmune liver disease, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalitis, polymyositis, dermatomyoma multiple endocrine insufficiency, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, cutaneous lupus erythematosus , Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hem anemia olfactory, pemphigus vulgaris, pemphigus, herpetiform dermatitis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia), male autoimmune infertility and male autoimmune infertility ankylosing, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture shdrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phosphoid smokers of farmer, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, chronic autoimmune active hepatitis, bird breeding lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrous alveolitis, pulmonary interstitial disease, nodular erythema , gangrenous pyoderma, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arthritis , schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behget's disease, Caplan's syndrome, Kawasaki disease, dengue, encephalomyelitis, endocarditis, endocarditis, encephalocarditis elevated diutine, psoriasis, fetal erythroblastosis, eosinoffic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterocratic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purple, graft versus host disease transplantation, cardiomyopathy, Eaton-Lambert's syndrome, recurrent polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal insufficiency.
[00294] In some embodiments, autoimmune disease and a disorder of B lymphocytes (eg, systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis and type I diabetes), Th1 lymphocytes (eg, rheumatoid arthritis, multiple sclerosis , psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2 lymphocytes (for example, atopic dermatitis, systemic lupus erythematosus, atopic erythematosus, atopic rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1 lymphocytes or Th2 lymphocytes. In some embodiments, the autoimmune disorder is an immune disorder mediated by T cells.
[00295] In some embodiments, the amount of Conjugate administered ranges from about 0.01 to about 10 mg / kg per dose. In some embodiments, the amount of Conjugate administered ranges from about 0.01 to about 5 mg / kg per dose. In some embodiments, the amount of Conjugate administered ranges from about 0.05 to about 5 mg / kg per dose. In some embodiments, the amount of Conjugate administered varies from about 0.1 to about 5 mg / kg per dose. In some embodiments, the amount of Conjugate administered ranges from about 0.1 to about 4 mg / kg per dose. In some embodiments, the amount of Conjugate administered ranges from about 0.05 to about 3 mg / kg per dose. In some embodiments, the amount of Conjugate administered ranges from about 0.1 to about 3 mg / kg per dose. In some modalities, the amount of Conjugate administered varies from about 0.1 to about 2 mg / kg per dose. Includes Other Forms Unless otherwise stated, the well-known ionic, salt, solvate, and protected forms of these substitutes are included in the above. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO-), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N + HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as the conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-O-), a salt or solvate thereof, as well as conventional protected forms. Salts
[00296] It may be convenient or desirable to prepare, purify, and / or handle a corresponding salt of the active compound, for example, a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berg, and col., J. Pharm. Sci., 66, 1-19 (1977).
[00297] For example, if the compound is anionic, or has a functional group that can be anionic (for example, -COOH can be -COO-), then a salt with a suitable cation can be formed. Examples of suitable inorganic cations include, but are not limited to, alkali metal tones, such as Na + and K +, alkaline earth cations, such as Ca2 + and Mg2 +, and other cations, such as Al + 3. Examples of suitable organic cations include, but are not limited to, ammonium fonts (i.e., NH4 +) and substituted ammonium tones (e.g., NH3R +, NH2R2 +, NHR3 +, NR4 +). Examples of some suitable substituted ammonium shades are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium fon is N (CH3) 4+.
[00298] If the compound is cationic, or has a functional group that can be cationic (for example, -NH2 can be -NH3 +), then a salt with a suitable anion can be formed. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, iodic acid, sulfuric, sulfurous, metric, nitrous, phosphoric, and phosphorous.
[00299] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphor sulfonic, cinnamic, hydrochloric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glycptonic, glyconic, glutamic, glycolic, hydroximalic, hydroxynaphthalene, carboxylic, isethionic, lactatic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, prophenyl, phenyl , pyruvic, salicylic, stearic, succinic, sulfanphilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Solvates
[00300] It may be convenient or desirable to prepare, purify, and / or handle a corresponding solvate of the active compound. The term "solvate" is used in this document in the conventional sense to refer to a solute complex (for example, active compound, active compound salt) and solvent. If the solvent is water, the solvate can be conveniently referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, etc. Carbinolamines
[00301] The invention includes compounds where a solvent is joined via the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (RAOH, where RA is C1-4 alkyl):

[00302] These forms can be called the carbinolamine and ether carbinolamine forms of PBD. The balance of these equiKbrios depends on the conditions in which the compounds are found, as well as on the nature of the portion properly said.
These particular compounds can be isolated in solid form, for example, by lyophilization. Isomers
[00304] Certain compounds may exist in one or more geometrical, optical, enantiomeric, diastereoisomeric, epimeric, atrophic, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis and trans forms; E and Z forms; forms c, t and r; endo and exo forms; R, S, and meso forms; forms D and L; d and l forms; (+) and (-) forms; keto, enol, and enolate forms; syn and anti forms; syncline and anticline forms; forms a and P; axial and equatorial forms; boat, chair, spiral, envelope, and half chair shapes; and their combinations, hereinafter collectively referred to as "isomers" (or "isomeric forms").
[00305] Note that, except as discussed below for tahomeric forms, are specifically excluded from the term "isomers", as used in this document, structural (or constitutional) isomers (ie, isomers that differ in the bonds between atoms, instead of merely the position of the atoms in space). For example, a reference to a methoxy group, -OCH3, is not to be interpreted as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be interpreted as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include the structurally isomeric forms that fall into this class (eg, C1-7 alkyl includes n-propyl and isopropyl; butyl includes n-, a iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
[00306] The exclusion mentioned above does not concern tautomeric forms, for example, keto, enol, and enolate forms, as, for example, in the following tautomeric pairs: keto / enol (illustrated below), imine / enamine, amide / imino alcohol, amidine / amidine, nitrous / oxime, thiocetone / enethiol, N-nitrous / hydroxy, and nitro / acit nitro.

