targeted pyrrolobenzodiazepine conjugates, pharmaceutical composition, use thereof for treatment of

专利摘要:
TARGETED PYROCHOBENZODIAZEPINE CONJUGATES Conjugates are provided which comprise PDBs conjugated to a targeting agent and methods of using such PBDs.
公开号:BR112012026801B1
申请号:R112012026801-5
申请日:2011-04-15
公开日:2021-05-04
发明作者:Peter Senter；Patrick Burke；Scott Jeffrey；Phillip Wilson Howard
申请人:Medimmune Limited；Seattle Genetics, Inc；
IPC主号:

专利说明:

[001] The present invention relates to targeted pyrrolobenzodiazepine (PBD) conjugates, in particular, pyrrolobenzodiazepine dimers that are conjugated to a targeting agent through the C2 position of one of the monomers. 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 PBD antitumor antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, several naturally occurring PBDs have been described, and more than 10 synthetic routes have been developed for a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abeimycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chykamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)) , DC-81 (Japanese Patent 58-180,487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)) , mazetramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., 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, et al. ., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., col., J. Antibiotics, 25,

[003] They differ in the number, type and position of the substituents, both in their aromatic A rings and in the C rings of pyrrole, and in the degree of saturation of the C ring. In ring B there is an imine (N=C), or an carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center responsible for alkylating DNA. All known natural products have an (S) configuration at the chiral C11a position, which provides them with a left-to-right rotation when viewed from the C-ring to the A-ring. isohelicity with the secondary groove of the B-form DNA, resulting in a small fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc Chem. Res., 19, 230-237 (1986)). The ability of PBDs to form an adduct in the secondary sulcus enables them to interfere with DNA processing, hence their use as antitumor agents.
The biological activity of these molecules can be potentiated by joining two PBD units together through their hydroxyl functionalities at C8/C' through a flexible alkylene linker (Bose, DS, et al., 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 sequence-selective DNA lesions, such as palindromic 5'-Pu-GATC-Py-3' strand crosslinking (Smellie, M., et al., Biochemistry, 42 , 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147), which is believed to be primarily responsible for its biological activity. An example of a PBD dimer, SG2000 (SJG-136):
(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, MC, et al., Cancer Research, 64, 6700-6706 (2004); Hartley, JA, et al., Cancer Research, 64, 6693-6699 (2004)).
[005] Due to the way in which these highly potent compounds act in the cross-linking of DNA, PBD dimers have been prepared symmetrically. That is, both dimer monomers are the same. This synthetic route provides direct synthesis, either by constructing the dimer portion of PBD simultaneously having already formed the dimer linkage, or by reacting the already constructed monomer portions of PBD with the dimer linking group. These synthetic approaches have limited the options for preparing target conjugates containing PBDs. Due to the observed potency of PBD dimers, however, there is a need for PBD dimers that are conjugable to target agents for use in targeted therapy. Disclosure of the Invention
[006] The present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein the PBD monomer has a substitute at the C2 position that provides an anchor for binding the compound to the targeting agent . The present invention also relates to Conjugates comprising dimers of PBDs conjugated to a targeting agent, wherein the PBD monomers of the dimer are different. One of the PBD monomers has a surrogate at the C2 position that provides an anchor for binding the compound to the target agent. The Conjugates described herein have potent cytostatic and/or cytotoxic activity against cells that express a target molecule, such as cancer cells or immune cells. These conjugates show good potency with reduced toxicity compared to drug compounds free of corresponding dimers.
[007] In some embodiments, the Conjugates have the following formula I: where L is a binding unit (i.e., a targeting agent), LU is a Ligand unit and D is a Drug unit comprising a dimer of PBD. The index p is an integer from 1 to 20. Consequently, Conjugates comprise a linker unit covalently linked to at least one Drug unit through a Linker unit. The binding unit, described more fully below, is a targeting agent that binds to a target moiety. The binding moiety can, for example, specifically bind a cellular component (a Cell Binding Agent) or target molecules of interest. Therefore, the present invention also provides methods for treating, for example, various cancers and autoimmune diseases. These methods include the use of Conjugates in which the binding moiety is a targeting agent that specifically binds to a target molecule. The binding moiety can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.
[008] In a first aspect, the Conjugates comprise a Conjugate of formula I (supra), wherein the Drug unit comprises a PBD dimer of the following formula II:
on what:
where A is an aryl group of C5-7, X is a group that can be activated for conjugation to the Linker unit, where X is selected from the group comprising: -O-, -S-, -C(O )O-, -C(O)-, -NHC(O)-, and -N(RN)-, wherein RN is selected from the group comprising H, C1-4 alkyl, and (C2H4O)mCH3 , where m is 1 to 3, 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 bond simple, O, S and NH and n is 1 to 3; or (ii) Q1 is -CH=CH-, and Q2 is a single bond; R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-alkylene of C1-3; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo; wherein R and R' are independently selected from optionally substituted C1-12 alkyl, optionally substituted C3-20 heterocyclyl and optionally substituted C5-20 aryl; R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn and halo; or: (a) R10 is H, and R11 is OH or ORA, where RA is C1-4 alkyl; (b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached; or (c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a pharmaceutically acceptable monovalent cation; R" is a C3-12 alkylene group whose chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, by 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, wherein, if R11 and R11' are SOzM, M may represent a pharmaceutically acceptable divalent cation.
[009] In a second aspect, the use of a Conjugate of formula I is provided for the manufacture of a drug for the treatment of a proliferative disease or autoimmune disease. In a third related aspect, the use of Formula I Conjugate is provided for the treatment of a proliferative disease or an autoimmune disease.
[0010] In another aspect, the use of a Conjugate of formula I to deliver a PBD dimer, or a salt or solvate thereof, at a target location is provided.
[0011] One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays that can be conveniently used to assess the activity offered by a particular compound are described in the examples below.
The term "proliferative disease" refers to an unwanted or uncontrolled cell proliferation of excessive or abnormal cells that is unwanted, such as neoplastic or hyperplastic growth, whether in vitro or in vivo.
[0013] Examples of proliferative conditions include, but are not limited to, benign, premalignant, and malignant cell proliferation, including, but not limited to, neoplasms and tumors (eg, histiocytoma, glioma, astrocytoma, osteoma), cancers ( eg 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 (eg, connective tissue) disorders, and atherosclerosis. Other cancers of interest include, but are not limited to, hematologic; malignancies 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.
[0014] Examples of autoimmune diseases include the following: rheumatoid arthritis, autoimmune demyelinating diseases (eg, multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis , glomerulonephritis, autoimmune hepatologic disease, inflammatory bowel disease (eg, Crohn's disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine insufficiency, Schmidt syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute hypocutaneous lupus erythematosus, parathyroidism , Dressler syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, anemia h emolytic, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia), female infertility, autoimmune and male autoimmune ankylosing, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, lung of farmer, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird breeders' lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, nodose erythema , pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, arthritis temporal, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, daytime, psoriasis, fetal erythroblastosis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal insufficiency.
[0015] In some modalities, the autoimmune disease is 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 (eg, atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve Th1 lymphocyte or Th2 lymphocyte disorders. In some embodiments, the autoimmune disorder is a T-cell mediated immune disorder.
[0016] In a fourth aspect, the present invention comprises a method of preparing the Conjugates of formula I.
The dimeric PBD compounds for use in the present invention are prepared by different strategies from those previously used in the production of symmetrical dimeric PBD compounds. In particular, the present inventors have developed a method that involves adding each aryl C2 substitute to a symmetrical PBD dimer core in separate process steps. Accordingly, a sixth aspect of the present invention provides a method of preparing a Conjugate of formula I, which comprises at least one of the steps of the method described herein. Brief Description of Figures
Figures 1-6 show the effect of the conjugates of the present invention on tumors. Definitions
[0019] When a trade name is used herein, the reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the branded product , unless otherwise indicated by the context. Liaison Agent and Targeting Agent
The terms "binding agent" and "targeting agent" as used herein refer to a molecule, for example, protein, polypeptide or peptide, that specifically binds to a target molecule. a full-length antibody, an antigen-binding fragment of a full-length antibody, another agent (protein, polypeptide, or peptide), which includes an antibody light and/or heavy chain variable region that specifically binds to target molecule, or an Fc fusion protein comprising an extracellular domain of a protein, peptide or polypeptide which binds to the target molecule and which is joined to an Fc region, domain or portion thereof of an antibody.
The terms "specifically binds" and "specific binding" refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (eg 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 its affinity for binding to a molecule. non-specific (eg BSA, casein), with the exception of the predetermined molecule or a closely related molecule. Pharmaceutically acceptable cations
Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.
[0023] The pharmaceutically acceptable cation can be inorganic or organic.
[0024]Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not imitated, alkali metal ions such as Na+ and K+. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca2+ and Mg2+. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e., NH4+) and substituted ammonium ions (eg, NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions 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 like lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+. Substitutes
[0025]The term "optionally substituted", as used in this document, refers to a head group which may be unsubstituted or which may be substituted.
[0026] Unless otherwise indicated, the term "substituted", as used herein, refers to a core group that contains one or more substitutes. The term "substitute" is used herein in the conventional sense and refers to a chemical moiety that is covalently attached to, or if appropriate, fused to, a head group. A wide variety of substitutes are well known, and methods for their formation and introduction into a variety of main groups are also well known.
[0027]Examples of substitutes are described in more detail below.
[0028]C1-12 Alkyl: The term "C1-12 alkyl", as used herein, refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having a 1 to 12 carbon atoms, which can be aliphatic or alicyclic, and which can be saturated or unsaturated (eg, partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the subclasses alkenyl, alkynyl, cycloalkyl, etc., discussed below.
[0029] 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) .
[0030]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).
[0031]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).
[0032]C2-12 Alkenyl: The term "C2-12 alkenyl", as used herein, refers to an alkyl group having one or more carbon-carbon double bonds.
[0033]Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2 ), isopropenyl (1-methylvinyl, -C(CH3)=CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).
[0034]C2-12 alkynyl: The term "C2-12 alkynyl", as used herein, refers to an alkyl group having one or more carbon-carbon triple bonds.
[0035]Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C=CH) and 2-propynyl (propargyl, -CH2-C=CH).
[0036]C3-12 Cycloalkyl: The term "C3-12 cycloalkyl", as used herein, refers to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing one hydrogen atom from an alicyclic ring atom of a cyclic (carbocyclic) hydrocarbon compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms .
[0037]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), cycloeptane (C7 ), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene ( C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and polycyclic saturated hydrocarbon compounds: norcarane (C7), norpinane (C7), norbornane (C7).
[0038] 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, which moiety which has from 3 to 20 ring atoms, of which 1 to 10 are ring heteroatoms. Preferably, each ring has 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms.
[0039] In this context, suffixes (eg C3-20, C3-7, C5-6, etc.) mean the number of atoms in the ring, or the range of number of atoms in the ring, whether carbon atoms , whether they are heteroatoms. For example, the term "C5-6 heterocyclyl" as used herein refers to a heterocyclyl group having 5 or 6 ring atoms.
[0040]Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from: N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (eg, 3-pyrroline, 2 ,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazol) (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), oxepin ( C7); S1: thyrane (C3), thiethane (C4), thiolane (tetrahydrothiophene) (C5), thiano (tetrahydrothiopyran) (C6), thiepane (C7); O2: dioxolane (C5), dioxane (C6), and dioxepane (C7); O3: trioxane (C6); N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazol) (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 oxathiane (thioxane) (C6); and, N1O1S1: oxathiazine (C6).