[00307] Note that compounds with one or more isotope substitutions are specifically included in the term "isomer". For example, H can be in any isotopic form, including 1H, 2H (D), and 3H (T); C can be in any isotopic form, including 12C, 13C, and 14C; O can be in any isotopic form, including 16O and 18O; It's similar.
[00308] Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including racemics (in whole or in part) and other mixtures thereof. Methods for the preparation (for example, asymmetric synthesis) and separation (for example, fractional crystallization and chromatographic media) of such isomeric forms are known in the art or are readily obtained by adapting the methods taught in this document, or known methods, in a known manner. General synthetic routes
[00309] The synthesis of PBD compounds is discussed extensively in the references below, which are incorporated into this document by reference: a) WO 00/12508 (pages 14 to 30); b) WO 2005/023814 (pages 3 to 10); c) WO 2004/043963 (pages 28 to 29); and d) WO 2005/085251 (pages 30 to 39). Synthesis route
[00310] The compounds of the present invention, where R10 and R11 form a double nitrogen-carbon bond between the nitrogen and carbon atoms to which they are attached, can be synthesized from a compound of Formula 2:
where R2, R6, R7, R9, R6 ', R7', R9 ', R12, X, X' and R ”are as defined for compounds of formula I, ProtN and a nitrogen protecting group for synthesis and ProtO and a protected oxygen group for the synthesis or an oxo group, by deprotecting the imine bond by standard methods.
[00311] The compound produced can be in its form of carbinolamine or ether of carbinolamine, depending on the solvents used. For example, if ProtN is Alloc and ProtO is an oxygen protecting group for synthesis, then deprotection is conducted using the palladium to remove the N10 protecting group, followed by the elimination of the oxygen protecting group for the synthesis. If ProtN is Troc and ProtO is an oxygen protecting group for synthesis, then deprotection is performed using a pair of Cd / Pd to produce the compound of formula (I). If ProtN is SEM, or an analogous group, and ProtO is an oxo group, then the oxo group can be removed by reduction, which results in a protected carbinolamine intermediate, which can then be treated to remove the protective group of SEM, followed by the elimination of water. The reduction of the compound of Formula 2 can be carried out, for example, through Ktio tetraborohydride, while a suitable means for removing the SEM protecting group and treatment with sHica gel.
[00312] The compounds of formula 2 can be synthesized from a compound of formula 3a:
where R2, R6, R7, R9, R6 ', R7', R9 ', X, X' and R ”are as defined for compounds of formula 2, by coupling an organometallic derivative comprising R12, such as an organoboro derivative. The organoboro derivative can be a boronate or boronic acid.
[00313] The compounds of formula 2 can be synthesized from a compound of formula 3b:
where R12, R6, R7, R9, R6 ', R7', R9 ', X, X' and R ”are as defined for the compounds of formula 2, by coupling an organometallic derivative comprising R2, such as a derivative of organoboro. The organoboro derivative can be a boronate or boronic acid.
[00314] The compounds of formulas 3a and 3b can be synthesized from a compound of formula 4:
where R2, R6, R7, R9, R6 ', R7', R9 ', X, X' and R ”are as defined for the compounds of formula 2, by coupling approximately a single equivalent (for example, 0.9 or 1 to 1.1 or 1.2) of an organometallic derivative, such as an oroganoboro derivative, comprising R2 or R12.
[00315] The couplings described above are normally performed in the presence of a palladium catalyst, for example, Pd (PPh3) 4, Pd (OCOCH3) 2, PdCl2, Pd2 (dba) 3. The coupling can be carried out under standard conditions, or it can also be carried out under microwave conditions.
[00316] The two coupling steps are normally performed sequentially. They can be performed with or without purification between the two steps. If no purification is carried out, then both steps can be carried out in the same reaction vessel. Purification is normally required after the second coupling step. Purification of the compound from unwanted by-products can be performed by column chromatography or ion exchange separation.
[00317] The synthesis of the compounds of formula 4, where ProtO is an oxo group and ProtN and SEM, is described in detail in WO 00/12508, which is incorporated in this document by reference. In particular, reference is made to scheme 7 on page 24, where the aforementioned compound is designated as intermediate P. This synthesis method is also described in WO 2004/043963.
[00318] The synthesis of the compounds of formula 4, where ProtO is a protected oxygen group for the synthesis, is described in WO 2005/085251, which synthesis is incorporated in this document by reference.
[00319] The compounds of formula I, where R10 and R10 'are H and R11 and R11' are SOzM, can be synthesized from the compounds of formula I, where R10 and R11 form a nitrogen-carbon double bond between the atoms of nitrogen and carbon to which they are attached, by the addition of the bisulfite salt or the appropriate sulfinate salt, followed by an appropriate purification step. Additional methods are described in GB 2 053 894, which is incorporated herein by reference. Nitrogen protecting groups for shtese
[00320] Nitrogen protecting groups for synthesis are well known in the art. In the present invention, the protecting groups of particular interest are the carbamate nitrogen protecting groups and the hemiaminal nitrogen protecting groups.
[00321] The nitrogen protecting groups of carbamate have the following structure:
where R'10 and R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, TW and Wuts, GM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated in this document. by reference.
[00322] Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.
[00323] The other possible groups are nitrobenzyloxycarbonyl (for example, 4-nitrobenzyloxycarbonyl) and 2- (phenylsulfonyl) ethoxycarbonyl.
[00324] Protective groups that can be removed with the palladium catalyst are not preferred, for example, Alloc.
[00325] Hemiaminal nitrogen protecting groups have the following structure:
where R'10 and R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups by Greene, TW and Wuts, GM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated in this document by reference. The groups disclosed in this document can be applied to the compounds of the present invention. Such groups include, but are not limited to SEM, MOM, MTM, MEM, BOM, nitro or methoxy-substituted BOM, CI3CCH2OCH2-. Protected oxygen group for synthesis
[00326] The protected oxygen group for synthesis is well known in the art. A large number of suitable oxygen protecting groups are described on pages 23 to 200 of Greene, T.W. and Wuts, G.M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated by reference in this document.
[00327] Classes of particular interest include syphilic ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates.
Preferred oxygen protecting groups include acetates, TBS and THP. Pharmaco Conjugate Synthesis
[00329] Conjugates can be prepared as previously described. Binders having a maleimidyl group (A), a peptide group (L1) and a self-destructive group (L2) can be prepared as described in United States Patent No. 66,214,345. Linkers having a maleimidyl group (A) and a peptide group (L1) can be prepared as described in WO 2009-0117531. Other binders can be prepared according to the references cited here or as known to those skilled in the art.
[00330] Binder-Pharmaco compounds can be prepared according to methods known in the art. Linking of X substitutes to the amide base (from the PDB dimer Pharmaco unit) to activate groups from the Liganentes unit can be performed according to the methods generally described in United States Patent No. 6,214,345 and 7,498,298; and WO 2009-0117531, or as otherwise known to those skilled in the art.
[00331] Antibodies can be conjugated to Pharmaceutical Linker Compounds as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg / ml) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris (carboxyethyl) phosphine hydrochloride (TCEP) at 37 ° C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5'-dithiobis (2-nitrobenzoic acid) and allowed to continue until the desired level of thiols / mAb is reached. The antibody is reduced and then cooled to 0 ° C and changed with 1.5 equivalent of maleimide binder-drug per thiol antibody. After 1 hour, the reaction is quenched by the addition of 5 equivalents of N-acetyl cysteine. Drug binder is quenched and removed by gel filtration on a PD-10 column. The ADC is then sterile filtered through a 0.22 µm syringe filter. The protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with a correlation for the contribution of drug absorbance at 280 nm. Chromatography with size exclusion can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the sharply cooled drug-ligand levels - NAC. Additional Preferences
[00332] The following preferences can apply to all aspects of the invention as described above, or can relate to a single aspect. Preferences can be combined together in any combination.
[00333] In some embodiments, R6 ', R7', R9 ', R10', R11 'and Y' are preferably the same as R6, R7, R9, R10, R11 and Y, respectively. Dimer connection
[00334] Y and Y 'are preferably O.
[00335] R "is preferably a C3-7 alkylene group with no substitute. More preferably, R" is a C3, C5 or C7 alkylene. Most preferably R ”is a C3 or C5 alkylene.
[00336] R6 to R9
[00337] R9 and preferably H.
[00338] R6 is preferably selected from H, OH, OR, SH, NH2, nitro and halo, and is most preferably H or halo, and most preferably is H.
[00339] R7 is preferably selected from H, OH, OR, SH, SR, NH2, NHR, NRR ', and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from from optionally substituted C1-7 alkyl groups, C3-10 heterocyclyl and C5-10 aryl groups. R may more preferably be a C 1-4 alkyl group, which may or may not be substituted. A substitute of interest and a C5-6 aryl group (eg, phenyl). Particularly preferred substitutes at positions 7 are OMe and OCH2Ph. Other substitutes of particular interest are dimethylamino (i.e. -NMe2); - (OC2H4) qOMe, where q is 0 to 2; nitrogen-containing C6 heterocyclyls, including morpholino, piperidinyl and N-methylpiperazinyl.
[00340] These preferences apply to R9 ', R6' and R7 ', respectively. R2
[00341] A in R2 can be a phenyl group or a C5-7 heteroaryl group, for example, furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl.
[00342] X and a group selected from the list comprising: OH, SH, CO2H, COH, N = C = O, NHNH2, CONHNH2
and NHRN, where RN is selected from the group comprising H and C1-4 alkyl. X can preferably be: OH, SH, CO2H, -N = C = O or NHRN, and can more preferably be: OH, SH, CO2H, -N = C = O or NH2. Particularly preferred groups include OH, SH and NH2, with NH2 being the most preferred group.
[00343] Q2-X can be on any of the ring atoms available from the C5-7 aryl group, but preferably on an atom of the ring that is not adjacent to the bond to the rest of the compound, ie, it is preferably p or ya binding to the rest of the compound. Therefore, where C5-7 aryl group (A) for phenyl, the substitute (Q2-X) is preferably in the meta or para positions, and most preferably it is in the para position.
[00344] In some modalities, Q1 is a simple link. In these modes, Q2 is selected from a simple link and -Z- (CH2) n-, where Z is selected from a simple link, O, S and NH is 1 to 3. In some of these modalities , Q2 is a simple link. In other modalities, Q2 and -Z- (CH2) n-. In these embodiments, Z may be O or S and n may not be 1 or n may be 2. In other embodiments, Z may be a single bond and may not be 1.
[00345] In other modalities, Q1 and -CH = CH-.
[00346] In some embodiments, R2 can be -A-CH2-X and -A-X. In these modalities, X can be OH, SH, CO2H, COH and NH2. In particularly preferred embodiments, X can be NH2. R12
[00347] R12 is selected from: (a) aliphatic saturated C1-5 alkyl; (b) saturated C3-6 cycloalkyl; (ç)
, where each of R21, R22 and R23 is independently taught from H, saturated C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the group R12 is not more than 5; (d)
, where one of R25a and R25b is H and the other is selected from: phenyl, whose phenyl is optionally substituted by a group selected from methyl halo, methoxy; pyridyl; and thiophenyl; and is)
where R24 is selected from: H; saturated C1-3 alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, whose phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
[00348] When R12 and C1-5 saturated aliphatic alkyl, this can be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it can be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these modalities, this can be methyl. In other embodiments, it can be butyl or pentyl, which can be straight or branched.
[00349] When R12 and saturated C3-6 cycloalkyl, this can be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, this may be cyclopropyl.
[00350] When R12 and
each of R21, R22 and R23 and independently selected from H, saturated C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the group R12 is not more than 5. In some embodiments, the total number of carbon atoms in group R12 is not more than 4 or not more than 3.
[00351] In some modalities, one from R21, R22 and R23 and H, with the other two groups being selected from H, saturated C1-3 alkyl, C2-3 alkenyl, C2- alkynyl 3 and cyclopropyl.
[00352] In other modalities, two of R21, R22 and R23 are H, with the other group being selected from H, saturated C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
[00353] In some modalities, groups that are not H are selected from methyl and ethyl. In some of these modalities, groups that are not H are methyl.
[00354] In some modalities, R21 and H.
[00355] In some modalities, R22 and H.
[00356] In some modalities, R23 and H.
[00357] In some modalities, R21 and R22 are H.
[00358] In some modalities, R21 and R23 are H.
[00359] In some modalities, R22 and R23 are H.
[00360] When R12 and '
, one from R25a and R25b and H and the other is selected from: phenyl, whose phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group that is not H is phenyl optionally substituted. If the optional substitute for phenyl and halo, it is preferably fluorine. In some embodiments, the phenyl group is unsubstituted.
[00361] When R12 and
R24 and selected from: H; saturated C1-3 alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, whose phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the optional substitute for phenyl and halo, it is preferably fluorine. In some embodiments, the phenyl group is unsubstituted.
[00362] In some modalities, R24 is selected from H, methyl, ethyl, ethylene and ethynyl. In some of these modalities, R24 is selected from H and methyl. M and z
[00363] It is preferred that M and M 'are pharmaceutically acceptable monovalent cations, and are more preferably Na +
[00364] z and preferably 3.
[00365] Particularly preferred compounds of the present invention are of the formula la:
where R12a is selected from (a)
(B)
(ç)
(d)
(and)
the amino group is in the meta or para positions of the phenyl group. 3rd aspect
[00366] The preferences expressed above for the first aspect may apply to compounds of this aspect, where appropriate.
[00367] When R10 is a nitrogen protecting group of carbamate, it can preferably be Teoc, Fmoc and Troc, and can more preferably be Troc.
[00368] When R11 is O-ProtO, where ProtO is an oxygen protecting group, ProtO can preferably be TBS or THP, and can more preferably be TBS.
[00369] When R10 for a hemiaminal nitrogen protecting group, it can preferably be MOM, BOM or SEM, and can more preferably be SEM.
[00370] Preferences for compounds of formula I apply as appropriate for D in the sixth aspect of this invention. Examples General Experimental Methods
[00371] Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and the concentrations (c) are given in g / 100 ml. Melting points were measured using a digital melting point device (Electrothermal). The IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. The 1H and 13C NMR spectra were acquired at 300 K using a Bruker Advance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are described in relation to TMS (6 = 0.0 ppm), and signals are specified as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets ), ddd (double voice doubles) or m (multiplete), with the coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using an Aguas Micromass ZQ instrument coupled to an Aguas HPLC 2695 with an Aguas PDA 2996. Aguas Micromass ZQ parameters used were: Capillary Tube (kV) 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C), 100; Desolvation temperature (° C), 200; Cone flow (L / h), 50: Desolvation flow (L / h), 250. High resolution mass spectroscopy (HRMS) data were recorded on a Micromass QTQF Global from Aguas in positive W mode, using metal-coated borosilicate tips to introduce samples into the instrument. Thin Layer Chromatography (TLC) was performed on aluminum silica gel plates (Merck 60, F254), and flash chromatography used silica gel (Merck 60, 230-400 mesh ASTM). Except for HOBt (NovaBiochem) and solid supported reagents (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied, without further purification. The anhydrous solvents were prepared by distillation under a dry nitrogen atmosphere, in the presence of an appropriate drying agent, and were stored on 4A molecular sieves or sodium filament. The ether of oil refers to the fraction that boils at 40-60 ° C.
[00372] Compound 1b was synthesized as described in WO 00/012508 (compound 210), which is incorporated herein by reference.
[00373] General LC / MS conditions: HPLC (Alliance 2695 from Aguas) was run using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid) . Gradient: initial composition 5% B for 1.0 min, then 5% B to 95% B within 3 min. The composition was maintained for 0.5 min at 95% B, and then returned to 5% B in 0.3 minutes. The running time of the total gradient is equal to 5 min. Flow rate 3.0 mL / min, 400 LIL were divided by means of a zero dead volume T handle that passes the mass spectrometer. Detection range of the wavelength: 220 to 400 nm. Type of function: diode arrangement (535 scans). Column: Phenomenex® Onyx Monolithic C18 50 x 4.60 mm
[00374] Specific LC / MS conditions for compounds protected by both a Troc group and a TBDMs: The chromatographic separation of the compounds protected by Troc and TBDMS was performed on an HPLC Alliance 2695 system from Aguas using an Onyx inverted phase column Monolitic (3 um, 50 x 4.6 mm) from Phenomenex Corp. Mobile phase A consisted of 5% acetonitrile - 95% water containing 0.1% formic acid, and mobile phase B consisted of 95% acetonitrile - 5% water containing 0.1% formic acid. After 1 min at 5% B, the proportion of B was increased to 95% B for the next 2.5 min and maintained at 95% B for 1 min more, before returning to 95% B at 10 rebalance for about 50 more seconds, giving a total running time of 5.0 min. The flow rate was maintained at 3.0 ml / min.
[00375] LC / MS conditions, for example 4: HPLC (Waters Alliance 2695) was performed using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B for 2.0 min, increasing to 50% B for 3 min. The composition was maintained for 1 min at 50% B, before raising to 95% B for 1 minute. The gradient composition then dropped to 5% B for 2.5 minutes and was maintained at this percentage for 0.5 minutes. The running time of the total gradient is equal to 10 min. Flow rate 1.5 mL / min, 400pL was divided through a T handle with a dead volume of zero that passes through the mass spectrometer. Wavelength detection range: 220 to 400 nm. Type of function: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50 x 4.60 mm Synthesis of key intermediates
(a) 1, r - [[(Propane-1,3-diyl) di6xi] bis [(5-met6xi-2-nitro-1,4-phenylene) carbonyl]] bis [(2S, 4R) -methyl- 4-hydroxypyrrolidine-2-carboxylate] (2a)
[00376] Method A: A catalytic amount of DMF (2 drops) was added to a stirred solution of nitro-acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride (0.95 mL, 1.36 g, 10.7 mmoles) in dry THF (20 ml). The reaction mixture was allowed to stir for 16 hours at room temperature, and the solvent was removed by evaporation in vacuo. The resulting residue was redissolved in dry THF (20 ml) and the acid chloride solution was added dropwise to a stirred mixture of (2S, 4R) -methyl-4-hydroxypyrrolidine-2- hydrochloride carboxylate (859 mg, 4.73 mmol) and TEA (6.6 mL, 4.79 g, 47.3 mmol) in THF (10 mL), at -30 ° C (dry ice / ethylene glycol), under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for an additional 3 hours, after which time TLC (CHCl3 / MeOH 95: 5 v / v) and LC / MS (2.45 min (ES +) m / z (relative intensity) 721 ([M + H] + -, 20)) revealed product formation. The excess THF was removed by rotary evaporation and the resulting residue was dissolved in DCM (50 ml). The organic layer was washed with 1N HCl (2 x 15 ml), saturated NaHCO3 (2 x 15 ml), H2O (20 ml), brine (30 ml) and dried (MgSO4). Filtration and evaporation of the solvent gave the crude product as a dark colored oil. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96: 4 v / v CHCl3 / MeOH) isolated the pure amide 2a as an orange glass (840 mg, 54%).
[00377] Method B: Oxalyl chloride (9.75 ml, 14.2 g, 111 mmoles) was added to a stirred suspension of nitro-acid 1a (17.3 g, 37.1 mmoles) and DMF (2 ml) in anhydrous DCM (200 ml). After the initial effervescence, the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. The conversion to the acid chloride was confirmed by treating a sample of the reaction mixture with MeOH and the resulting bis-methyl ester was observed by LC / MS. Most of the solvent was removed by evaporation in vacuo, the resulting concentrated solution was dissolved again in a minimum amount of dry DCM and ground with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40 ° C. Solid acid chloride was added, in portions, over a period of 25 minutes, to a stirred suspension of (2S, 4R) -methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (15.2 g, 84.0 mmoles ) and TEA (25.7 ml, 18.7 g, 185 mmoles) in DCM (150 ml), at -40 ° C (dry ice / CH3CN). Immediately, the reaction was complete, as assessed by LC / MS (2.47 min (ES +) m / z (relative intensity) 721 ([M + H] + -, 100)). The mixture was diluted with DCM (150 ml) and washed with 1N HCl (300 ml), saturated NaHCO3 (300 ml), brine (300 ml), filtered (through a phase separator) and the solvent evaporated in vacuo to give pure 2a product as an orange solid (21.8 g, 82%).
[00378] Analytical data: [a] 22D = -46.1 ° (c = 0.47, CHCl3); 1H NMR (400 MHz, CDCI3) (rotamers) 5 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H, J = 11.1 Hz), 2,482.30 (m, 4H), 2.29-2.04 (m, 4H); 13C NMR (100 MHz, CDCl3) (rotamers) 5 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7, 108.2, 69.7, 65, 1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IV (ATR, CHCl 3) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211, 1177, 1072, 1050, 1008, 871 cm-1; MS (ES +) m / z (relative intensity) 721 ([M + H] + - 47), 388 (80); HRMS [M + H] + - C31H36N4O16 theoretical m / z 721.2199, found (ES +) m / z 721.2227.
[00379] (a) 1, r - [[(Pentane-1,5-diyl) di6xi] bis [(5-met6xi-2-nitro-1,4-phenylene) carbonyl]] bis [(2S, 4R) -methyl-4-hydroxypyrrolidine-2-carboxylate] (2b)
[00380] Preparation from 1b according to Method B provided the product pure as an orange foam (75.5 g, 82%).
[00381] Analytical Data: (ES +) m / z (relative intensity) 749 ([M + H] + -, 100). (b) 1,1 '- [[(Propane-1,3-diyl) di6xi] bis (11aS, 2R) -2- (hydr6xi) -7-met6xi- 1,2,3,10,11,11a- hexahydro-5H-pyrrolo [2,1-c] [1,4] -benzodiazepin-5,11-dione] (3a)
[00382] Method A: A suspension of 10% Pd / C (7.5 g, 10% w / w) in DMF (40 ml) was added to a solution of nitroester 2a (75 g, 104 mmoles) in DMF (360 ml). The suspension was hydrogenated in a Parr hydrogenation apparatus for 8 hours. The progress of the reaction was monitored by LC / MS (2.12 min (ES +) m / z (relative intensity) 597 ([M + H] +., 100), (ES-) m / z (relative intensity) 595 ([M + H] + -, 100) after the hydrogen absorption has stopped The solid Pd / C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 1 kPa (10 mbar)), 40 ° C, to provide a dark oil containing traces of DMF and residual charcoal The residue was digested in EtOH (500 mL) at 40 ° C in a water bath (rotary evaporator bath) and the resulting suspension was filtered through celite and washed with ethanol (500 mL) to give a clear filtrate.Hydrazine hydrate (10 mL, 321 mmol) was added to the solution and the reaction mixture was heated to reflux. formation of a white precipitate and the reflux was allowed to continue for another 30 minutes.The mixture was allowed to cool to room temperature and the precipitate was recovered by filtration, washed with diethyl ether (2 * 1 volume of precipitate) and dried in a vacuum desiccant to provide 3a (50 g, 81%).
[00383] Method B: A solution of nitro-ester 2a (6.80 g, 9.44 mmoles) in MeOH (300 mL) was added to the Raney® mousse (4 tips of the large spatula of a ~ 50% slurry) in H2O) and anti-impact granules, in a 3-neck round-bottom flask. The mixture was heated to reflux and then treated, drop by drop, with a solution of hydrazine hydrate (5.88 ml, 6.05 g, 188 mmoles) in MeOH (50 ml), at which point it was observed a vigorous effervescence. When the addition was complete (~ 30 minutes), additional Raney® melon was added carefully until effervescence ceased and the initial yellow color of the reaction mixture was discharged. The mixture was heated to reflux for about 30 minutes more, at which point the reaction was considered complete by TLC (CHCl3 / MeOH at 90:10 v / v) and LC / MS (2.12 min (ES +) m / z ( relative intensity) 597 ([M + H] +., 100)). The reaction mixture was allowed to cool to approximately 40 ° C and then the excess nickel was removed by filtration through a sintering funnel, without suctioning the vacuum. The filtrate was reduced in volume by evaporation in vacuo, at which point a colorless precipitate formed, which was collected by filtration and dried in a vacuum desiccant to provide 3a (5.40 g, 96%).
[00384] Analytical data: [a] 27D = + 404 ° (c = 0.10, DMF); 1H NMR (400 MHz, DMSO-d6) 5 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H, J = 3.98 Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H , J = 12.1, 3.60 Hz), 3.43 (dd, 2H, J = 12.0, 4.72 Hz), 2.67-2.57 (m, 2H), 2.26 ( p, 2H, J = 5.90 Hz), 1.99-1.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6) 5 169.1, 164.0, 149.9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8, 54.4 , 53.1, 33.5, 27.5; IV (pure ATR) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm-1; MS (ES +) m / z (relative intensity) 619 ([M + Na] +., 10), 597 ([M + H] +., 52), 445 (12), 326 (11); HRMS [M + H] +. Theoretical C29H32N4O10 m / z 597.2191, found (ES +) m / z 597.2205.
[00385] (b) 1, 1 '- [[(Pentane-1,5-diyl) dioxy] bis (11 aS, 2R) -2- (hydr6xi) -7-met6xi- 1,2,3,10, 11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] -benzodiazepin-5,11-dione] (3b)
[00386] Preparation from 2b according to Method A provided the product as a white solid (22.1 g, 86%).
[00387] Analytical data: MS (ES-) m / z (relative intensity) 623.3 ([M - H] -., 100);
[00388] (c) 1,1 '- [[(Propane-1,3-diyl) di6xi] bis (11aS, 2R) -2- (tert-butyldimethylsilyl6xi) -7-met6xi-1,2,3,10 , 11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] - benzodiazepin-5,11-dione] (4a)
[00389] TBSCl (317 mg, 2.1 mmoles) and imidazole (342 mg, 5.03 mmoles) were added to a cloudy solution of tetralactam 3a (250 mg, 0.42 mmol) in anhydrous DMF (6 mL ). The mixture was allowed to stir under a nitrogen atmosphere for 3 hours, after which time the reaction was considered complete, as assessed by LC / MS (3.90 min (ES +) m / z (relative intensity) 825 ([M + H ] +., 100)). The reaction mixture was poured onto ice (~ 25 ml) and allowed to warm to room temperature, with stirring. The resulting white precipitate was collected by vacuum filtration, washed with H2O, diethyl ether and dried in a vacuum desiccant to provide 4th purple (252 mg, 73%).
[00390] Analytical data: [a] 23D = + 234 ° (c = 0.41, CHCl3); 1H NMR (400 MHz, CDCl3) 5 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H, J = 5, 38 Hz), 4.21 - 4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H), 2.85-2.79 (m, 2H ), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H), 0.08 (s, 12H); 13C NMR (100 MHz, CDCl3) 5 170.4, 165.7, 151.4, 146.6, 129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56 , 3, 55.7, 54.2, 35.2, 28.7, 25.7, 18.0, -4.82 and -4.86; IV (ATR, CHCl 3) 3235, 2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099, 1033 cm-1; MS (ES +) m / z (relative intensity) 825 ([M + H] +., 62), 721 (14), 440 (38); HRMS [M + H] +. Theoretical C41H60N4O10Si2 m / z 825.3921, found (ES +) m / z 825.3948.
[00391] (c) 1, 1 '- [[(Pentane-1,5-diyl) dioxy] bis (11 aS, 2R) -2- (tert-butyldimethylsilyloxy) -7-methoxy-1,2,3, 10,11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] - benzodiazepin-5,11-dione] (4b)
[00392] Preparation from 3b according to the method described above provided the product as a white solid (27.3 g, 93%).
[00393] Analytical data: MS (ES +) m / z (relative intensity) 853.8 ([M + H] +., 100), (ES-) m / z (relative intensity) 851.6 ([M - H] -., 100.
[00394] (d) 1,1 '- [[(Propane-1,3-diyl) dioxy] bis (11aS, 2R) -2- (tert-butyldimethylsilyloxy) -7-methoxy-10 - ((2- ( trimethylsilyl) ethoxy) methyl) -1,2,3,10,11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] -benzodiazepin-5,11-dione] (5a)
[00395] A solution of n-BuLi (4.17 ml of a 1.6 M solution in hexane, 6.67 mmoles) in anhydrous THF (10 ml) was added dropwise to a stirred suspension. of tetralactam 4a (2.20 g, 2.67 mmoles) in anhydrous THF (30 mL), at -30 ° C (dry ice / ethylene glycol), under a nitrogen atmosphere. The reaction mixture was allowed to stir at this temperature for 1 hour (now a reddish orange color), at which point a solution of SEMCl (1.18 mL, 1.11 g, 6.67 mmoles) in anhydrous THF (10 ml) was added dropwise. The reaction mixture was allowed to warm slowly to room temperature and was stirred for 16 hours under a nitrogen atmosphere. The reaction was considered complete, as assessed by TLC (EtO-Ac) and LC / MS (4.77 min (ES +) m / z (relative intensity) 1085 ([M + H] +., 100)). The THF was removed by evaporation in vacuo and the resulting residue dissolved in EtOAc (60 ml), washed with H2O (20 ml), brine (20 ml), dried (MgSO4), filtered and evaporated in vacuo to provide the product. gross. Purification by flash chromatography (80:20 v / v Hexane / EtOAc) gave SEM-protected tetralactam in N10 5a as an oil (2.37 g, 82%).
[00396] Analytical data: [a] 23D = + 163 ° (c = 0.41, CHCl3); 1H NMR (400 MHz, CDCl3) 5 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J = 9.98 Hz), 4.68 (d, 2H , J = 9.99 Hz), 4.57 (p, 2H, J = 5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s, 6H), 3.79 -3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J = 5.81 Hz), 2.03-1.90 (m, 2H ), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); 13C NMR (100 MHz, CDCl3) 5 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67 , 1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, -1.24, -4.73; IV (ATR, CHCl 3) 2951, 1685, 1640, 1606, 1517, 1462, 1433, 1360, 1247, 1127, 1065 cm -1; MS (ES +) m / z (relative intensity) 1113 ([M + Na] +., 48), 1085 ([M + H] +., 100), 1009 (5), 813 (6); HRMS [M + H] +. Theoretical C53H88N4O12Si4 m / z 1085.5548, found (ES +) m / z 1085.5542.
[00397] (d) 1,1 '- [[(Pentane-1,5-diyl) dioxy] bis (11aS, 2R) -2- (tert-butyldimethylsilyloxy) -7-methoxy-10 - ((2- ( trimethylsilyl) ethoxy) methyl) -1,2,3,10,11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] -benzodiazepin-5,11-dione] (5b)
[00398] Preparation from 4b according to the method described above provided the product as a light orange foam (46.9 g, 100%), used without further purification.
[00399] Analytical data: MS (ES +) m / z (relative intensity) 1114 ([M + H] +., 90), (ES-) m / z (relative intensity) 1158 ([M + 2Na] -. , 100).
[00400] (e) 1,1 '- [[(Propane-1,3-diyl) dioxy] bis (11aS, 2R) -2-hydroxy-7-methoxy-10- ((2- (trimethylsilyl) ethoxy) methyl) -1,2,3,10,11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] - benzodiazepin-5,11-dione] (6a)
[00401] A solution of TBAF (5.24 ml of a 1.0 M solution in THF, 5.24 mmoles) was added to a stirred solution of bisphilic ether 5a (2.58 g, 2.38 mmoles) ) in THF (40 mL) at room temperature. After stirring for 3.5 hours, analysis of the reaction mixture by TLC (CHCl3 / MeOH 95: 5 v / v) revealed the completion of the reaction. The reaction mixture was poured into a saturated NH 4 Cl solution (100 ml) and extracted with EtOAc (3 x 30 ml). The combined organic layers were washed with brine (60 ml), dried (MgSO 4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96: 4 v / v CHCl3 / MeOH) gave pure tetralactam 6a as a white foam (1.78 g, 87%).
[00402] Analytical data: [a] 23D = + 202 ° (c = 0.34, CHCl3); 1H NMR (400 MHz, CDCl3) 5 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J = 10.0 Hz), 4.72 (d, 2H , J = 10.0 Hz), 4.61 - 4.58 (m, 2H), 4.25 (t, 4H, J = 5.83 Hz), 4.20 - 4.16 (m, 2H) , 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH), 2.96-2.90 (m, 2H ), 2.38 (p, 2H, J = 5.77 Hz), 2.11-2.05 (m, 2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) 5 169.5, 165.9, 151.3, 147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67 , 2, 65.2, 56.5, 56.2, 54.1, 35.2, 29.1, 18.4, -1.23; IV (ATR, CHCl 3) 2956, 1684, 1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm -1; MS (ES +) m / z (relative intensity) 885 ([M + 29] +., 70), 857 ([M + H] +., 100), 711 (8), 448 (17); HRMS [M + H] +. Theoretical C41H60N4O12Si2 m / z 857.3819, found (ES +) m / z 857.3826.
[00403] (e) 1,1 '- [[(Pentane-1,5-diyl) dioxy] bis (11aS, 2R) -2-hydroxy-7-methoxy-10- ((2- (trimethylsilyl) ethoxy) methyl) -1,2,3,10,11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] - benzodiazepin-5,11-dione] (6b)
[00404] Preparation from 5b according to the method described above provided the product as a white foam (15.02 g).
[00405] Analytical data: MS (ES +) m / z (relative intensity) 886 ([M + H] +., 10), 739.6 (100), (ES-) m / z (relative intensity) 884 ( [M - H] -., 40).
[00406] (f) 1,1 '- [[(Propane-1,3-diyl) dioxy] bis [(11aS) -11-sulfo-7-methoxy-2-oxo-10- ((2- (trimethylsilyl ) ethoxy) methyl) 1,2,3,10,11,11a-hexahydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5,11-dione]] (7a)
[00407] Method A: A 0.37 M sodium hypochlorite solution (142.5 mL, 52.71 mmoles, 2.4 eq) was added dropwise to a vigorously stirred mixture of diol 6a (18.8 g, 21.96 mmoles, 1 eq), TEMPO (0.069 g, 0.44 mmol, 0.02 eq) and 0.5 M solution of potassium bromide (8.9 mL, 4.4 mmoles, 0.2 eq) in DCM (115 ml), at 0 ° C. The temperature was maintained between 0 ° C and 5 ° C by adjusting the rate of attachment. The resulting yellow emulsion was stirred at 0 ° C to 5 ° C for 1 hour. TLC (EtOAc) and LC / MS [3.53 min. (ES +) m / z (relative intensity) 875 ([M + Na] +., 50), (ES-) m / z (relative intensity) 852 ([M - H] -., 100)] indicated that the reaction was complete.
[00408] The reaction mixture was filtered, the organic layer separated and the aqueous layer was backwashed with DCM (x 2). The combined organic portions were washed with brine (x 1), dried (MgSO4) and evaporated to give a yellow foam. Purification by flash column chromatography (elution of the gradient n-hexane / EtOAc at 35/65 v / v, n-hexane / EtOAc at 30/70 to 25/75 v / v) provided bis-ketone 7a as a foam white (14.1 g, 75%).
[00409] A sodium hypochlorite solution, reagent grade, available in 10-13% chlorine was used. This was assumed to be 10% (10 g NaClO in 100 g) and calculated to be 1.34 M in NaClO. A stock solution was prepared from there by diluting it to 0.37 M with water. This gave a solution of approximately pH 14. The pH was adjusted to 9.3 to 9.4 by the addition of solid NaHCO3. An akota of this stock was then used to give 2.4 mol eq. for the reaction.
[00410] When adding the bleach solution, an initial increase in temperature was observed. The rate of attachment was controlled to maintain the temperature between 0 ° C to 5 ° C. The reaction mixture formed a thick, lemon yellow emulsion.
[00411] Oxidation was an adaptation of the procedure described in Thomas Fey et al, J. Org. Chem., 2001, 66, 8154-8159.
[00412] Method B: Solid TCCA (10.6 g, 45.6 mmoles) was added, in portions, to a stirred solution of alcohol 6a (18.05 g, 21.1 mmoles) and TEMPO (123 mg, 0.78 mmol) in anhydrous DCM (700 mL) at 0 ° C (ice / acetone). The reaction mixture was stirred at 0 ° C, under a nitrogen atmosphere, for 15 minutes, after which time the TLC (EtOAc) and LC / MS [3.57 min (ES +) m / z (relative intensity) 875 ([M + Na] +., 50)] revealed the end of the reaction. The reaction mixture was filtered through celite and the filtrate was washed with saturated aqueous NaHCO3 (400 ml), brine (400 ml), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash column chromatography (EtOAc / Hexane at 80:20 v / v) provided bis ketone 7a as a foam (11.7 g, 65%).
[00413] Method C: A solution of anhydrous DMSO (0.72 mL, 0.84 g, 10.5 mmol) in dry DCM (18 mL) was added dropwise over a period of 25 min to a stirred oxalyl chloride solution (2.63 ml of a 2.0 M solution in DCM, 5.26 moles), under a nitrogen atmosphere, at -60 ° C (liquid N2 / CHCl3). After stirring at -55 ° C for 20 minutes, a slurry of substrate 6a (1.5 g, 1.75 mmol) in dry DCM (36 mL) was added dropwise over a period of 30 min to reaction mixture. After stirring for a further 50 minutes at -55 ° C, a solution of TEA (3.42 ml, 2.49 g, 24.6 mmoles) in dry DCM (18 ml) was added dropwise over a period of 20 min, the reaction mixture. The stirred reaction mixture was allowed to warm to room temperature (~ 1.5 h) and then diluted with DCM (50 ml). The organic solution was washed with 1 N HCl (2 x 25 ml), H2O (30 ml), brine (30 ml) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo gave the crude product, which was purified by flash column chromatography (EtOAc / Hexane at 80:20 v / v) to provide bis ketone 7a as a foam (835 mg, 56 %)
[00414] Analytical data: [a] 20D = + 291 ° (c = 0.26, CHCl3); 1H NMR (400 MHz, CDCl3) 5 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J = 10.1 Hz), 4.75 (d, 2H , J = 10.1 Hz), 4.60 (dd, 2H, J = 9.85, 3.07 Hz), 4.31 - 4.18 (m, 6H), 3.89-3.84 ( m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J = 19.2, 2.85 Hz), 2.76 (dd, 2H, J = 19, 2, 9.90 Hz), 2.42 (p, 2H, J = 5.77 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCI3) 5 206.8, 168.8, 165.9, 151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67 , 3, 65.6, 56.3, 54.9, 52.4, 37.4, 29.0, 18.4, -1.24; IV (ATR, CHCl 3) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm -1; MS (ES +) m / z (relative intensity) 881 ([M + 29] +., 38), 853 ([M + H] +., 100), 707 (8), 542 (12); HRMS [M + H] +. Theoretical C41H56N4O12Si2 m / z 853.3506, found (ES +) m / z 853.3502.
[00415] (f) 1,1 '- [[(Pentane-1,5-diyl) dioxy] bis [(11aS) -11-sulfo-7-methoxy-2-oxo-10- ((2- (trimethylsilyl ) ethoxy) methyl) 1,2,3,10,11,11a-hexahydro-5H-pyrrolo [2,1- c] [1,4] benzodiazepin-5,11-dione]] (7b)
[00416] Preparation from 6b according to Method C provided the product as a white foam (10.5 g, 76%).
[00417] Analytical data: MS (ES +) m / z (relative intensity) 882 ([M + H] +., 30), 735 (100), (ES-) m / z (relative intensity) 925 ([M + 45] -., 100), 880 ([M - H] -., 70).
[00418] (g) 1,1 '- [[(Propane-1,3-diyl) dioxy] bis (11aS) -7-methoxy-2- [[((trifluoromethyl) sulfonyl] oxy] -10 - ((2 - (trimethylsilyl) ethoxy) methyl) -1,10,11,11a-tetrahydro-5H-pyrrolo [2,1-c] [1,4] -benzodiazepin-5,11-dione] (8a)
[00419] Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmoles) was injected in a portion into a vigorously stirred solution of bis-ketone 7a (6.08 g, 7.1 mmoles) in dry DCM (180 mL), at -45 ° C (dry ice / acetonitrile cooling bath), under a nitrogen atmosphere. Anhydrous technical anhydride, obtained from a recently opened ampoule (7.2 ml, 12.08 g, 42.8 mmoles), was injected quickly, drop by drop, while maintaining the temperature at -40 ° C or below. The reaction mixture was allowed to stir at -45 ° C for 1 hour, at which point the TLC (n-hexane / EtOAc at 50/50 v / v) revealed the complete consumption of the starting material. The cold reaction mixture was immediately diluted with DCM (200 ml) and, with vigorous stirring, washed with water (1 x 100 ml), 5% solution of citric acid (1 x 200 ml), saturated NaHCO3 (200 ml), brine (100 ml) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo gave the crude product, which was purified by flash column chromatography (gradient elution: n-hexane / EtOAc at 90:10 v / v to n-hexane / EtOAc at 70 : 30 v / v) to give bisenol triflate 8a as a yellow foam (5.5 g, 70%).
[00420] Analytical data: [a] 24D = + 271 ° (c = 0.18, CHCl3); 1H NMR (400 MHz, CDCl3) 5 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J = 1.97 Hz), 5.51 (d, 2H , J = 10.1 Hz), 4.76 (d, 2H, J = 10.1 Hz), 4.62 (dd, 2H, J = 11.0, 3.69 Hz), 4.32-4 , 23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J = 16.3, 11, 0., 2.36 Hz), 2.43 (p, 2H, J = 5.85 Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H); 13C NMR (100 MHz, CDCl3) 5 167.1, 162.7, 151.9, 148.0, 138.4, 133.6, 120.2, 118.8, 111.9, 107.4, 78 , 6, 67.5, 65.6, 56.7, 56.3, 30.8, 29.0, 18.4, -1.25; IV (ATR, CHCl 3) 2958, 1690, 1646, 1605, 1517, 1456, 1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm-1; MS (ES +) m / z (relative intensity) 1144 ([M + 28] +., 100), 1117 ([M + H] +., 48), 1041 (40), 578 (8); HRMS [M + H] +. Theoretical C43H54N4O16Si2S2F6 m / z 1117.2491, found (ES +) m / z 1117.2465.
[00421] (g) 1,1 '- [[(Pentane-1,5-diyl) dioxy] bis (11aS) -7-methoxy-2- [[((trifluormethyl) sulfonyl] oxy] -10 - ((2 - (trimethylsilyl) ethoxy) methyl) -1,10,11,11a-tetrahydro-5H-pyrrolo [2,1-c] [1,4] -benzodiazepin-5,11-dione] (8b)
[00422] Preparation from 7b according to the method described above gave bisenol triflate as a light yellow foam (6.14 g, 82%).
[00423] Analytical data: (ES +) m / z (relative intensity) 1146 ([M + H] + -, 85). Example 1

[00424] (a) (S) -2- (4-aminophenyl) -7-met6xi-8- (3 - ((S) -7-met6xi-2- (trifluoromethylsulfonyl) -5.11-d / oxo- 10 - ((2- (trimethylsilyl) et6xi) methyl) -5,10,11,11 a-tetrahydro-1 H-pyrrolo [2,1 -c] [1,4] benzodiazepin-8-yl6xi) prop6xi) - 10 - ((2- (trimethylsilyl) et6xi) methyl) -1 H-pyrrolo [2,1-c] [1,4] benzodiazepine-5,11 (10H, 11aH) -dione (9)
[00425] Solid Pd (PPh3) 4 (20.18 mg, 17.46 mmol) was added to a stirred solution of triflate 8a (975 mg, 0.87 mmol), 4- (4.4.5.5 -tetramethyl-1,3,2-dioxaboralane-2-yl) aniline (172 mg, 0.79 mmol) and Na2CO3 (138 mg, 3.98 moles) in toluene (13 mL), EtOH (6.5 mL) and H2O (6.5 ml). The dark solution was allowed to stir under a nitrogen atmosphere for 24 hours, after which time the analysis by TLC (EtOAc) and LC / MS revealed the formation of the mono-coupled product and the presence of unreacted starting material. The solvent was removed by rotary evaporation, under reduced pressure, and the resulting residue divided between H2O (100 ml) and EtOAc (100 ml), after the final separation of the layers, the aqueous phase was extracted again with EtOAc ( 2 x 25 mL). The combined organic layers were washed with H2O (50 ml), brine (60 ml), dried (MgSO4), filtered and evaporated in vacuo to provide the crude Suzuki product. The crude Suzuki product was subjected to flash chromatography (40% EtOAc / 60% Hexane ^ 70% EtOAc, 30% Hexane). Removal of the excess eluent by rotary evaporation, under reduced pressure, provided the desired product (399 mg) in 43% yield.
[00426] 1H NMR: (CDCl3, 400 MHz) 5 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J = 8.5 Hz), 7.15 (t, 1H, J = 2.0 Hz), 6.66 (d, 2H, J = 8.5 Hz), 5.52 (d, 2H, J = 10 , 0 Hz), 4.77 (d, 1H, J = 10.0 Hz), 4.76 (d, 1H, J = 10.0 Hz), 4.62 (dd, 1H, J = 3.7 , 11.0 Hz), 4.58 (dd, 1H, J = 3.4, 10.6 Hz), 4.29 (t, 4H, J = 5.6 Hz), 4,003.85 (m, 8H ), 3.80 - 3.60 (m, 4H), 3.16 (ddd, 1H, J = 2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J = 2 , 2, 10.5, 16.1 Hz), 2.43 (p, 2H, J = 5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H) , 13C NMR: (CDCI3, 100 MHz) 5 □ 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147.6, 139 , 6, 134.8, 134.5, 127.9 (meth), 127.5, 125.1, 123.21, 121.5, 120.5 (meth), 120.1 (meth), 116, 4 (methyl), 113.2 (methyl), 108.7 (methyl), 79.8 (methyl), 79.6 (methyl), 68.7 (methyl), 68.5 (methyl), 67 , 0 (methyl), 66.8 (methyl), 58.8 (methyl), 58.0 (methyl), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3 (methyl), 19.7 (methyl), 0.25 (methyl).
[00427] (b) (S) -2- (4-aminophenyl) -7-methoxy-8- (3 - ((S) -7-methoxy-2- (4-methoxyphenyl) - 5,11-dioxo- 10 - ((2- (trimethylsilyl) ethoxy) methyl) -5,10,11,11a-tetrahydro-1H-pyrrolo [2,1- c] [1,4] benzodiazepin-8-yloxy) propoxy) -10- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrrolo [2,1-c] [1,4] benzodiazepine-5,11 (10H, 11aH) -dione (10)
[00428] A suspension of 4-amine triflate [see Patent 33], (210 mg, 0.198 mmol), methylboronic acid (50 mg, 0.835 mmol, 4.2 eq.), Silver oxide I (139 mg, 0.600 mmol., 3 eq.), tribasic potassium phosphate (252 mg, 1.2 eq w / w), triphenylsine (36.7 mg, 0.12 mmol, 0.6 eq.) and bis (benzonitrile) dichloro- palladium II (11.5 mg, 0.030 mmol, 0.15 eq.) was heated to 75 ° C in dry dioxane (8 mL) in a sealed tube under an inert atmosphere for 1.5 h. The reaction mixture was filtered through cotton and the filter pad was washed with ethyl acetate and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with 80% EtOAc: 20% hexane. Removal of excess eluent by rotary evaporation under reduced pressure gave the product as an off-white foam (100 mg, 0.11 mmol, 54% yield).
[00429] LC-MS RT 3.87 mins, 926 (M + H)
[00430] (c) (S) -2- (4-aminophenyl) -7-met6xi-8- (3 - ((S) -7-met6xi-2-methyl-5-oxo- 5.11 a-dihydro -1 H-pyrrolo [2,1 -c] [1,4] benzodiazepine-8-yl6xi) prop6xi) -1 H-pyrrolo [2,1 -c] [1,4] benzodiazepine-5 (11aH) -one (11)
The newly produced LiBH4 (44 mg, 2.0 mmoles, 20 eq.) Was added to a stirred solution of SEM-dilactam (90 mg, 0.1 mmol) in THF (8 mL) at room temperature. The reaction mixture was allowed to stir for 0.5 h, at which time LC-MS showed complete reaction. The reaction mixture was partitioned between water (50 ml) and chloroform (100 ml). The organic phase was washed with brine (50 ml), dried over magnesium sulfate and concentrated in vacuo. The resulting residue was treated with DCM (5 ml), EtOH (14 ml), H2O (7 ml) and silica gel (10 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was slowly filtered through a sintering funnel and the silica residue washed with 90% CHCl3: 10% MeOH (~ 250 mL) until the UV activity completely disappeared from the eluent. The organic phase was washed with H2O (50 ml), brine (60 ml), dried (MgSO4), filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (from 100% CHCl3: 0% MeOH to 96% CHCl3: 4% MeOH) to provide the PBD dimer (5 mg 8% yield).
[00432] LC-MS RT 2.30 mins, 634 (M + H)
[00433] 1H NMR (400 MHZ, CDCl3) □ □□□□□□ (d, J = 4.0 Hz, 1H), 7.73 (d, J = 4.0 Hz, 1H), 7.45 (s, 1H), 7.43 (s, 1H), 7.26 (bs, 1H), 7.14 (d, J = 8.5 Hz, 1H), 6.79 (s, 1H), 6.77 (s, 1H), 6.71-6 , 64 (m, 1H), 4.34-4.03 (m, 6H), 3.86 (s, 3H), 3.85 (s 3H), 3.55-3.37 (m, 1H), 3.36-3.19 (m, 1H), 3, 17-3.00 (m, 1H), 2.96-2.80 (m, 1H), 1.75 (s 3H). Example 2