[0041]Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lithifuranose, ribofuranose, and xylofuranose, and pyranoses (C6), such as allopyranose, altropyranose, glycopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
[0042] C5-20 aryl: The term "C5-20 aryl", as used herein, refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, moiety this one has from 3 to 20 atoms in the ring. Preferably, each ring has 5 to 7 ring atoms.
[0043] In this context, suffixes (eg 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 carbon atoms , whether they are heteroatoms. For example, the term "C5-6 aryl" as used herein refers to an aryl group having 5 or 6 ring atoms.
[0044] The ring atoms can all be carbon atoms, as in the "carboaryl groups".
[0045] Examples of carboaryl groups include, but are not limited to, those derived from benzene (ie, phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).
[0046] Examples of aryl groups comprising fused rings, at least one of which is an aromatic ring, include, but are not limited to, indane-derived groups (eg, 2,3-dihydro-1H-indene) (C9), indene (C9), isoindene (C9), tetralin (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceantrene (C16).
[0047] Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroaryl groups". Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from: N1: pyrrole (azole) (C5), pyridine (azine) (C6); O1: furan (oxol) (C5); S1: thiophene (thiol) (C5); N1O1: oxazol (C5), isoxazol (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).
[0048]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) (eg adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxol (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S); C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), 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); and, C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxatin (O1S1), phenazine (N2), phenoxazine (N1O1), pheno- thiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
[0049] The aforementioned groups, either alone or part of another substitute, may themselves optionally be substituted with one or more groups selected from themselves and the additional substitutes listed below.
[0050]Halo: -F, -Cl, -Br, and -I.
[0051]Hydroxy: -OH.
[0052]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 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7 alkyl group.
[0053]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).
Acetal: -CH(OR1)(OR2), where R1 and R2 are independently acetal substitutes, for example, a C1-7 alkyl group, a C320 heterocyclyl group, or a C5-20 aryl group , preferably a C1-7 alkyl group, or, in the case of a "cyclic" acetal group, R1 and R2 taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached. , form a heterocyclic ring having 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, -CH(OMe)2, -CH(OEt)2, and -CH(OMe)(OEt).
[0055]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).
[0056] Ketal: -CR(OR1)(OR2), where R1 and R2 are as defined for acetals, and R is a substitute for a ketal other than hydrogen, 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 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).
[0057] Hemiketal: -CR(OH)(OR1), where R1 is as defined for hemiacetals, and R is a substitute for a hemiketal other than hydrogen, eg a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiketal groups include, but are not limited to, -C(Me)(OH)(OMe), -C(Et)(OH)(OMe), -C(Me)(OH)(OEt), and - C(Et)(OH)(OEt).
[0058]Oxo (keto, -one): =O.
[0059]Thione (thioketone): =S.
[0060]Imino (imine): =NR, where R is an imino substitute, e.g. 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.
[0061]Formyl (carbaldehyde, carboxaldehyde): -C(=O)H.
[0062]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 alkanoyl ), a C3-20 heterocyclyl group (also referred to as a C320 heterocyclylacyl), or a C5-20 aryl group (also referred to as a 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).
[0063]Carboxy (carboxylic acid): -C(=O)OH.
[0064]Thiocarboxylic acid (thiocarboxylic acid): -C(=S)SH.
[0065]Thiolocarboxylic acid (thiolocarboxylic acid): -C(=O)SH.
[0066]Thionocarboxylic acid (thionocarboxylic acid): -C(=S)OH.
[0067]Imidic acid: -C(=NH)OH.
[0068] Hydroxamic acid: -C(=NOH)OH.
[0069] Ester (carboxylate, carboxylic acid ester, oxycarbonyl); -C(=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 C5-20 alkyl group C1-7. 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.
[0070]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.
[0071]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.
[0072]Amino: -NR1R2, where R1 and R2 are independently amino substitutes, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7 alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a "cyclic" amino group, R1 and R2 taken together with the atom of nitrogen to which they are attached form a heterocyclic ring having 4 to 8 ring atoms. Amino groups can be primary (-NH2), secondary (-NHR1), or tertiary (-NHR1R2), and in the 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 cyclic amino groups include, but are not limited to, aziridine, azetidine, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
[0073] Starch (carbamoyl, carbamyl, aminocarbonyl, carboxamide); - C(=O)NR1R2, where R1 and R2 are independently amino substitutes, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3, and - C(=O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure, as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinecarbonyl.
[0074]Thioamido (thiocarbamyl): -C(=S)NR1R2, where R1 and R2 are independently amino substitutes, as defined for the amino groups. Examples of amido groups include, but are not limited to, -C(=S)NH2, -C(=S)NHCH3, -C(=S)N(CH3)2, and -C(=S)NHCH2CH3.
[0075] 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-aryl group 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, maleimidyl, and phthalimidyl:

[0076]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.
[0077]Ureido: -N(R1)CONR2R3, where R2 and R3 are independently amino substitutes, as defined for the amino groups, and R1 is a ureido substitute, eg hydrogen, a 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 ureido groups include, but are not limited to, -NHCONH2, -NHCONHEt, -NHCONHEt, -NHCONMe2, -NHCONEt2, -NMeCONH2, -NMeCONHMe, -NMeCONHEt, -NMeCONMe2, and -NMeCONHEt2.
[0078]Guanidino: -NH-C(=NH)NH2.
[0079] Tetrazolyl: a five-element aromatic ring having four nitrogen atoms and one carbon atom,

[0080]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, and =NEt.
[0081]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.
[0082]Nitro: -NO2.
[0083]Nitrous: -NO.
[0084]Azide: -N3.
[0085]Cyano (nitrile, carbonitrile): -CN.
[0086]Isocyan: -NC.
[0087]Cyanate: -OCN.
[0088]Isocyanate: -NCO.
[0089]Thiocyano(thiocyanate): -SCN.
[0090]Isothiocyan (isothiocyanate): -NCS.
[0091] Sulfidril (thiol, mercapto): -SH.
[0092]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.
[0093] Disulfide: -SS-R, wherein 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 group C1-7 alkyl (also referred to herein as C1-7 alkyl disulfide). Examples of C1-7 alkyl disulfide groups include, but are not limited to, -SSCH3 and -SSCH2CH3.
[0094] Sulphine (sulfinyl, sulfoxide): -S(=O)R, where R is a sulphine 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 sulphine groups include, but are not limited to, -S(=O)CH3 and -S(=O)CH2CH3.
[0095]Sulfone (sulfonyl): -S(=O)2R, where R is a sulfone substitute, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5 aryl group -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 (esil), -S( =O)2C4F9 (nonafyl), -S(=O)2CH2CF3 (tresyl), -S(=O)2CH2CH2NH2 (tauryl), -S(=O)2Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (naphsyl), and 5-dimethylamino-naphthalen-1-yl sulfonate (dansyl).
[0096] Sulfinic acid (sulfino): -S(=O)OH, -SO2H.
[0097] Sulphonic acid (sulfo): -S(=O)2OH, -SO3H.
[0098] Sulfinate (sulfinic acid ester): -S(=O)OR; where R is a sulfinate 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 sulfinate groups include, but are not limited to, -S(=O)OCH3 (methoxysulfinyl; methyl sulfinate) and -S(=O)OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).
[0099] Sulfonate (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 group C5-20 aryl, 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).
[00100]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.
[00101] Sulfonyloxy: -OS(=O)2R, where R is a sulfonyloxy 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 sulfonyloxy groups include, but are not limited to, -OS(=O)2CH3 (mesylate) and -OS(=O)2CH2CH3 (esylate).
[00102]Sulfate: -OS(=O)2OR; where R is a sulfate 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 sulfate groups include, but are not limited to, -OS(=O)2OCH3 and -SO(=O)2OCH2CH3.
[00103]Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): -S(=O)NR1R2, wherein R1 and R2 are independently amino substitutes, as defined for the 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.
[00104] Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): -S(=O)2NR1R2, where R1 and R2 are independently amino substitutes, as defined for the 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.
[00105]Sulfamino: -NR1S(=O)2OH, where R1 is an amino substitute as defined for the amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=O)2OH and -N(CH3)S(=O)2OH.
[00106] Sulphonamino: -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 group C3-20 heterocyclyl, 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.
[00107] Sulfinamino: -NR1S(=O)R, where R1 is an amino substitute, as defined for the amino groups, and R is a sulfinamino substitute, for example, a C1-7 alkyl group, a group C3-20 heterocyclyl, or a C5-20 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.
[00108]Phosphine (phosphine): -PR2, where R is a phosphine substitute, for example, -H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group , preferably -H, a C1-7 alkyl group, or a C520 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.
[00109]Phosphorus: -P(=O)2.
[00110]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.
[00111]Phosphonic acid (phosphono): -P(=O)(OH)2.
[00112]Phosphonate (phosphono 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.
[00113] Phosphoric acid (phosphonooxy): -OP(=O)(OH)2.
[00114] Phosphate (phosphonooxy ester): -OP(=O)(OR)2, where R is a phosphate 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 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.
[00115] Phosphorous acid: -OP(OH)2.
[00116]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 C520 aryl group, 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.
[00117]Phosphoramidite: -OP(OR1)-NR22, where R1 and R2are phosphoramidite substitutes, 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 phosphoramidite groups include, but are not limited to, -OP(OCH2CH3)-N(CH3)2, -OP(OCH2CH3)-N(i-Pr)2, and -OP(OCH2CH2CN)-N(i-Pr )two.
[00118] Phosphoramidate: -OP(=O)(OR1)-NR22, where R1 and R2 are phosphoramidate substitutes, for example, -H, a C1-7 alkyl group (optionally substituted), 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 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
[00119]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, of a hydrocarbon compound having 3 to 12 carbon atoms (unless otherwise noted), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the subclasses alkenylene, alkynylene, cycloalkylene, etc., discussed below.
[00120]Examples of linear saturated C3-12 alkylene groups include, but are not limited to, -(CH2)n-, where n is an integer from 3 to 12, for example, -CH2CH2CH2- (propylene), -CH2CH2CH2CH2- (butylene), -CH2CH2CH2CH2CH2- (pentylene) and -CH2CH2CH2CH2CH2CH2CH2- (heptylene).
[00121]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-.
[00122]Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, -CH=CH-CH2-, -CH2-CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH =CH-CH2-CH2-, -CH=CH-CH2-CH=CH-, -CH=CH-CH2-CH2-CH=CH-, and -CH2-CEC-CH2-.
[00123]Examples of partially unsaturated branched C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2 -, -CH=CH-CH(CH3)- and -CEC-CH(CH3)-.
[00124]Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (eg, cyclopent-1,3-ylene), and cyclohexylene (eg, cycloex-1,4-ylene).
[00125]Examples of alicyclic partially unsaturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (eg, 4-cyclopenten-1,3-ylene), cyclohexenylene (for example, 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).
[00126] Oxygen Protecting Group: The term "oxygen protecting group" refers to a moiety 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 silyl ethers (eg TMS, TBDMS), substituted methyl ethers (eg THP) and esters (eg acetate).
[00127]Carbamate nitrogen protecting group: The term "carbamate nitrogen protecting group" refers to a moiety that masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure:
where R'10 is 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 herein by reference.