[00434] (a) 2- (3-aminophenyl) -11- (tert-butyldimethylsilyl6xi) -8- (5 - ((11S, 11aS) -11- (tert-butyldimethylsilyl6xi) -7-met6xi-5-oxo- 10 - ((2! 2,2-trichloroethoxy) carbonyl) -2- (trifluoromethylsulfonyl6xi) -5,10,11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepin- d / azep / n-8- / 76x /) pentyl6xi) -7-met6xi-5-oxo-11.11 a-dihydro-1 H-pyrrolo [2,1 - c] [1,4] benzodiazepine-10 (5H) (11S, 11aS) -2,2,2-trichloroethylcarboxylate 13
[00435] Solid 3-aminobenzeneboronic acid (60.3 mg) was added to a solution of Troc 12 protected bis triflate (Compound 44, WO 2006/111759) (600 mg, 0.41 mmol), sodium carbonate (65 mg, 0.61 mmoml) and palladium tetracis triphenylphosphine (0.012 mmol) in toluene (10.8 ml), ethanol (5.4 ml) and water (5.4 ml). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulfate. The excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution: EtO-Ac / hexane 20/80 ^ 30/70 ^ 40/60 ^ 60 / 40) to remove unreacted bis-triflate. Removal of excess eluent from selected fractions to generate the desired compound in 41% yield (230 mg, 0.163 mmol).
[00436] LC-MS RT 4.28 mins, 1411 (M + H); 1H-NMR (400 MHZ, CDCl3) □ □ 7.44 (bs, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 7.20 (s, 1H), 7.16 (t, J = 7.9 Hz, 1H), 6.84 - 6.73 (m, 3H), 6.70 (bs, 1H), 6.62 (dd, J = 7.9, 1.7 Hz, 1H), 6.66 - 6.58 (m, 2H), 5.25 (d, J = 12.0 Hz, 1H), 5.24 (d, J = 12.0 Hz, 1H), 4.24 (d, J = 12.0 Hz, 1H), 4.22 (d, J = 12.0 Hz, 1H), 4.17 - 4.07 (m, 2H), 4.08 - 3 , 89 (m, 10H), 3.43 - 3.28 (m, 2H), 2.85 (d, J = 1.65 Hz, 2H), 2.07 - 1.90 (m, 4H), 1.78 - 1.63 (m, 2H), 0.94 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.27 (s, 6H).
[00437] (b) 2- (3-aminophenyl) -11 - (tert-butyldimethylsilyloxy) -8- (5 - ((11S, 11 aS) -11 - (tert-butyldimethylsilyl6xi) -2-propenyl-7-met6xi -5-oxo-10 - ((2,2,2-trichloroethoxy) carbonyl) - 5,10,11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepindiazepin-8-yl6xi ) pentyl6xi) - 7-met6xi-5-oxo-11,11a-dihydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepine-10 (5H) - (11S, 11aS) -2 carboxylate, 2,2-trichloroethyl 14
[00438] Solid 1-propenyl boronic acid (7.1 mg, 0.084 mmol) was added to a solution of Troc 13 protected triflate (73 mg, 0.052 mmol), sodium carbonate (18 mg, 0.17 mmol) and palladium tetracis triphenylphosphine (3 mg) in toluene (1 ml), ethanol (0.5 ml) and water (0.5 ml). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. The excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was eluted through a plug of silica gel with ethyl acetate. Removing excess eluent from selected fractions yielded coupled product 14 (40 mg, 0.031 mmol, 59%).
[00439] LC-MS RT 4.38 mins, (1301, 1305, 1307, 1308, 1310 multi-mass due to chlorine isotopes)
[00440] (c) (S) -2- (3-aminophenyl) -8- (5 - ((S) -2-propenyl-7-met6xi-5-oxo-5,11a-dihydro-1H-pyrrole [ 2,1-c] [1,4] benzodiazepine-8-yl6xi) pentyl6xi) -7-met6xi-1H-pyrrolo [2,1-c] [1,4] benzodiazepine-5 (11aH) -one 15
[00441] Cadmium / lead pair (100 mg, Q Dong et al. Tetrahedron Letters vol 36, issue 32, 5681-5682, 1995) was added to a solution of the Suzuki product 14 (40 mg, 0.029 mmol) in THF (1 ml) and ammonium acetate (1N, 1 ml) and the reaction mixture was allowed to stir for 1 hour. The reaction was filtered through something to remove particles and break the emulsion. The reaction mixture was partitioned between chloroform and water, the phases separated and the aqueous phase extracted with chloroform. The combined organic layers were washed with brine and dried over magnesium sulfate. Rotary evaporation under reduced pressure yielded the crude product which was subjected to column chromatography (silica gel, 1% MeOH / 5% CHCl 3). Removal of excess eluent by rotary evaporation under reduced pressure yielded the desired enzyme product 15 (9 mg 0.013 mmol 43%)
[00442] LC-MS RT 2.80 mins, 689 (M + H)
[00443] 1H-NMR (400 MHZ, CDCl3) □ □□□□□□ (d, J = 3.9 Hz, 1H), 7.82 (d, J = 3.9 Hz, 1H), 7.52 (s, 1H), 7.49 (s, 1H), 7.45 (bs, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.92 (bs, 1H), 6 , 84-6.76 (m, 3H), 6.72 (bs, 1H), 6.60 (dd, J = 7.9, 1.9 Hz, 1H), 6.26 (d, J = 15 , 3 Hz, 1H), 5.67-5.51 (m, 1H), 4.46-4.35 (m, 1H), 4.34-4.24 (m, 1H), 4.20- 4.00 (m, 4H), 3.94 (s, 3H), 3.93 (s 3H), 3.62-3.44 (m, 1H), 3.43-3.23 (m, 2H ), 3.19-3.02 (m, 1H), 2.06-1.89 (m, 4H), 1.84 (d, J = 6.5 Hz, 3H), 1.76-1, 62 (m, 2H). Example 3

[00444] (a) (S) -2- (3-aminophenyl) -7-met6xi-8- (3 - ((S) -7-met6xi-2- (trifluoromethylsulfonyl) -5.11-dioxo-10- ((2- (trimethylsilyl) ethoxy) methyl) -5,10,11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepin-8-yloxy) propoxy) -10 - (( 2- (trimethylsilyl) ethoxy) methyl) -1H-pyrrolo [2,1-c] [1,4] benzodiazepine-5,11 (10H, 11aH) -dione 16
[00445] Pd (PPh3) 4 solid (20 mg, 17.8 □ moles) was added to the stirred solution of triflate 8a (2.5 g, 2.24 mmoles), 3-aminobenzenoboronic acid (291 mg, 2 , 12 mmoles) and Na2CO3 (356 mg, 3.35 mmoles) in toluene (20 ml), EtOH (10 ml) and H2O (10 ml). The solution was allowed to stir under a nitrogen atmosphere for 3 hours at room temperature, after which time analysis by TLC (EtOAc) and LC / MS revealed the formation of the desired mono-coupled product and the presence of non- reacted. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue divided between H2O (100 ml) and EtOAc (100 ml), after any separation of the layers, the aqueous phase was extracted again with EtOAc (2 x 25 ml) . The combined organic layers were washed with H2O (50 ml), brine (60 ml), dried (MgSO4), filtered and evaporated in vacuo to provide the crude Suzuki product. The crude Suzuki product was subjected to flash chromatography (30% EtOAc / 70% Hexane ^ 80% EtOAc, 20% Hexane). Removal of excess eluent by rotary evaporation under reduced pressure yielded the desired product (1 g) in 42% yield.
[00446] LC-MS, 4.17 minutes, ES + 1060.19.
[00447] (b) (S) -2- (3-aminophenyl) -7-methoxy-8- (3 - ((S) -7-methoxy-2-methyl-5,11- dioxo-10 - (( 2- (trimethylsilyl) ethoxy) methyl) -5,10,11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepin-8-yloxy) propoxy) -10 - ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrrolo [2,1-c] [1,4] benzodiazepine-5.11 (10H, 11aH) -dione 17
[00448] A suspension of 3-anilino triflate, (50 mg, 47.2 Dmoles), methylboronic acid (8.47 mg, 141 Dmoles, 3 eq.), Silver (I) oxide (21.8 mg, 94.3 □ moles, 2 eq.), Tribasic potassium phosphate (60 mg, 1.2 eq w / w), triphenylsine (5.78 mg, 18.9 Dmoles, 0.4 eq.) And bis (benzonitrile ) dichloro-palladium II (1.81 mg, 4.7 Dmoles, 0.1 eq.) was heated to 67 ° C in dry dioxane (2 mL) in a sealed tube under an inert atmosphere for 3 hrs. The reaction mixture was filtered through cotton and the filter pad was washed with ethyl acetate and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with 80% EtOAc: 20% hexane. Removal of excess eluent by rotary evaporation under reduced pressure gave the product as an off-white foam (18 mg, 19.4 Dmoles, 41% yield). The reaction was subsequently repeated on a large scale to generate 250 mg of the 2-methyl product.
[00449] LC-MS 3.88 mins, 925.86 (M + H)
[00450] (c) (S) -2- (3-aminophenyl) -7-met6xi-8- (3 - ((S) -7-met6xi-2-methyl-5-oxo- 5.11 a-dihydro -1 H-pyrrolo [2,1 -c] [1,4] benzodiazepine-8-yl6xi) prop6xi) -1 H-pyrrolo [2,1 -c] [1,4] benzodiazepine-5 (11aH) -one 18
[00451] Freshly produced LiBH4 (20.6 mg, 0.95 mmol, 3.5 eq.) Was added to a stirred solution of SEM-dilactam (250 mg, 0.27 mmol) in THF (4 mL) at temperature environment. The reaction mixture was allowed to stir for 1.0 h, at which time LC-MS showed complete reaction. The excess LiBH4 was quenched with acetone (c. 1 ml) to 0 ° C (ice bath). The reaction mixture was partitioned between water (50 ml) and 10% methanol in chloroform (100 ml). The organic phase was washed with brine (50 ml), dried over magnesium sulfate and concentrated in vacuo.
The resulting residue was treated with 10% methanol in chloroform (c. 50 ml) and silica gel (20 g). The viscous mixture was allowed to stir at room temperature for 5 days. The mixture was slowly filtered through a sintered funnel and the silica residue washed with 90% CHCl 3: 10% MeOH (~ 250 mL) until the UV activity from the eluent completely disappeared. The organic phase was washed with H2O (50 ml), brine 60 ml), dried (MgSO4), filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (gradient from 100% CHCl3: 0% MeOH to 96% CHCl3: 4% MeOH) to provide the PBD 18 dimension. Example 4 Part (i)