[00128]Hemiaminal nitrogen protecting group: The term "hemiaminal nitrogen protecting group" refers to a group having the following structure:
where R'10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, TW and Wuts, GM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein document by reference. Detailed Description of the Invention
[00129] The present invention provides Conjugates comprising a PBD dimer connected to a linker unit through a linker unit. In one embodiment, the Ligand unit includes an Elongation unit (A), a Specificity unit (L1), and a Spacer unit (L2). The Linker unit is connected at one end to the Linker unit and at the other end to the PBD dimer compound.
[00130] In one aspect, such Conjugate is shown below in formula Ia: L-(A1a-L1s-L2y-D)p(Ia) wherein: L is the linker unit; and -A1a-L1s-L2y- is a Ligand (LU) unit, where: -A1- is an Elongation unit, a is 1 or 2, L1 - is a Specificity unit, s is an integer that varies from 1 to 12, - L2- is a Spacer unit, y is 0, 1 or 2; - D is a PBD dimer; and p is from 1 to 20.
[00131]The drug charge is represented by p, the number of drug molecules per binding unit (for example, an antibody). Drug loading can range from 1 to 20 Drug Units (D) per binding Unit (eg Ab or mAb). For compositions, p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20.
[00132] In some embodiments, p is from about 1 to about 8 drug units per binder unit. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is from about 2 to about 8 Drug units per binding unit. In some embodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4 units of Drug per unit binding. In some embodiments, p is about 2, about 4, about 6, or about 8 units of Drug per unit binding.
[00133] The average number of Drug units per binding unit in a preparation from a conjugation reaction can be characterized by conventional means such as mass spectroscopy, ELISA and HPLC assay. The quantitative distribution of Conjugates in terms of p can also be determined. In some cases, separation, purification and characterization of homogeneous Conjugates, where p is a given value, from Conjugates with other drug loadings can be achieved by means such as reverse phase HPLC or electrophoresis.
[00134] In another aspect, such Conjugate is shown below in formula Ib:
also illustrated as: L - (A1a-L2y (-L1s) -D)p(Ib) where: L is the linker moiety; and - A1a-L1s(L2y)- is a Linker unit (LU), where: - A1- is an Elongation unit linked to an Elongation unit (L2), a is 1 or 2, L1 - is a unit of Specificity linked to an Elongation unit (L2), s is an integer ranging from 0 to 12, -L2- is a Spacer unit, y is 0, 1 or 2; -D is a PBD dimer; ep is from 1 to 20. Preferences
[00135] The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. Preferences can be combined together or in any combination.
[00136] 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.
[00137] In one embodiment, the binding moiety (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 connection to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is an Elongation unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit , which is a covalent bond, a self-destructive group or together with -OC(=O)- forms a self-destructive group, and L2 is optional.
[00138] In another embodiment, the binding 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: CBA — A1a — L1s — L2y — * where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a unit of Specificity, A1 is a Elongation unit connecting L1 to Cell Bonding Agent, L2 is a Spacer unit which is a covalent bond or a self-destructive group, and a is 1 or 2, s is 0, 1 or 2, and y is 0 or 1 or 2 .
[00139] In the modalities illustrated above, L1 may be a cleavable Specificity unit, and may be referred to as a "trigger" which, when cleaved, activates a self-destructive group (or self-destructive groups) L2, when one 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 Drug unit (D).
[00140] In another embodiment, the binding 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 Drug linkage point (D), CBA is the Cellular Bonding Agent, L1 is a Specificity unit connected to L2, A1 is an Elongation unit connecting L2 to the Cellular Bonding Agent, L2 is a group self-destructive, and a is 1 or 2, s is 1 or 2, and y is 1 or 2.
[00141] In the various modalities discussed here, the nature of L1 and L2 can vary widely. These groups are chosen based on their characteristics, which may 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 such that it is cleaved by the action of enzymes present at the target site (eg, the target cell). L1 units that are cleavable by changes in pH (eg, unstable base or acid), temperature, or after irradiation (eg, photolabile) can also be used. L1 units that are cleavable under conditions of reduction or oxidation may also find use in Conjugates.
[00142] In some embodiments, L1 may comprise an amino acid or a contiguous sequence of amino acids. The amino acid sequence can be the target substrate for an enzyme.
[00143] 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 cathepsin.
[00144]In a modality, L2 is present and together with -C(=O)O- forms a self-destructive group or self-destructive groups. In some embodiments, -C(=O)O- is also a self-destructive group.
[00145] In one modality, where L1 is cleavable by the action of an enzyme and L2 is present, the enzyme cleaves the bond between L1 and L2, whereby the self-destructive group(s) ) release the Medicine unit.
[00146]L1 and L2, where present, 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-, -NHC(=O)NH, and -O- (a glycosidic bond).
[00147] An amino group of L1 that connects to L2 may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example, a lysine amino acid side chain.
[00148] A carboxyl group of L1 that connects to L2 can be the C-terminus of an amino acid or can be derived from a carboxyl group of an amino acid side chain, for example, an amino acid side chain of glutamic acid .
[00149]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.
[00150] In one modality, -C(=O)O- and L2 together form the group:
where the asterisk indicates the point of connection to the Medicine unit, the wavy line indicates the point of connection to L1, Y is -N(H)-, -O-, -C(=O)N(H)- or - C(=O)O-, and en is 0 to 3. The phenylene ring is optionally substituted with one, two or three substitutes as described herein.
[00151] In one embodiment, Y is NH.
[00152] In one embodiment, n is 0 or 1. Preferably, n is 0.
[00153] Where Y is NH and n is 0, the self-destructive group can be referred to as the p-aminobenzylcarbonyl (PABC) linker.
[00154] The self-destruct group will allow the release of the Drug unit (ie, the asymmetric PBD) when a remote location in the ligand is activated, proceeding along the lines shown below (for n=0):
where the asterisk indicates the Drug binding, L* is the activated form of the remaining portion of the ligand and the Drug unit released is not shown. These groups have the advantage of separating the medicine activation site.
[00155]In another modality, -C(=O)O- and L2 together form a group selected from:
where the asterisk, wavy line, Y, and n are as defined above. Each phenylene ring is optionally substituted with one, two or three substitutes as described herein. In one embodiment, the phenylene ring having the Y-substitute is optionally substituted and the phenylene ring not having the Y-substitute is unsubstituted.
[00156] In another modality, -C(=O)O- and L2 together form a group selected from:
where the asterisk, wavy line, Y, and n are as defined above, E is O, S, or NR, D is N, CH, or CR, and F is N, CH, or CR.
[00157] In one modality, D is N.
[00158] In one embodiment, D is CH.
[00159]In one modality, E is O or S.
[00160]In one modality, F is CH.
[00161] In a preferred embodiment, the covalent bond between L1 and L2 is an unstable (e.g., cleavable) cathepsin bond.
[00162] In one embodiment, L1 comprises a dipeptide. The amino acids in the dipeptide can be any combination of natural amino acids and unnatural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the ligand is an unstable cathepsin ligand, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide is then a recognition site for cathepsin.
[00163] In one embodiment, the group -X1-X2- in dipeptide, -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 is citrulline. In such a dipeptide, -NH- is the amino group of X1, and CO is the carbonyl group of X2.
[00164] Preferably, the group -X1-X2- in dipeptide, -NH-X1-X2-CO-, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, - Ala-Lys-, and -Val-Cit-.
More preferably, the -X1-X2- group in the dipeptide, -NH-X1-X2-CO-, is -Phe-Lys-, Val-Cit or -Val-Ala-.
[00166]Other dipeptide combinations of interest include: -Gly-Gly-, -Pro-Pro-, and -Val-Glu-.
[00167] Other dipeptide combinations can be used, including those described by Dubowchik et al., which are incorporated herein by reference.
[00168] 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 dipeptide sequence comprising a Lys residue protected by Boc side chain is cleavable by cathepsin.
Protective 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.
[00170]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.
[00171]In one modality, -X2- is indirectly connected to the Medicine unit. In such a modality, the Spacer unit L2 is present.
[00172] In one embodiment, the dipeptide is used in combination with a self-destructive group(s) (the Spacer unit). The self-destructive group(s) can be connected to -X2-.
[00173]Where a self-destruct group is present, -X2- is directly connected to the self-destruct group. In one modality, -X2- is connected to the Y group of the self-destructive group. Preferably, the -X2-CO- group is connected to Y, where Y is NH.
[00174]-X1- is directly connected to A1. In one mode, -X1- is directly connected to A1. Preferably, the NH-X1- group (the amino terminus of X1) is connected to A1. A1 may comprise the -CO- functionality to thereby form an amide bond with -X1-.
[00175] In one modality, L1 and L2 together with -OC(=O)- comprise the group -X1-X2-PABC-. The PABC group is directly connected to the Medicine unit. In one example, the self-destructive group and the dipeptide together form the -Phe-Lys-PABC- group, which is illustrated below:
where the asterisk indicates the point of attachment to the Drug 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.
[00176] Alternatively, the self-destructive group and the dipeptide together form the -Val-Ala-PABC- group, which is illustrated below:
where the asterisk and wavy line are as defined above.
[00177] In another modality, L1 and L2 together with -OC(=O)- represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group, and E is a group that is susceptible to cleavage to thereby activate a group self-destructive.
[00178]E 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 (eg β-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a β-glucuronidase.
[00179]The Y group can be a covalent bond.
[00180]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-.
[00181] The Y group is preferably -NH-, -CH2_, -O-, and -S-.
[00182] In some modalities, L1 and L2 together with -OC(=O)- represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A, Y is a covalent bond or a functional group, and E is glucuronic acid (eg, β-glucuronic acid). Y is preferably a functional group selected from -NH-.
[00183] In some modalities, L1 and L2 together represent:
where the asterisk indicates the point of attachment to the remainder of L2 or the Medicine unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group, and E is glucuronic acid (eg, β- acid glucuronic). Y is preferably a functional group selected from -NH-, -CH2_, -O-, and -S-.
[00184] In some additional embodiments, Y is a functional group as defined above, the functional group is linked to the amino acid, and the amino acid is linked to the A1 Elongation unit. In some embodiments, the amino acid is β-alanine. In such an embodiment, the amino acid is equivalently considered part of the Elongation unit.
[00185]The Specificity unit L1 and the binding unit are indirectly connected through the Elongation unit.
[00186]L1 and A1 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-.
[00187] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one embodiment, n is 5.
[00188] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one embodiment, n is 5.
[00189] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00190] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00191] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one embodiment, n is 5.
[00192] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one embodiment, n is 5.
[00193] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00194] In one modality, the A1 group is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00195] In one embodiment, the connection between the linker unit and A1 is through a thiol residue of the linker unit and a maleimide group of A1.
[00196] In one modality, the connection between the binding unit and A1 is:
where the asterisk indicates the point of attachment to the remainder of A1, L1, L2 or D, and the wavy line indicates the point of attachment to the remainder of the linker unit. In this embodiment, the S atom is typically derived from the linker moiety.
[00197] In each of the above embodiments, an alternative functionality can be used in place of the malemide-derived group shown below:
where the wavy line indicates the point of attachment to the linking unit as above, and the asterisk indicates attachment to the remaining portion of the A1 group, or to L1, L2 or D.
[00198] In one embodiment, the maleimide-derived group is replaced with the group:
where the wavy line indicates the point of attachment to the linker unit, and the asterisk indicates attachment to the remaining portion of the A1 group, or to L1, L2 or D.
[00199] In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the linker moiety (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=.