[00453] (a) Alternate Synthesis of (S) -2- (4-Aminophenyl) -7-met6xi-8- (3 - ((S) -7-met6xi-2- (trifluoromethylsulfonyl) -5.11-dioxo -10 - ((2- (trimethylsilyl) et6xi) methyl) -5,10,11,11a tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepin-8-yl6xi) prop6xi) -10 - (((2- (trimethylsilyl) et6xi) methyl) -1 H-pyrrolo [2,1 -c] [1,4] benzodiazepine-5.11 (10H, 11 aH) -dione (9)
[00454] Tetracis (triphenylphosphine) palladium (0) (208 mg) was added to triflate (8a) (5 g), 4-anilinaboronic acid (0.93 g) and sodium carbonate (0.62 g) in a mixture of toluene (60 ml), ethanol (30 ml) and water (10 ml). The reaction mixture was allowed to stir for 3 days at room temperature. The reaction mixture was washed with water, brine and dried over magnesium sulfate. After filtration, the excess solvent was removed by rotary evaporation under reduced pressure. The crude coupling product was purified by flash column chromatography (silica gel; Gradient: 100% hexane to 100% ethyl acetate). Pure fractions were combined and removal of excess eluent yielded the pure product as a solid (2.2 g, 93% yield, LC / MS 8.05 mins, m / z ES + 1060).
[00455] (b) (S) -2- (4-aminophenyl) -7-met6xi-8- (3 - ((S) -7-met6xi-2- (phenyl-vinyl) - 5,11-dioxo- 10 - ((2- (trimethylsilyl) et6xi) methyl) -5,10,11,11a-tetrahydro-1H-pyrrolo [2,1- c] [1,4] benzodiazepin-8-yl6xi) prop6xi) -10- ((2- (trimethylsilyl) et6xi) methyl) -1H-pyrrolo [2,1-c] [1,4] benzodiazepine-5,11 (10H, 11aH) -dione (20)
[00456] A mixture of triflate 9 (0.5 g), trans-2-phenylvinylboronic acid (0.091 g), triethylamine (0.38 g) and tetracis (triphenylphosphine) palladium (0) (30 ml) in ethanol (3 mL), toluene (6 mL) and water (1mL) was irradiated with microwave for 8 minutes at 80 ° C in a sealed microwave flask. The reaction mixture was diluted with dichloromethane, washed with water and dried over magnesium sulfate. The excess solvent was removed by rotary evaporation under reduced pressure to generate the crude product which was used without further purification in the next reaction. Retention time 8.13 mins, ES + 1014.13.
[00457] (c) (S) -2- (4-aminophenyl) -7-met6xi-8- (3 - ((S) -7-met6xi-2- (phenyl-vinyl) -5- oxo-5, 11 α-dihydro-1 H-pyrrolo [2,1-c] [1,4] benzodiazepine-8-yl6xi) prop6xi) -1 H-pyrrolo [2,1 - c] [1,4] benzodiazepine-5 ( 11aH) -one (21)
[00458] A solution of superhydride in THF (1 M, 1.2 ml) was added by syringe to a solution of the crude Suzuki product (0.477 g) in THF (10 ml) at - 78 ° C (water bath) dry ice / acetone). The reaction mixture was allowed to stir at -78 ° C for 20 minutes, after which time the reaction was quenched with water. The reaction mixture was extracted with ethyl acetate and the organic layer washed with brine and dried over magnesium sulfate. Removal of excess solvent by rotary evaporation under reduced pressure yielded the crude SEM-carbinolamine which was dissolved in dichloromethane (3 ml), ethanol (6 ml) and water (1 ml) and stirred with silica gel for 2 days. The reaction mixture was filtered, the excess solvent evaporated by rotary evaporation under reduced pressure and the residue subjected to flash column chromatography (3% methanol in chloroform). Pure fractions were combined and the excess eluent removed by rotary evaporation under reduced pressure to generate compound 21 (0.75 mg, 22% yield in 3 steps). Retention time 5.53 mins, ES + 721.99. Part (ii)