[00200] In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the linker unit, is selected from:
where the wavy line indicates both the point of attachment to the linking unit and the attachment to the remainder of the A1 group, and the asterisk indicates the other of the point of attachment to the linking unit or the attachment to the remainder of the A1 group.
[00201]Other groups suitable for connecting L1 to Cell Linker are described in WO 2005/082023.
[00202] In one embodiment, the Elongation unit A1 is present, the Specificity unit L1 is present, and the Spacer unit L2 is absent. In this way, L1 and the Medicine unit are directly connected through a link. Equivalently in this modality, L2 is a bond.
[00203]L1 and D 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-.
[00204] In one embodiment, L1 and D are preferably connected by a bond selected from: -C(=O)NH-, and -NHC(=O)-.
In one embodiment, L1 comprises a dipeptide and one end of the dipeptide is linked to D. As described above, the amino acids in the dipeptide can be any combination of natural amino acids and unnatural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the ligand is an unstable cathepsin ligand, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide, then, is a recognition site for cathepsin.
[00206] In one embodiment, the group -X1-X2- in dipeptide, -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 is citrulline. In such a dipeptide, -NH- is the amino group of X1, and CO is the carbonyl group of X2.
[00207] Preferably, the group -X1-X2- in dipeptide, -NH-X1-X2-CO-, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, - Ala-Lys-, and -Val-Cit-.
More preferably, the -X1-X2- group in the dipeptide, -NH-X1-X2-CO-, is -Phe-Lys- or -Val-Ala-.
[00209] Other dipeptide combinations of interest include: -Gly-Gly-, -Pro-Pro-, and -Val-Glu-.
[00210]Other dipeptide combinations can be used, including those described above.
[00211] In one modality, L1-D is: - -NH-X1-X2-CO-NH- * where -NH-X1-X2-CO is the dipeptide, -NH- is part of the Medicine unit, the asterisk indicates the point of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remainder of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
[00212] In one embodiment, the dipeptide is valine-alanine and L1-D is:
where the asterisk, -NH-, and the wavy line are as defined above.
[00213] In one embodiment, the dipeptide is phenylalanine-lysine and L1-D is:
where the asterisk, -NH-, and the wavy line are as defined above.
[00214] In one embodiment, the dipeptide is valine-citrulline.
[00215] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one embodiment, n is 5.
[00216] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one embodiment, n is 5.
[00217] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00218] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, more preferably 3 or 7.
[00219] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one modality, n is 5.
[00220] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the binding unit, and n is 0 to 6. In one modality, n is 5.
[00221] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00222] In one modality, the A1-L1 groups are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10 , 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00223] In one modality, the L-A1-L1 groups are:
where the asterisk indicates the point of attachment to D, S is a sulfur group of the linker unit, the wavy line indicates the point of attachment to the rest of the linker unit, and n is 0 to 6. In one embodiment, n is 5.
[00224] In one modality, the L-A1-L1 groups are:
where the asterisk indicates the point of attachment to D, S is a sulfur group of the linker unit, the wavy line indicates the point of attachment to the remainder of the linker unit, and n is 0 to 6. In one embodiment, n is 5.
[00225] In one modality, the L-A1-L1 groups are:
where the asterisk indicates the point of attachment to D, S is a sulfur group of the linker unit, the wavy line indicates the point of attachment to the remainder of the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment , n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00226] In one modality, the L-A1-L1 groups are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the linker unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, more preferably 4 or 8.
[00227] In one modality, the L-A1-L1 groups 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 binding unit, and n is 0 to 6. In one mode, n is 5.
[00228] In one modality, the L-A1-L1 groups 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 binding unit, and n is 0 to 6. In one mode, n is 5.
[00229] In one modality, the L-A1-L1 groups 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 linker unit, n is 0 or 1, m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00230] In one modality, the L-A1-L1 groups 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 linker unit, n is 0 or 1, m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, more preferably 4 or 8.
[00231] In one modality the Elongation unit is an acetamide unit, having the formula:
where the asterisk indicates the point of attachment to the remainder of the Elongation unit, L1 or D, and the wavy line indicates the point of attachment to the binding unit.
[00232] In other embodiments, Binding-Drug compounds are provided for conjugation to a binding unit. In one embodiment, the Ligand-Drug compounds are designed to bind to a Cellular Binding Agent.
[00233] In one modality, the Drug-Binder compound has the formula:
where the asterisk indicates the point of attachment to the Drug unit, G1 is an Elongation group (A1) to form a connection to a bonding unit, L1 is a Specificity unit, L2 (the Spacer unit) is a covalent bond or together with -OC(=O)- forms a self-destructive group(s).
[00234] In another embodiment, the Drug-Binder compound has the formula: G1-L1-L2- * where the asterisk indicates the point of connection to the Drug unit, G1 is an Elongation unit (A1) to form a connection to a linker unit, L1 is a Specificity unit, L2 (the Spacer unit) is a covalent bond or a self-destructive group(s).
[00235]L1 and L2 are as defined above. References to the connection to A1 can be constructed here as referring to a connection to G1.
[00236] In one embodiment, where L1 comprises an amino acid, the side chain of that amino acid may be protected. Any suitable protective 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 can be orthogonal to other protecting groups on the molecule, where present.
[00237] Suitable protective groups for amino acid side chains include those groups described in the Novabiochem Catalog 2006/2007. Protective groups for use in an unstable cathepsin ligand are also discussed in Dubowchik et al.
[00238] 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 protecting group. A Boc protecting group is most preferred.
[00239]Functional group G1 forms a connecting group when reacting with a linker unit (for example, a Cell Binding Agent).
[00240] In one embodiment, the functional group G1 is or comprises an amino, carboxylic acid, hydroxy, thiol, or maleimide group for reaction with an appropriate group on the linker moiety. In a preferred embodiment, G1 comprises a maleimide group.
[00241] In one embodiment, the G1 group is 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.
[00242] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one modality, n is 5.
[00243] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one modality, n is 5.
[00244] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
[00245] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
[00246] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one modality, n is 5.
[00247] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one modality, n is 5.
[00248] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
[00249] In one modality, the G1 group is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
[00250] In each of the above modalities, an alternative functionality can be used in place of the maleimide group shown below:
where the asterisk indicates binding to the remaining portion of the G group.
[00251] In one embodiment, the maleimide-derived group is replaced with the group:
where the asterisk indicates binding to the remaining portion of the G group.
[00252] In one embodiment, the maleimide group is replaced with a group selected from: -C(=O)OH, -OH, -NH2, -SH, -C(=O)CH2X, where X is Cl, Br or I, -CHO, -NHNH2 -C=CH, and -N3 (azide).
[00253] In one embodiment, L1 is present, and G1 is -NH2, -NHMe, -COOH, -OH or -SH.
[00254] In a modality, where L1 is present, G1 is -NH2 or -NHMe. Either group can be the N-terminus of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is -NH2, and L1 is an amino acid sequence -X1-X2-, as defined above.
[00256]In one modality, L1 is present and G1 is COOH. This group can be the C-terminus of an L1 amino acid sequence.
[00257]In one modality, L1 is present and G1 is OH.
[00258]In one modality, L1 is present and G1 is SH.
[00259]Group G1 can be convertible from one functional group to another. In one modality, L1 is present and G1 is -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 (eg, N-succinimide forms) of those G1 groups comprising maleimide shown above.
[00260]The G1 group can therefore be converted to a functional group that is more suitable for reaction with a linker moiety.
[00261] As noted above, in one modality, L1 is present and G1 is -NH2, -NHMe, -COOH, -OH or -SH. In an additional embodiment, these groups are provided in a chemically protected form. The chemically protected form is therefore a precursor to the Linker which is provided with a functional group.
[00262]In one embodiment, G1 is -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:
[00263] Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.
[00264] Preferably, where G1 is -NH2, it is protected with an Alloc or Fmoc group.
[00265] In one modality, where G1 is -NH2, it is protected with an Fmoc group.
[00266]In one embodiment, the protective group is the same as the carbamate protective group of the capping group.
[00267] In one embodiment, the protective group is not the same as the carbamate protective group of the capping group. In this embodiment, it is preferable that the protective group be removable under conditions that do not remove the carbamate protective group from the capping group.
[00268]The chemical protecting group can be removed to provide a functional group to form a connection to a linker unit. Optionally, this functional group can then be converted to another functional group as described above.
[00269] In one embodiment, the active group is an amine. This amine is preferably the N-terminal amine of a peptide, and may be the N-terminal amine of preferred dipeptides of the invention.
[00270]The active group can be reacted to provide the functional group that is intended to form a connection to a linker unit.
[00271] In other embodiments, the Ligand unit is a precursor to the Ligand unit having an active group. In this modality, the Ligand unit comprises the active group, which is protected by means of a protection group. The protective group can be removed to provide the Linker unit having an active group.
[00272] Where the active group is an amine, the protecting group may be an amine protecting group, such as those described in Green and Wuts.
[00273] The protecting group is preferably orthogonal to other protecting groups, where present, in the Linker unit.
[00274] In one embodiment, the protective group is orthogonal to the capping group. In this way, the active group protective group is removable retaining the capping group. In other embodiments, the protective group and capping group are removable under the same conditions as those used to remove the capping group.
[00275] In one modality, the Binder unit is:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of the Ligand unit, as applicable, or the point of attachment to G1. Preferably, the wavy line indicates the point of attachment to G1.
[00276] In one modality, the Linker unit is:
where the asterisk and wavy line are as defined above.
[00277]Other functional groups suitable for use in forming a connection between L1 and the Cell Binding Agent are described in WO 2005/082023. binding unit
The binding moiety 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 linker moiety can be a protein, polypeptide or peptide. In some embodiments, the linker moiety can be a cyclic polypeptide. These Linker moieties may include antibodies or a fragment of an antibody that contains at least one target molecule binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target .
[00279]Examples of linker units include those agents described for use in WO 2007/085930, which is incorporated herein.
[00280] In some embodiments, the binding unit is a Cell Binding Agent that binds to an extracellular target in a cell. Such 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 can 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).
[00281] In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; fully human antibody; or a single chain antibody. In one embodiment, the antibody is a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab', F(ab')2 and Fv fragments.
The antibody may be a diabody, a domain antibody (DAB) or a single chain antibody.
[00283] In one embodiment, the antibody is a monoclonal antibody.
[00284] 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 antigens. Examples of such antigens known in the art include, but are not limited to, those tumor associated antigens defined in WO 2005/082023. See, for example, pages 41-55.
[00285] In some embodiments, Conjugates are designed to target tumor cells through their cell surface antigens. Antigens can be cell surface antigens that are over-expressed or expressed at abnormal cell types or times. Preferably, the target antigen is expressed only on proliferating cells (preferably tumor cells); however, this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between healthy and proliferative tissue.
[00286] Antibodies have been raised to target specific tumor-related antigens including:
[00287] Crypto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (Prostate Specific Membrane Antigen), BCMA, E-Selectin, EphB2, Melanotransferin, Muc16 and TMEFF2.
[00288]The Linker unit is connected to the Linker unit. In one embodiment, the linker unit is connected to A, where present, of the linker unit.
[00289] In one embodiment, the connection between the binding unit and the binding unit is through the thioether bond.
[00290] In one embodiment, the connection between the linker unit and the linker unit is through the disulfide bond.
[00291] In one embodiment, the connection between the binding unit and the binding unit is through an amide bond.
[00292] In one embodiment, the connection between the binding unit and the binding unit is via an ester bond.
[00293] In one embodiment, the connection between the linker unit and the linker is formed between a thiol group of a cysteine residue of the linker unit and a maleimide group of the linker unit.
[00294]The cysteine residues of the linker unit may be available for reaction with the functional group of the linker unit to form a connection. In other embodiments, for example, where the linker moiety is an antibody, the antibody's thiol groups may participate in interchain disulfide bonds. These interchain bonds can be converted to free thiol groups by, for example, treating the antibody with DTT prior to reaction with the functional group of the Linker moiety.
[00295] In some embodiments, the cysteine residue is one introduced into the light or heavy chain of an antibody. Positions for insertion of cysteine by substitution into antibody light or heavy chains include those described in United States Published Application No. 2007-0092940 and International Patent Publication WO2008070593, which are incorporated herein. Treatment Methods
The Conjugates 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 Conjugate of formula I. The term "therapeutically effective amount" is an amount sufficient to show benefit to a patient. Such benefit may be at least the improvement of at least one symptom. The actual amount of a Conjugate administered and the rate and course of time of administration will depend on the nature and severity of what is being treated. Prescription of treatment, eg decisions about dosage, is within the responsibility of general practitioners and other physicians.
[00297] 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 ranges 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 embodiments, the amount of Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose. In some embodiments, the amount of Conjugate administered ranges from about 0.01 to about 1 mg/kg per dose.
[00298] A Conjugate can be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the condition being treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, for example, medications; surgery; and radiation therapy.
[00299] Pharmaceutical compositions according to the present invention, and for use according to the present invention, may comprise, in addition to the active ingredient, ie, a Conjugate of formula I, an excipient, vehicle, buffer, stabilizer 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 may be oral, or by injection, eg, cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. A tablet can comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule can comprise a solid carrier such as gelatin.
[00301] For intravenous, cutaneous or subcutaneous injection, or injection at the site of the condition, 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 Sodium Chloride Injection, Ringer's Injection, Lacquered Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as required. Includes Other Forms
[00302] Unless otherwise indicated, 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 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
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound (the Conjugate), for example, a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berg, et al., J. Pharm. Sci., 66, 1-19 (1977).
[00304]For example, if the compound is anionic, or has a functional group that might be anionic (for example, the -COOH might 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 ions 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 ion (i.e., NH4+) and substituted ammonium ions (eg, NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions 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 ion is N(CH3)4+.
[00305] If the Conjugate is cationic, or has a functional group that might be cationic (eg -NH2 can be -NH3+), then a salt can be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorus.
[00306] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetioxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic , fumaric, glycceptonic, glyconic, glutamic, glycolic, hydroxymaleic, carboxylic hydroxynaphthalene, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, propionic, phenyl, panionic, pyruvic, salicylic, stearic, succinic, sulphanilic, 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
[00307] It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the Conjugate(s). The term "solvate" is used in this document in the conventional sense to refer to a complex of solute (eg, Active Conjugate, Salt of Active Conjugate) and solvent. If the solvent is water, the solvate may conveniently be referred to as a hydrate, for example a monohydrate, a dihydrate, a trihydrate, etc. Carbinolamines
[00308] The invention includes the Conjugates where a solvent joins through the imine bond of the PBD portion, which is illustrated below to a PBD monomer, where the solvent is water or an alcohol (RAOH, where RA is alkyl of C1-4):

[00309] These forms can be called the carbinolamine and carbinolamine ether forms of PBD. The balance of these equilibria depends on the conditions under which the compounds are found, as well as on the nature of the portion itself.
[00310] These particular compounds can be isolated in solid form, for example, by lyophilization. isomers
[00311] Certain compounds may exist in one or more geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis and trans forms; E and Z shapes; c, t and r forms; endo and exo forms; R, S, and meso forms; D and L shapes; d and l shapes; (+) and (-) forms; keto, enol, and enolate forms; syn and anti forms; syncline and anticlinal forms; α and β forms; axial and equatorial shapes; boat, chair, spiral, envelope, and half chair shapes; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").
[00312] Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (ie, isomers that differ in the bonds between atoms, rather than merely by the position of 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 structurally isomeric forms falling within that class (eg, C1-7 alkyl includes n-propyl and isopropyl; butyl includes n-, a iso-, to sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
[00313] The above-mentioned exclusion does not refer to tautomeric forms, for example, the keto, enol, and enolate forms, as, for example, in the following tautomeric pairs: keto/enol (shown below), imine/enamine, amide/ imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/acinitro.

[00314] Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. 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.
[00315] Unless otherwise indicated, a reference to a particular Conjugate includes all such isomeric forms, including racemic (wholly or partially) and other mixtures thereof. Methods for the preparation (eg, asymmetric synthesis) and separation (eg, fractional crystallization and chromatographic means) of such isomeric forms are known in the art or are readily obtained by adaptation of the methods taught in this document, or from known methods, in a known manner. General synthetic routes
The synthesis of PBD dimer compounds is discussed extensively in the following references, which discussions are incorporated herein 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
[00317] The Conjugates of the present invention, in which R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached, can be synthesized from a compound of Formula 2 compound:
where R2, R6, R7, R9, R6', R7', R9', R12, X, X' and R" are as defined for compounds of formula III, ProtN is a nitrogen protecting group for synthesis and ProtO is a protected oxygen group for synthesis or an oxo group, by deprotection of the imine bond by standard methods.
[00318] The compound produced can be in its carbinolamine or carbinolamine ether form, 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 palladium to remove the N10 protecting group, followed by elimination of the oxygen protecting group for synthesis. If ProtN is Troc and ProtO is an oxygen protecting group for the synthesis, then deprotection is carried out using a Cd/Pd pair 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 SEM protecting group. , followed by the elimination of water. Reduction of the compound of compounded Formula 2 can be carried out, for example, via lithium tetraborohydride, while a suitable means of removing the SEM protecting group is treatment with silica gel.
[00319] Compounds of compound formula 2 can be synthesized from a compound of compound formula 3a:
where R2, R6, R7, R9, R6', R7', R9', X, X' and R" are as defined for compounds of compound formula 2, by coupling an organometallic derivative comprising R12, such as a derivative of organoboron. The organoboron derivative can be a boronate or boronic acid.
[00320] Compounds of compound formula 2 can be synthesized from a compound of compound formula 3b:
R" are as defined for compounds of compound formula 2, by coupling an organometallic derivative comprising R2, such as an organoboron derivative. The organoboro derivative can be a boronate or boronic acid.
[00321] Compounds of compound 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 compounds of compound formula 2, by coupling approximately a single equivalent (eg, 0.9 or 1 to 1.1 or 1.2) of an organometallic derivative, such as an oroganoboron derivative, comprising R2 or R12.
[00322] The couplings described above are normally carried out in the presence of a palladium catalyst, for example, Pd(PPh3)4, Pd(OCOCH3)2, PdCl2, or Pd2(dba)3. Coupling can be carried out under standard conditions, or it can also be carried out under microwave conditions.
[00323]The two coupling steps are normally carried out sequentially. They can be carried out 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 usually required after the second coupling step. Purification of the compound from unwanted by-products can be carried out by column chromatography or ion exchange separation.
The synthesis of compounds of compound formula 4, wherein ProtO is an oxo group and ProtN is SEM, is described in detail in WO 00/12508, which is incorporated herein by reference. In particular, reference is made to scheme 7 on page 24, in which the above-mentioned compound is designated as intermediate P. This method of synthesis is also described in WO 2004/043963.
[00325] The synthesis of compounds of compound formula 4, wherein ProtO is a protected oxygen group for the synthesis, is described in WO 2005/085251, which synthesis is incorporated herein by reference.
[00326] Compounds of formula I, wherein R10 and R10' are H and R11 and R11' are SOzM, can be synthesized from compounds of formula I, wherein R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached, by addition of the appropriate bisulfite salt or 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 Synthesis
[00327] 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 hemi-aminal nitrogen protecting groups.
[00328] Carbamate nitrogen protecting groups have the following structure:
where R'10 is 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 herein by reference. .
Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.
[00330] Other possible groups are nitrobenzyloxycarbonyl (eg 4-nitrobenzyloxycarbonyl) and 2-(phenylsulfonyl)ethoxycarbonyl.
[00331] Protecting groups that can be removed with palladium catalyst are not preferred, eg Alloc.
[00332] The hemiaminal nitrogen protecting groups have the following structure:
where R'10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, TW and Wuts, GM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein document by reference. The groups disclosed herein can be applied to compounds for use in the present invention. Such groups include, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro or BOM substituted with methoxy and Cl3CCH2OCH2-. Protected Oxygen Group for Synthesis
[00333] 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 herein by reference.
Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates.
[00335]Preferred oxygen shielding groups include acetates, TBS and THP. Additional Preferences
[00336] The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. Preferences can be combined together in any combination.
[00337] In some embodiments, R6', R7', R9', R10', R11' and Y' are preferably the same as R6, R7, R9, R10, R11 and Y, respectively. Dimer bond
[00338]Y and Y' are preferably O.
[00339]R" is preferably a C3-7 alkylene group with no substitute. More preferably, R" is a C3, C5 or C7 alkylene. R6 to R9
[00340]R9 is preferably H.
[00341]R6 is preferably selected from H, OH, OR, SH, NH2, nitro and halo, and is more preferably H or halo, and most preferably is H.
[00342]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, wherein R is preferably selected from optionally substituted C1-7 alkyl, 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 is a C5-6 aryl group (eg phenyl). Particularly preferred 7-position substitutes are OMe and OCH2Ph.
[00343]These preferences apply to R9’, R6’ and R7’, respectively. R2
[00344]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. In other embodiments, A is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
[00345]X is a group selected from the list comprising: O-, -S-, -C(O)O-, -C(O)-, -NH(C=O)- and -N(RN )-, wherein RN is selected from the group comprising H and C1-4 alkyl. X may preferably be: -O-, -S-, -C(O)O-, -NH(C=O)- or -NH-, and may more preferably be: O-, -S-, or -NH -, and most preferably it is -NH-.
[00346]Q2-X can be on any of the available ring atoms of the aryl group of C5-7, but is preferably on a ring atom that is not adjacent to the bond to the remainder of the compound, ie, it is preferably β or y binding to the remainder of the compound. Therefore, where the aryl group of C5-7 (A) is phenyl, the substitute (Q2-X) is preferably in the meta or para positions, and more preferably is in the para position.
[00347]In some modes, Q1 is a simple link. In these modalities, Q2 is selected from a single bond and -Z-(CH2)n-, where Z is selected from a single bond, O, S and NH and is from 1 to 3. In some of these modalities , Q2 is a simple link. In other embodiments, Q2 is -Z-(CH2)n-. In these modalities, Z can be O or S and n can be 1 or n can be 2. In other modalities, Z can be a single bond and n can be 1.
[00348] In other embodiments, Q1 is -CH=CH-.
[00349] In some embodiments, R2 can be -A-CH2-X and -A-X. In these embodiments, X can be -O-, -S-, -C(O)O-, -NH(C=O)- and -NH-. In particularly preferred embodiments, X can be -NH-. R12
[00350]R12 may be an aryl group of C5-7. A C5-7 aryl group can be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophene and pyridyl. In some embodiments, R12 is preferably phenyl. In other embodiments, R12 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
[00351]R12 can be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group can be attached to the PBD nucleus through any available ring position. For example, quinolinyl can be quinolin-2-yl, quinolin-3-yl, quinolin-4-yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these, quinolin-3-yl and quinolin-6-yl may be preferred. Isoquinolinyl can be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4-yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl, and isoquinolin -8-ila. Of these, isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
[00352]R12 can contain any number of surrogate groups. It preferably contains from 1 to 3 substituted groups, with 1 and 2 being more preferred, and individually substituted groups being more preferred. Substitutes can be in any position.