[00459] (a) (S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido acid ) propanoic (23)
[00460] A suspension of dipepUdeide (22) (0.1 g, 0.54 mmol, 1 eq.) And maleimidohexanoic acid ester succinimide (0.165 g, 0.54 mmol, 1 eq.) In anhydrous DMF ( 5 mL) was stirred at room temperature for 24 hours, at which time LCMS indicated 50% conversion to a new product. The reaction mixture was diluted with anhydrous DMF (5 ml) and the reaction was allowed to proceed for an additional 24 hours. The solvent was evaporated under reduced pressure to give a colorless residue. Diethyl ether (60 mL) was added and the mixture was sonicated for 5 min, the ether was decanted and the process was repeated (x 2). The final ethereal portion was filtered to isolate the product (23) as a white powder that was dried under vacuum (0.105 g, 52%). Analytical Data: RT 2.28 min; MS (ES +) m / z (relative intensity) 382 ([M + H] +., 90), MS (ES-) m / z (relative intensity) 380 ([M - H]) -., 100).
[00461] (b) 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -N - ((S) -1 - ((((S) -1 - ((4 - (((S) -7- metdx / -8- (3 - ((((S) -7-metdxi-5-oxo-2 - ((E) -styryl) -5,11a-dihydro-pyrrole [2, 1- c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -5-oxo-5,11a-dihydro-pyrrolo [2,1- c] [1,4] benzodiazepin-2-yl) phenyl ) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) hexanamide (24)
[00462] The asymmetric PBD dimer (21) (0.019 g, 26 pmoles, 1 eq.) Was added to a solution of the linker (23) (0.0121 g, 31.6 pmoles, 1.2 eq.) And EEDQ (0.0098 g, 39.6 pmoles, 1.5 eq.) In a mixture of anhydrous DCM / MeOH (3 ml / 0.5 ml) under an argon atmosphere. The resulting solution was stirred at room temperature for 5 hours, at which time LCMS indicated 50% conversion to a new product. The reaction mixture was diluted with anhydrous DCM (2 ml) and the reaction was allowed to continue for another 18 hours. The solvent was evaporated under reduced pressure and the residue purified by flash column chromatography [100% DCM for 94% DCM / 6% MeOH in 1% increments] to give the product as a yellow solid (5, 2 mg, 18%). Analytical Data: RT 3.10 min; MS (ES +) m / z (relative intensity) 1085 ([M + H] +., 90). Example 5

[00463] (a) (S) -2- (4-aminophenyl) -8- (3 - (((S) -2-cyclopropyl-7-met6xi-5,11-dioxo- 10 - ((2- ( trimethylsilyl) et6xi) methyl) -5,10,11,11a-tetrahydro-pyrrolo [2,1-c] [1,4] benzodiazepin-8-yl) 6xi) prop6xi) -7-met6xi-10 - ((2 - (trimethylsilyl) et6xi) methyl) -1H-pyrrolo [2,1- c] [1,4] benzodiazepine-5,11 (10H, 11aH) -dione (25)
[00464] A suspension of silver (I) oxide (0.441 g), tribasic potassium phosphate (1.177 g), triphenylarsine (0.116 g), cyclopropylboronic acid (0.206 g) and starting material 9 (0.5 g) in dioxane (15 mL) under an argon atmosphere was heated to 71 ° C. A catalytic amount of palladium (II) bis (benzonitrile chloride) (0.036 g) was added and the reaction mixture was allowed to stir for 2 hours and 10 mins at 71 ° C. The reaction mixture was filtered through celite and the filter pad was washed with ethyl acetate (400 ml). The organic solution was extracted with water (2 x 600 ml) and brine (600 ml) and dried over magnesium sulfate. Removal of the organic solvent by rotary evaporation under reduced pressure yielded the crude product which was purified by silica gel gravity chromatography (ethyl acetate only as eluent). Removal of excess eluent by rotary evaporation under reduced pressure yielded product 25 as a yellow solid (145 mg, 32% yield). LCMS RT 3.92 mins, ES + 952.06
[00465] (b) (S) -2- (4-aminophenyl) -8- (3 - (((S) -2-cyclopropyl-7-methoxy-5-oxo-5,11a- dihydro-pyrrole [2 , 1-c] [1,4] benzodiazepin-8-yl) oxy) propoxy) -7-methoxy-pyrrolo [2,1-c] [1,4] benzodiazepin-5 (11aH) -one (26)
[00466] A super hydride solution (0.361 ml, 1M in THF) was added in drops for 5 minutes to a solution of SEM dilictam 25 (0.137 g) in anhydrous tetrahydrofuran (5 ml) under an argon atmosphere at - 78 ° C. LCMS after 35 minutes revealed that the reaction was complete and excess super hydride was cooled down sharply with water (4 mL) followed by brine (4 mL). The aqueous solution was extracted with a mixture of dichloromethane / methanol (9: 1, 2 x 16 ml) and the organic layer dried over magnesium sulfate. Solvent was removed by rotary evaporation under reduced pressure and the crude product was taken up in a mixture of ethanol, dichloromethane and water (8: 3: 1, 15 ml) and treated with silica gel. The slurry was allowed to stir for 4 days. The mixture was filtered through a sinter, washing with dichloromethane / methanol (9: 1, 140 ml) until the product finished eluting. The organic layer was washed with brine (2x 250 ml) and then dried over magnesium sulfate. Rotary evaporation under reduced pressure yielded the crude product which was subjected to flash column chromatography (silica gel; gradient of 100% methanol / 5% dichloromethane). Removing excess eluent yielded product 26 (23 mg, 25% yield). LCMS RT 2.42, ES + 659.92 Example 6