[00353] Where R12 is an aryl group of C5-7, a single substitute is preferably on a ring atom that is not adjacent to the bond to the remainder of the compound, ie, it is preferably β or y to the bond to the remainder of the compound. compound. Therefore, where the aryl group of C5-7 is phenyl, the substitute is preferably in the meta or para positions, and more preferably is in the para position.
Where R12 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may contain any number of substitutes at any position on the quinoline or isoquinoline rings. In some modalities, it contains one, two, or three surrogates, and these can be over either the proximal and distal rings or over both (if more than one surrogate). R12 Substitutes
[00355]If a substitute over R12 is halo, it is preferably F or Cl, more preferably Cl.
[00356] If a substitute over R12 is ether, it may in some embodiments be an alkoxy group, eg a C1-7 alkoxy group (eg methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (eg, phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example, by an amino group (for example dimethylamino).
[00357] If a substitute over R12 is C1-7 alkyl, it may preferably be a C1-4 alkyl group (for example, methyl, ethyl, propyl, butyl).
[00358] If a substitute over R12 is a C3-7 heterocyclyl, it may, in some embodiments, be a C6 nitrogen-containing heterocyclyl group, eg, morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups can be attached to the rest of the PBD portion via the nitrogen atom. These groups can be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen-containing heterocyclyl group is piperazinyl, said additional substitute may be on the second ring nitrogen atom.
[00359] If a substitute on R12 is bis-oxy-C1-3 alkylene, it is preferably bis-oxy-methylene or bis-oxy-ethylene.
Particularly preferred substitutes for R12 include methoxy, ethoxy, fluorine, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl. Another particularly preferred substitute for R12 is dimethylaminopropyloxy. R12 groups
Particularly preferred substituted R12 groups include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl , 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl , 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R12 group is 4-nitrophenyl. M and z
It is preferred that M and M' are pharmaceutically acceptable monovalent cations, and are more preferably Na+.
[00363]z is preferably 3. Examples General Experimental Methods
[00364] Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. 1H and 13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. The chemical shifts are described in relation to the TMS ( ppm = 0.0 ppm), and the signals are specified as s (single), d (double), t (triplet), dt (double triplet), dd (doublet of doublets ), ddd (double doublet of doublets) or m (multiplet), with the coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using an Águas Micromass ZQ instrument coupled to an Águas 2695 HPLC with an Águas PDA 2996. The parameters of the Micromass ZQ da Águas 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 an Águas Global Micromass QTQF in positive W mode, using metal-coated borosilate glass tips to introduce the 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. Anhydrous solvents were prepared by distillation under an atmosphere of dry nitrogen, in the presence of an appropriate drying agent, and stored over 4A molecular sieves or sodium filament. Petroleum ether refers to the fraction that boils at 40-60°C.
Compound 1b was synthesized as described in WO 00/012508 (compound 210), which is incorporated herein by reference.
[00366] General LC/MS conditions: The 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 over 1.0 min, then 5% B to 95% B within 3 min. The composition was held for 0.5 min at 95% B, then returned to 5% B in 0.3 min. The running time of the total gradient is equal to 5 min. Flow 3.0 mL/min, 400 μL were divided by means of a zero dead volume T-piece that passes through the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: Diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50 x 4.60 mm.
[00367] LC/MS conditions specific for compounds protected by either a Troc group or a TBDMs: The chromatographic separation of the compounds protected by Troc and TBDMS was performed on an Alliance 2695 HPLC system from Águas using an Onyx reversed phase column Monolitic (3 µm particles, 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% of in 10 sec. - equilibration for a further 50 s, giving a total run time of 5.0 min. The flow rate was maintained at 3.0 mL/min.
[00368] LC/MS conditions specific to compound 33: The LC was run on an Águas 2767 Sample Manager coupled with an Águas 2996 photodiode array detector and an Águas single quadrupole ZQ mass spectrometer Waters. The column used was Luna Phenyl-Hexyl 150 x 4.60 mm, 5 µm, Part No 00F-4257-E0 (Phenomenex). The mobile phases employed were: Mobile phase A: 100% HPLC grade water (0.05% triethylamine), pH = 7 Mobile phase B: 20% HPLC grade water and 80% HPLC grade acetonitrile (0.05% of triethylamine), pH = 7
[00369] The gradients used were:

[00370]Mass Spectrometry was performed in positive ion mode and SIR (selective ion monitor) and the monitored ion was m/z = 727.2. Summary of key intermediaries
(a)1,1'-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate] (2a)
[00371] Method A: A catalytic amount of DMF (2 drops) was added to a stirred solution of the nitro acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride (0.95 mL, 1.36 g, 10.7 mmol) 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-carboxylate hydrochloride ( 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)) showed product formation. 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 solvent evaporation 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 colored glass (840 mg, 54%).
[00372] Method B: Oxalyl chloride (9.75 mL, 14.2 g, 111 mmol) was added to a stirred suspension of the nitro acid 1a (17.3 g, 37.1 mmol) and DMF (2 mL) in anhydrous DCM (200 mL). After initial effervescence, the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. Conversion to the acid chloride was confirmed by treating a sample of the reaction mixture with MeOH and the resulting bis-methyl ester observed by LC/MS. Most of the solvent was removed by evaporation in vacuo, the resulting concentrated solution was re-dissolved in a minimal amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with ice-cold diethyl ether and dried for 1 hour in a vacuum oven at 40°C. The 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 mmol) in DCM (150 mL) at -40°C (dry ice/CH 3 CN). Immediately, the reaction was complete as judged 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 NaHCO 3 (300 ml), brine (300 ml), filtered (through a phase separator) and the solvent evaporated in vacuo to give pure product 2a as an orange solid (21.8 g, 82%).
[00373] Analytical data: [a]22D = -46.1° (c = 0.47, CHCte); 1H NMR (400 MHz, CDCl 3 ) (rotamers) δ 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) δ 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; IR (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]+. theoretical C31H36N4O16 m/z 721.2199, found (ES+) m/z 721.2227. (a)1,1'-[[(Pentane-1,5-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate] (2b)
Preparation from 1b according to Method B provided pure product as an orange foam (75.5 g, 82%).
[00375] Analytical Data: (ES+) m/z (relative intensity) 749 ([M + H]+., 100). (b) 1,1'-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a- hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3a)
[00376] Method A: A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) was added to a solution of the nitro-ester 2a (75 g, 104 mmoles) in DMF (360 ml). The suspension was hydrogenated in a Parr hydrogenation apparatus for 8 hours. Reaction progress 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 hydrogen uptake had stopped. The solid Pd/C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 1 kPa (10 mbar)), at 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 over a water bath (rotary evaporator bath) and the resulting suspension was filtered through of 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. After 20 minutes, the formation of a white precipitate and reflux was allowed to continue for a further 30 minutes. The mixture was allowed to cool to room temperature and the precipitate was recovered by filtration, washed with ether. diethyl ether (2*1 volume of precipitate) and dried in a vacuum desiccator to provide 3a (50 g, 81%).
[00377] Method B: A solution of the nitro-ester 2a (6.80 g, 9.44 mmol) in MeOH (300 mL) was added to Raney® Nickel (4 large spatula tips 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 dropwise with a solution of hydrazine hydrate (5.88 mL, 6.05 g, 188 mmol) in MeOH (50 mL), at which point effervescence was observed. vigorous. When the addition was complete (~30 minutes) additional Raney® nickel was added carefully until effervescence had ceased and the initial yellow color of the reaction mixture was discharged. The mixture was heated to reflux for a further 30 minutes, at which point the reaction was judged complete by TLC (CHCl3/MeOH 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 excess nickel removed by filtration through a sinter funnel, without vacuum suction. 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 desiccator to give 3a (5.40 g, 96%).
[00378] Analytical data: [a]27D = +404° (c = 0.10, DMF); 1H NMR (400 MHz, DMSO-d6) δ 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 ( mp, 2H, J = 5.90 Hz), 1.99-1.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 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; IR (ATR, pure) 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. (b) 1,1'-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a- hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3b)
Preparation from 2b according to Method A provided the product as a white solid (22.1 g, 86%).
[00380] Analytical data: MS (ES-) m/z (relative intensity) 623.3 ([M - H]-., 100). (c) 1,1'-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11, 11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4a)
The TBSCl (317 mg, 2.1 mmol) and the imidazole (342 mg, 5.03 mmol) were added to a cloudy solution of the 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 evaluated by LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M + H ]+., 100)). The reaction mixture was poured onto ice (~25ml) 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 vacuum desiccating to provide pure 4a (252 mg, 73%).
[00382] Analytical data: [α]23D = +234° (c = 0.41, CHCl3); 1H NMR (400 MHz, CDCl 3 ) δ 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) δ 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; IR (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. (c) 1,1'-[[(Pentane-1,5-diyl)dioxy]bis(11aS,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)
Preparation from 3b according to the method described above provided the product as a white solid (27.3 g, 93%).
[00384] Analytical data: MS (ES+) m/z (relative intensity) 853.8 ([M + H]+., 100), (ES-) m/z (relative intensity) 851.6 ([M - H]-., 100. (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)
A solution of n-BuLi (4.17 mL of a 1.6 M solution in hexane, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise to a stirred suspension of the 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 an atmosphere of nitrogen. 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 (60ml), washed with H 2 O (20ml), brine (20ml), dried (MgSO 4 ), filtered and evaporated in vacuo to give the crude product. Purification by flash chromatography (80:20 v/v Hexane/EtOAc) gave the N10 SEM protected tetralactam 5a as an oil (2.37 g, 82%).
[00386] Analytical data: [α]23D = +163° (c = 0.41, CHCl3); 1H NMR (400 MHz, CDCl 3 ) δ 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); 13 C NMR (100 MHz, CDCl3) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67 .1.6, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, -1.24, -4.73; IR (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]+. C53H88N4O12Si4 theoretical m/z 1085.5548, found (ES+) m/z 1085.5542. (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)
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.
[00388]Analytical data: MS (ES+) m/z (relative intensity) 1114 ([M + H]+., 90), (ES-) m/z (relative intensity) 1158 ([M + 2Na]-. , 100). (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)
[00389] 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 the bis-silyl ether 5a (2.58 g, 2.38 mmoles) ) in THF (40 ml) at room temperature. After stirring for 3.5 hours, TLC analysis of the reaction mixture (CHCl3/MeOH 95:5 v/v) revealed the completion of the reaction. The reaction mixture was poured into saturated NH 4 Cl solution (100 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine (60ml), dried (MgSO4), filtered and evaporated in vacuo to give the crude product. Purification by flash chromatography (gradient elution: 100% CHCl 3 to 96:4 v/v CHCl 3 /MeOH) gave pure tetralactam 6a as a white foam (1.78 g, 87%).
[00390]Analytical data: [α]23D = +202° (c = 0.34, CHCl3); 1H NMR (400 MHz, CDCl 3 ) δ 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); 13 C NMR (100 MHz, CDCl 3 ) , 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; IR (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]+. C41H60N4O12Si2 theoretical m/z 857.3819, found (ES+) m/z 857.3826. (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)
[00391] Preparation from 5b according to the method described above provided the product as a white foam (15.02 g).
[00392] Analytical data: MS (ES+) m/z (relative intensity) 886 ([M + H]+., 10), 739.6 (100), (ES-) m/z (relative intensity) 884 ( [M - H] -., 40). (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)
[00393] 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 potassium bromide solution (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 addition rate. 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.
The reaction mixture was filtered, the organic layer separated and the aqueous layer backwashed with DCM (x 2). The combined organic portions were washed with brine (x1), dried (MgSO4 ) and evaporated to give a yellow foam. Purification by flash column chromatography (gradient elution n-hexane/EtOAc 35/65 v/v, n-hexane/EtOAc 30/70 to 25/75 v/v) provided the bis-ketone 7a as a foam white (14.1 g, 75%).
[00395] A reagent grade sodium hypochlorite solution was used, available in 10-13% chlorine. This was assumed to be 10% (10 g NaClO in 100 g) and calculated to be 1.34 M NaClO. A stock solution was prepared from this 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 NaHCO 3 . An aliquot of this stock was then used to give 2.4 mol eq. for the reaction.
[00396] Upon addition of the bleach solution, an initial increase in temperature was observed. The addition rate was controlled to maintain the temperature between 0°C to 5°C. The reaction mixture formed a thick lemon yellow emulsion.
The oxidation was an adaptation of the procedure described in Thomas Fey et al, J. Org. Chem., 2001, 66, 8154-8159.
[00398] 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 TLC (EtOAc) and LC/MS [3.57 min (ES+) m/z (relative intensity) 875 ([M + Na]+., 50)] revealed the completion of the reaction. The reaction mixture was filtered through celite and the filtrate was washed with saturated aqueous NaHCO 3 (400 ml), brine (400 ml), dried (MgSO 4 ), filtered and evaporated in vacuo to give the crude product. Purification by flash column chromatography (80:20 v/v EtOAc/Hexane) provided bis ketone 7a as a foam (11.7 g, 65%).
[00399] Method C: A solution of anhydrous DMSO (0.72 ml, 0.84 g, 10.5 mmoles) in dry DCM (18 ml) was added dropwise over a period of 25 min. stirred solution of oxalyl chloride (2.63 mL of a 2.0 M solution in DCM, 5.26 moles) under a nitrogen atmosphere at -60°C (liq N 2 /CHCl 3 ). 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 30 min period to the 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. 20 min period to 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 1N HCl (2 x 25 mL), H2O (30 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of solvent in vacuo provided the crude product, which was purified by flash column chromatography (80:20 v/v EtOAc/Hexane) to afford the bis-ketone 7a as a foam (835 mg, 56 %)
[00400] Analytical data: [a]20D = +291° (c = 0.26, CHCl3); 1H NMR (400 MHz, CDCl 3 ) δ 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); 13 C NMR (100 MHz, CDCl 3 ) δ 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; IR (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]+. C41H56N4O12Si2 theoretical m/z 853.3506, found (ES+) m/z 853.3502. (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)
Preparation from 6b according to Method C provided the product as a white foam (10.5 g, 76%).
[00402] 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). (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)
Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in one portion to 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 an atmosphere of nitrogen. Anhydrous triflic anhydride, obtained from a freshly opened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injected rapidly, dropwise, 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 TLC (n-hexane/EtOAc 50/50 v/v) revealed complete consumption of starting material. The ice-cold reaction mixture was immediately diluted with DCM (200 mL) and, with vigorous stirring, washed with water (1 x 100 mL), 5% citric acid solution (1 x 200 mL), saturated NaHCO3 (200 mL) , brine (100 ml) and dried (MgSO4 ). Filtration and solvent evaporation in vacuo provided the crude product, which was purified by flash column chromatography (gradient elution: n-hexane/EtOAc 90:10 v/v to n-hexane/EtOAc 70:30 v/v) to give bis-enol triflate 8a as a yellow foam (5.5 g, 70%).
[00404] Analytical data: [α]24D = +271° (c = 0.18, CHCl3); 1H NMR (400 MHz, CDCl 3 ) δ 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); 13 C NMR (100 MHz, CDCl 3 ) δ 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; IR (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]+. C43H54N4O16Si2S2F6 theoretical m/z 1117.2491, found (ES+) m/z 1117.2465. (g) 1,1'-[[(Pentane-1,5-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] (8b)
Preparation from 7b according to the method described above gave bis-enol triflate as a light yellow foam (6.14 g, 82%).
[00406] Analytical data: (ES+) m/z (relative intensity) 1146 ([M + H]+., 85). Example 1
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 (9)
Solid Pd(PPh3)4 (20.18 mg, 17.46 mmoles) was added to a stirred solution of the triflate 8a (975 mg, 0.87 mmol), 4-(4,4,5.5 -tetramethyl-1,3,2-dioxaboralan-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 analysis by TLC (EtOAc) and LC/MS revealed the formation of the monocoupled product and the presence of unreacted starting material. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue partitioned between H2O (100ml) and EtOAc (100ml), after final separation of the layers, the aqueous phase was extracted again with EtOAc (2x 25 ml). The combined organic layers were washed with H2O (50 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to give the crude Suzuki product. The crude Suzuki product was flash chromatographed (40% EtOAc/60% Hexane ^ 70% EtOAc, 30% Hexane). Removal of excess eluent by rotary evaporation under reduced pressure provided the desired product 9 (399 mg) in 43% yield.
1H NMR: (CDCl3, 400 MHz) δ 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) , 13 C NMR: (CDCl3, 100 MHz) δ δ 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 (methine), 127.5, 125.1, 123.21, 121.5, 120.5 (methine), 120.1 (methine), 116, 4 (methine), 113.2 (methine), 108.7 (methine), 79.8 (methylene), 79.6 (methylene), 68.7 (methylene), 68.5 (methylene), 67 .0 (methylene), 66.8 (methylene), 58.8 (methine), 58.0 (methine), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3 (methylene), 19.7 (methylene), 0.25 (methyl). (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)
The solid Pd(PPh3)4 (10 mg, 8.69 μmoles) was added to a stirred solution of the mono-triflate 9 (230 mg, 0.22 mmol) in toluene (3 mL), EtOH (10 mL) ), with 4-methoxyphenyl boronic acid (43 mg, 0.28 mmol), Na2CO3 (37 mg, 0.35 mmol) in H2O (1.5 mL) at room temperature. The reaction mixture was allowed to stir under an atmosphere of nitrogen for 20 h, at which point the reaction was judged complete as judged by LC/MS and TLC (EtOAc). The solvent was removed by rotary evaporation under reduced pressure in vacuo and the resulting residue partitioned between EtOAc (75ml) and H 2 O (75ml). The aqueous phase was extracted with EtOAc (3 x 30 ml) and the combined organic layers washed with H 2 O (30 ml), brine (40 ml), dried (MgSO 4 ), filtered and evaporated to give the crude product. The crude product was purified by flash chromatography (60% Hexane: 40% EtOAc → 80% EtOAc: 20% Hexane) to provide the pure dimer as an orange foam. Removal of excess eluent under reduced pressure provided the desired product 10 (434 mg) in 74% yield.
[00410]-1H NMR: (CDCl3, 400 MHz) δ D7.38 (s, 2H), 7.34 (d, 2H, J = 8.8 Hz), 7.30 (bs, 1H), 7. 26-7.24 (m, 3H), 7.22 (d, 2H, J = 8.5 Hz), 6.86 (d, 2H, J = 8.8 Hz), 6.63 (d, 2H , J = 8.5 Hz), 5.50 (d, 2H, J = 10.0 Hz), 4.75 (d, 1H, J = 10.0 Hz), 4.74 (d, 1H, J = 10.0 Hz), 4.56 (td, 2H, J = 3.3, 10.1 Hz), 4.27 (t, 2H, J = 5.7 Hz), 4.00-3, 85 (m, 8H), 3.80 (s, 3H), 3.77-3.60 (m, 4H), 3.20-3.00 (m, 2H), 2.42 (p, 2H, J = 5.7 Hz), 0.96 (t, 4H, J = 8.3 Hz), 0.00 (s, 18H), 13 C NMR: (CDCl3, 100 MHz) δ □ 169.8, 169 ,7, 162.9, 162.7, 160.6, 152.7, 152.6, 149.0, 147.5, 134.8, 127.8 (methine), 127.4, 126.8, 125.1, 123.1, 123.0, 121.5 (methine), 120.4 (methine), 116.4 (methine), 115.5 (methine), 113.1 (methine), 108.6 (methine), 79.6 (methylene), 68.5 (methylene), 66.9 (methylene), 58.8 (methine), 57.6 (methoxy), 56.7 (methoxy), 32.8 ( methylene), 30.3 (methylene), 19.7 (methylene), 0.0 (methylene). (c) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro -1H-pyrrolo[2,1-c] [1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5(11aH)-one (11 )
The newly produced LiBH4 (183 mg, 8.42 mmoles) was added to a stirred solution of SEM-diIactam 10 (428 mg, 0.42 mmoI) in THF (5 mL) and EtOH (5 mL) in the room temperature. After 10 minutes, a delayed vigorous effervescence was observed, requiring the reaction vessel to be placed in an ice bath. After removal from the ice bath, the mixture was allowed to stir at room temperature for 1 hour. LC/MS analysis at this point revealed the total consumption of starting material, with very little mono-reduced product. The reaction mixture was poured onto ice (100 ml) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3 x 30 mL) and the combined organic layers washed with H 2 O (20 mL), brine (30 mL) and concentrated in vacuo. The resulting residue was treated with DCM (5ml), EtOH (14ml), H2O (7ml) and silica gel (10g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was slowly filtered through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (~250 mL) until UV activity completely disappeared from the eluent. The organic phase was washed with H 2 O (50ml), brine (60ml), dried (MgSO 4 ), filtered and evaporated in vacuo to give the crude material. The crude product was purified by flash chromatography (97% CHCl 3 : 3% MeOH) to provide pure C 2 /C 2 aryl PBD dimer (185 mg), 61% yield.
[00412]-1H NMR: (CDCl3, 400 MHz) δ D7.88 (d, 1H, J = 4.0 Hz), 7.87 (d, 1H, J = 4.0 Hz), 7.52 ( s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.20 (d, 2H, J = 8.5 Hz), 6.89 (d, 2H) , J = 8.8 Hz), 6.87 (s, 1H), 6.86 (s, 1H), 6.67 (d, 2H, J = 8.5 Hz), 4.40-4.20 (m, 6H), 3.94 (s, 6H), 3.82 (s, 3H), 3.61-3.50 (m, 2H), 3.40-3.30 (m, 2H), 2.47-2.40 (m, 2H), 13 C-NMR: (CDCl3, 100 MHz) δ □ □□□□ (imine methine), 161.3, 161.1, 159.3, 156.0, 151.1, 148.1, 146.2, 140.3, 126.2 (methine), 123.2, 122.0, 120.5 (methine), 119.4, 115.2 (methine), 114 .3 (methine), 111.9 (methine), 111.2 (methine), 65.5 (methylene), 56.2 (methoxy), 55.4 (methoxy), 53.9 (methine), 35.6 (methylene), 28.9 (methylene).