[00467] (S) -2- (4-aminophenyl) -7-met6xi-8- (3 - ((((S) -7-met6xi-5,11-dioxo-10 - ((2- (t / 7methylsil / l) et6x /) methyl) -2 - ((trimethylsilyl) ethynyl) -5,10,11,11 a-tetrahydro-pyrrolo [2,1 - c] [1,4] benzodiazepin-8-yl) 6xi) prop6xi) -10 - ((2- (trimethylsilyl) et6xi) methyl) -pyrrole [2,1 - c] [1,4] benzodiazepine-5,11 (10H, 11aH) -dione (27)
[00468] A mixture of 9 (0.150 g, 0.14 mmol), CuI (0.003 g, 0.014 mmol, 0.1 eq), Pd (PPh3) 4 (0.0162 g, 0.014 mmol, 0.1 eq) and PPh3 (0.007 g, 0.028 mmol, 0.2 eq) was dissolved in piperidine (9 mL) in the presence of molecular sieves under an argon atmosphere. Ethinyltrimethylsilane (0.06 ml, 0.42 mmol, 3 eq) was added to the mixture at 70 ° C and the reaction mixture was allowed to stir overnight. The solvent was removed by rotary evaporation under reduced pressure and the resulting brown solid purified by flash column chromatography (silica gel, 90% EtOAc, 10% hexane). Compound 27 was obtained as an orange solid (0.043 g, 30%); Rf 0.69 [EtOAc]; LC-MS (5 min) 4.28 min, ES + 1008.28. Example 7
6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -N - ((S) -1 - ((((S) -1 - ((4 - ((S) - 7-met6xi-8- (3- ((((S) -7-met6xi-2-methyl-5-oxo-5,11a-dihydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepin- 8- yl) 6xi) prop6xi) -5-oxo-5,11a-dihydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepin-2-yl) phenyl) amino) -1-oxopropan-2 -yl) amino) -3-methyl-1-oxobutan-2-yl) hexanamide (30)
[00469] To a mixture of 23 carboxylic acid (8 mg, 21 umoles) in methanol / 5% dichloromethane was added EEDQ (6.1 mg, 24.6 umoles) and the mixture was stirred for 15 minutes under nitrogen at room temperature. The resulting mixture was added to 11 (12 mg, 18.9 umoles) and stirred for 3 hours under nitrogen. The reaction mixture was aspirated directly onto a 1 mm radial chromatotron plate and eluted with a gradient of 1 to 4% methanol in dichloromethane. Fractions containing product were concentrated under reduced pressure to give 9.4 mg (50%) of 30 as a yellow solid: MS (ES-) m / z 997.18 (M + H) +
6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -N - ((S) -1 - ((((S) -1 - ((3 - ((S) - 7-met6xi-8- (3- ((((S) -7-met6xi-2-methyl-5-oxo-5,11a-dihydro-pyrrolo [2,1-c] [1,4] benzo diazepin-8 - yl) 6xi) prop6xi) -5-oxo-5,11a-dihydro-pyrrolo [2,1-c] [1,4] benzodiazepin-2-yl) phenyl) amino) -1- oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) hexanamide (31)
[00470] Compound 31 was synthesized from compound 18 using the same method as in part (a) in 25% yield. Example 8: Determination of Free Drug In Vitro Cytotoxicity
[00471] The cells as detailed below were collected and placed in 96-well dark-walled plates at a density of 10,000 cells / well in 150 DL of medium. Serial dilutions of the test article (50 nL) were added and incubation was carried out for 92 hours at 37 ° C. After addition of the test compound, the cultures were incubated for 96 hours at 37 ° C. Resazurin (0.25 mM, 50 DL, Sigma, St. Louis, MO) in medium was added and incubation continued for 4 h. The plates were read on a Fusion HT microplate reader (Packard, Meriden, CT) using an excitement wavelength of 525 nm and an emission wavelength of 590 nm. Data for all trials were reduced using GraphPad Prism Version 4 for Windows (GraphPad Software, San Diego, CA). The IC50 concentrates compared to untreated control cells were determined using parameter 4 curve adjustments.
[00472] The IC 50 (nM) values for compound 15 are:

[00473] The same method was also used to determine the activity of compounds 11 and 18:
Alternative Cell Assay
[00474] The cells were placed in 150 pL of growth medium per well in 96-well plates with a light bottom and dark walls (Costar, Corning) and allowed to sit for 1 hour in the biological cabin before placing in a 37 ° incubator. C, 5% CO2. The following day, 4X drug stock concentration was prepared, and then titrated as 10-fold serial dilutions producing dose curves of 8 curved points and added to 50 µl per well in duplicate. The cells were then incubated for 48 hours at 37oC, 5% CO2. Cyto-toxicity was measured by incubation with 100 µL of Cell Titer Glo solution (Promega) for 1 hour, and then luminescence was measured on a Fusion HT plate reader (Perkin Elmer). The data were processed with Excel (Microsoft) and GraphPad (Prism) to produce dose response curves and IC50 values were generated and the data collected.

[00475] Example 9: Preparation of PDB Dimer Conjugates
[00476] Antibody-drug conjugates were prepared as previously described (see Doronina et al., Nature Biotechnology, 21, 778-784 (2003)) or as described below.
[00477] hIgGI antibodies developed with introduced cisterns: CD70 antibodies containing a cysteine residue at position 239 of the heavy chain (h1F6d) were totally reduced by adding 10 equivalents of TCEP and EDTA to 1 mM and adjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). After 1 hour of incubation at 37 ° C, the reaction was cooled to 22 ° C and 30 equivalents of dehydroascorbic acid were added to selectively reoxidate native disulfides, while leaving system 239 in the reduced state. The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction was allowed to proceed for 1 hour at 22 ° C. The pH of the solution was then raised again to 7.4 by the addition of 1 M Tris buffer (pH 9.0). 3.5 equivalents of the PBD drug binder in DMSO were placed in a suitable container for dilution with propylene glycol prior to addition to the reaction. To maintain the solubility of the PBD drug binder, the antibody alone was first diluted with propylene glycol for a final concentration of 33% (for example, if the antibody solution was in a 60 mL reaction volume, 30 ml of propylene glycol were added). This same volume of propylene glycol (30 ml in this Example) was then added to the PBD drug binder as a diluent. After mixing, the PBD drug binder solution in propylene glycol was added to the antibody solution to effect the conjugation; the final concentration of propylene glycol is 50%. The reaction was allowed to proceed for 30 minutes and then quenched by adding 5 equivalents of N-acetyl cysteine. The ADC was then purified by ultrafiltration through a 30 kD membrane. (Note that the concentration of propylene glycol used in the reaction can be reduced for any particular PBD, since its sole purpose is to maintain the solubility of the drug binder in the aqueous medium). Example 10: Determination of In Vitro Cytotoxicity of Conjugate
[00478] The cells as detailed below were collected and placed in 96-well dark-walled plates at a density of 10,000 cells / well in 150 DL of medium. Serial dilutions of the test article (50 nL) were added and incubation was carried out for 92 hours at 37 ° C. After addition of the test compound, the cultures were incubated for 96 hours at 37 ° C. Resazurin (0.25 mM, 50 DL, Sigma, St. Louis, MO) in medium was added and incubation continued for 4 h. The plates were read on a Fusion HT microplate reader (Packard, Meriden, CT) using an excitement wavelength of 525 nm and an emission wavelength of 590 nm. Data for all trials were reduced using GraphPad Prism Version 4 for Windows (GraphPad Software, San Diego, CA). IC50 concentrations compared to untreated control cells were determined using parameter 4 curve adjustments. The antibody used was CD70 antibody (humanized 1F6, see United States Order No. N ° 2009-148942) having res ^ cisternal duo at position 239 of the amino acid heavy chain (according to the EU numbering system) (indicated as h1F6d).
[00479] IC50 (nM) values for ADCs of compound 31 are:

[00480] Note: Maximum inhibition (Inhib. Max.b) =% inhibition in the upper concentration of 100% untreated. Example 11: Determination of In Vivo Cytoxicity of Selected Conjugates
[00481] The following study was conducted according to the Animal Care and Use Committee in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The antibodies used were an antibody having introduced cysteine residues at position 239 (S239C) in the heavy chains and conjugated to compound 31, and a non-binding control conjugated to the same compound 31.
[00482] Treatment studies were conducted in an anUgene + xenograft model. Tumor cells were implanted subcutaneously in scid mice. The mice were randomized to study groups (n = 6). The composite ADC 31 or control ADCs were dosed according to a q4dx4 schedule. The tumor volume as a function of time was determined using the formula (L x W2) / 2. The animals were euthanized when the tumor volume reached 1000 mm3.
[00483] Referring to Figure 1, the ADC of compound 31 was dosed at 0.1 (□), 0.3 (■) and 1 (■) mg / kg. A non-binding control, conjugated to compound 31, was administered in the same doses (0.1 (A), 0.3 (A) and 1 (A) mg / kg). All three doses of the conjugated Ab-compound 31 had better activity than the non-binding control conjugate. Untreated tumors are shown by *

权利要求:
Claims (18)
[0001]
1. Compound CHARACTERIZED by the fact that it has formula I:
[0002]
2. Compound, according to claim 1, CHARACTERIZED by the fact that R7 is a C1-4 alkyloxy group.
[0003]
3. Compound, according to claim 1 or 2, CHARACTERIZED by the fact that R '' and C3-7 alkylene.
[0004]
4. A compound according to any one of claims 1 to 3, CHARACTERIZED by the fact that R12 is selected from: a) methyl, ethyl or propyl; b) cyclopropyl; c) a group of formula:
[0005]
A compound according to any one of claims 1 to 4, CHARACTERIZED by the fact that R10 and R11 form a nitrogen-carbon double bond.
[0006]
6. Compound according to any one of claims 1 to 5, CHARACTERIZED by the fact that R6 ', R7', R9 ', R10', R11 'and Y' are the same as R6, R7, R, R10, R11 and Y respectively.
[0007]
7. Composed, according to claim 1, CHARACTERIZED by the fact that it has the structure:
[0008]
8. Use of a compound, as defined in any one of claims 1 to 7, CHARACTERIZED by the fact that it is in the manufacture of a medicine to treat a proliferative disease.
[0009]
9. Compound according to any one of claims 1 to 7, CHARACTERIZED by the fact that it is for use in the treatment of a proliferative disease
[0010]
10. Compound CHARACTERIZED by the fact that it has formula II:
[0011]
11. Conjugate CHARACTERIZED by the fact that it has the formula III: L - (LU-D) p (I) where L is a linker unit that is an antibody or an antigen-binding fragment of an antibody, LU is a Binder unit with the following structure:
[0012]
12. Conjugate, according to claim 11, CHARACTERIZED by the fact that m and 0.
[0013]
13. Conjugate, according to claim 11 or 12, CHARACTERIZED by the fact that dipepUdeo is selected from: -Phe-Lys-; -Val-Ala-; -Val-Lys-; -Ala-Lys-; and -Val-Cit.
[0014]
14. Conjugate, according to claim 13, CHARACTERIZED by the fact that dipepUdeo is selected from -Phe-Lys-; -Val-Cit-; and -Val-Ala-.
[0015]
15. Conjugate, according to any one of claims 11 to 14, CHARACTERIZED by the fact that A1 is:
[0016]
16. Conjugate, according to claim 15, CHARACTERIZED by the fact that n and 5.
[0017]
17. Use of a conjugate, as defined in any of claims 11 to 16, CHARACTERIZED by the fact that and in the manufacture of a medication for the treatment of cancer.
[0018]
18. Conjugate according to any of claims 11 to 16, CHARACTERIZED by the fact that it is for use in the treatment of cancer.

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法律状态:
2017-11-21| B25A| Requested transfer of rights approved|Owner name: SEATTLE GENETICS, INC (US) , SPIROGEN SARL (CH) |

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2017-12-12| B25A| Requested transfer of rights approved|Owner name: SEATTLE GENETICS, INC (US) , MEDIMMUNE LIMITED (GB) Owner name: SEATTLE GENETICS, INC (US) , MEDIMMUNE LIMITED (GB |

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2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-11-19| B07E| Notice of approval relating to section 229 industrial property law|

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2019-12-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-04-14| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2020-07-21| B09A| Decision: intention to grant|

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2020-12-01| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/04/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US32445310P| true| 2010-04-15|2010-04-15|

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US61/324,453|2010-04-15|

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PCT/US2011/032668|WO2011130616A1|2010-04-15|2011-04-15|Pyrrolobenzodiazepines used to treat proliferative diseases|

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