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((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)pentyloxy)-10-((2 -(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5,11(10H,11aH)-dione (12)
[00413] The solid Pd(PPh3)4 (32 mg, 27.7 μmoles) was added to a stirred solution of the bis-triflate 8b (1.04 g, 0.91 mmol) in toluene (10 mL), EtOH (5 mL), with 4-methoxyphenyl boronic acid (0.202 g, 1.32 mmol), Na2CO3 (0.169 g, 1.6 mmol) in H2O (5 mL) at 30°C. The reaction mixture was allowed to stir under a nitrogen atmosphere for 20 hours. Additional solid 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (0.203 g, 0.93 mmol) and Na2CO3 (0.056 g, 0.53 mmol) were added , followed by solid Pd(PPh3)4 (10 mg, 8.6 µmoles). The reaction mixture was allowed to stir under a nitrogen atmosphere for a further 20 hours. LC/MS indicated the formation of the desired product. EtOAc (100 mL) and H2O (100 mL) were added, the aqueous was separated and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with H 2 O (100 ml), brine (100 ml), dried (MgSO 4 ), filtered and evaporated to give a dark brown oil. The oil was dissolved in DCM and loaded onto a 10 g SCX-2 cartridge, pre-equilibrated with DCM (1 vol). The cartridge was washed with DCM (3 vol), MeOH (3 vol) and the crude product eluted with 2M NH3 in MeOH (2 vol). Flash chromatography (50% n-hexane: 50% EtOAc → 20% n-hexane: 80% EtOAc) provided pure dimer 12 as a yellow foam (0.16 g, 34%).
[00414] Analytical data: [a]23D = +388° (c = 0.22, CHCl3); 1 H-NMR: (CDCl3, 400 MHz) δ 07.39 (s, 2H), 7.35 (d, 2H, J = 12.8 Hz), 7.32 (bs, 1H), 7.26-7 .23 (m, 5H), 6.89 (d, 2H, J = 8.8 Hz), 6.66 (d, 2H, J = 8.5 Hz), 5.55 (d, 2H, J = 10.0 Hz), 4.73 (d, 1H, J = 10.0 Hz), 4.72 (d, 1H, J = 10.0 Hz), 4.62 (td, 2H, J = 3 .2, 10.4 Hz), 4.15 - 4.05 (m, 4H), 4.00-3.85 (m, 8H), 3.82 (s, 3H), 3.77-3, 63 (m, 4H), 3.20-3.05 (m, 2H), 2.05 - 1.95 (m, 4H), 1.75 - 1.67 (m, 2H) 1.01 - 0 .95 (m, 4H), 0.03 (s, 18H); MS (ES+) m/z (relative intensity) 1047 ([M + H]+., 45). (b) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro -1H-pyrrolo[2,1-c] [1,4]benzodiazepine-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5(11aH)-one (13 )
The newly produced LiBH4 (66 mg, 3.04 mmol) was added to a stirred solution of SEM-dilactam 12 (428 mg, 0.42 mmol) in THF (3 mL) and EtOH (3 mL) to 0°C (ice bath). The ice bath was removed and the reaction mixture was allowed to reach room temperature (vigorous effervescence). After 2 hours, LC/MS analysis indicated complete consumption of starting material. The reaction mixture was poured onto ice (50ml) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3 x 50 mL) and the combined organic layers washed with H 2 O (50 mL), brine (50 mL), dried (MgSO 4 ) and concentrated in vacuo. The resulting residue was treated with DCM (2 mL), EtOH (5 mL), H2O (2.5 mL) and silica gel (3.7 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (~250 mL) until UV activity completely disappeared from the eluent. The organic phase was dried (MgSO4), filtered and evaporated in vacuo to give the crude material. The crude product was purified by flash chromatography (99.5% CHCl3 : 0.5% MeOH to 97.5% CHCl3 : 2.5% MeOH, in 0.5% increments) to provide the dimer of Aryl PBD in pure C2/C2' 13 (59 mg, 52%).
[00416] Analytical data: [α]28D = +760° (c = 0.14, CHCl3); 1H NMR (400 MHz, CDCl 3 ) δ 7.89 (d, 1H, J = 4.0 Hz), 7.87 (d, 1H, J = 4.0 Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.22 (d, 2H, J = 8.4 Hz), 6.91 (d, 2H, J = 8. 8 Hz), 6.815 (s, 1H), 6.81 (s, 1H), 6.68 (d, 2H, J = 8.4 Hz), 4.45 - 4.35 (m, 2H), 4 0.2-4.0 (m, 4H), 3.94 (s, 6H), 3.85 - 3.7 (s, 3H), 3.65 - 3.50 (m, 2H), 3.45 - 3.3 (m, 2H), 2.05 - 1.9 (m, 4H), 1.75 - 1.65 (m, 2H); MS (ES+) (relative intensity) 754.6 ([M + H]+., 100), (ES-) (relative intensity) 752.5 ([M - H]-., 100). Example 3
(a)(S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10-( (2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2 -(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5,11(10H,11aH)-dione (14)
The solid Pd(PPh3)4 (41 mg, 0.036 mmol) was added to a stirred solution of the bis-triflate 8a (1 g, 0.9 mmol) in toluene (10 mL), EtOH (5 mL), with thien-2-yl boronic acid (149 mg, 1.16 mmol), Na2CO3 (152 mg, 1.43 mmol), in H2O (5 mL). The reaction mixture was allowed to stir under an atmosphere of nitrogen overnight at room temperature. The solvent was removed by evaporation in vacuo and the resulting residue partitioned between H 2 O (100ml) and EtOAc (100ml). The aqueous layer was extracted with EtOAc (2 x 30 mL) and the combined organic layers washed with H2O (50 mL), brine (50 mL), dried (MgSO4), filtered and evaporated in vacuo to give the crude product, which was purified by flash chromatography (80 hexane: 20 EtOAc 50 hexane: 50 EtOAc) to provide dimer 14 (188 mg, 20% yield).
[00418] Analytical data: LC-MS TR 4.27 min, 1051 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ 7.36 (s, 1H), 7.31 (bs, 1H), 7.27 (bs, 1H), 7.26-7.23 (m, 2H), 7.227.17 (m, 1H), 7.12 (bs, 1H), 7.02-6.96 (m, 2H), 5.50 (d, J = 10.0 Hz, 2H), 7.75 (d, J = 10.0 Hz, 2H), 4.65-4.55 (m, 2H), 4.37-4.13 (m, 4H), 4.00-3.85 (m, 8H) ), 3.8-3.6 (m, 4H), 3.20-3.10 (m, 2H), 2.50-2.35 (m, 2H), 1.0-0.9 (m, , 4H), 0 (s, 18H). (b) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-trifluoromethanesulfonyloxy)-5,11-dioxo-10-(( 2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5,11(10H,11aH)-dione (15)
The solid Pd(PPh3)4 (7.66 mg, 6.63 µmoles) was added to a stirred cloudy solution of 14 (174 mg, 0.17 mmol), Na2CO3 (28 mg, 0.22 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (47 mg, 0.22 mmol) in toluene (2-5 mL), EtOH (1 .25 mL) and H2O (125 mL) at room temperature. The reaction mixture was allowed to stir under an atmosphere of N2 for 24 hours, at which point the reaction was considered complete by the largest peak of LC/MS (at 3.97 min, FW = 1016, M+Na) and TLC (EtOAc ). The solvent was removed by evaporation in vacuo and the resulting residue partitioned between EtOAc (60ml) and H2O (30ml). The layers were separated and the organic phase was washed with H 2 O (20ml), brine (30ml), dried (MgSO 4 ), filtered and evaporated in vacuo to give the crude product, 123mg, 75% yield.
[00420] Analytical data: LC-MS TR 3.98 min, 100% area, 994 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ 7.40 (d, J = 5.3 Hz, 2H), 7.30 (t, J =1.70 Hz, 1H), 7.297.27 (m, 3H) , 7.25 (d, J = 8.5 Hz, 2H), 7.21 (dd, J = 1.4, 4.73 Hz, 1H), 7.03-6.97 (m, 2H), 6.66 (d, J = 8.5 Hz, 2H), 5.52 (d, J = 10.0 Hz, 2H), 4.78 (d, J = 10.0 Hz, 1H), 4. 77 (d, J = 10.0 Hz, 1H), 4.62 (dd, J = 3.4, 10.5 Hz, 1H), 4.59 (dd, J = 3.40, 10.6 Hz , 1H), 4.30 (t, J = 5.85 Hz, 4H), 3.85-4.03 (m, 8H), 3.84-3.64 (m, 6H), 3.18 ( ddd, J = 2.2, 10.5, 16.0 Hz, 1H), 3.11 (ddd, J = 2.2, 10.5, 16.0 Hz, 1H), 2.44 (p, J = 5.85 Hz, 2H), 0.98 (t, J = 1.5 Hz, 4H), 0 (s, 18H). (c) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-aminophenyl)-5-oxo-5,11a - dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2.1-c] [1,4]benzodiazepine-5(11aH)-one (16)
The newly produced LiBH4 (47 mg, 2.22 mmol) was added to a stirred solution of SEM-dilactam 15 (110 mg, 0.11 mmol) in dry THF (3 mL) and EtOH (3 mL), at 0°C (ice bath). The ice bath was removed and the reaction mixture stirred under a N2 atmosphere for 1 hour. Reaction analysis by LC/MS analysis revealed significant formation of the desired product (Peak at 2.57 min) (I=69.32), FW=702, M+H) and semi-imine. The reaction mixture was allowed to stir for a further 1 hour, after which time no further reaction progress was observed by LC/MS. The reaction mixture was poured onto ice, stirred and allowed to warm to room temperature. After partitioning between DCM (50 ml) and water (50 ml), the aqueous phase was extracted with DCM (3 x 20 ml). The combined organic layers were washed with H 2 O (50 ml), brine (50 ml) and the solvent removed by evaporation in vacuo under reduced pressure.
The resulting residue was dissolved in DCM (5 mL), EtOH (15 mL) and H2O (7 mL), then treated with silica gel (5 g). The reaction was allowed to stir at room temperature for 48 h. The silica was removed by filtration through a sintering funnel and the residue rinsed with 90:10 CHCl3:MeOH (100 mL). H2O (50 mL) was added to the filtrate and the layers were separated (after stirring). The aqueous layer was extracted with CHCl 3 (2 x 30 mL) and H 2 O (50 mL), brine (50 mL), dried (MgSO 4 ), filtered and evaporated in vacuo to give the crude product. Flash chromatography (CHCl3 ^ 98% CHCl3 : 2% MeOH) provided the product (41 mg, 53%).
[00423] Analytical data: LC-MS TR 2.55 min, 702 (M + H). Example 4
(a)(S)-2-(4-methoxyphenyl)-7-methoxy-8-(3-((S)-7-methoxy-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)propyloxy)-10-((2-( trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5,11(10H,11aH)-dione (17)
Solid 4-methoxybenzeneboronic acid (0.388 g, 2.55 mmoles) was added to a solution of the SEM protected bis triflate (8a) (3.0 g, 2.69 mmoles), sodium carbonate (426 mg , 4.02 mmol) and tetrakis triphenylphosphine palladium (0.08 mmol) in toluene (54.8 mL), ethanol (27 mL) and water (27 mL). The reaction mixture was allowed to stir at room temperature for 3 hours. 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. 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 30/70^35/65^40/60^45 /55) to remove unreacted ob/s-triflate (0.6 g). Removal of excess eluent from selected fractions provided the product coupled with 4-methoxyphenyl (1.27 g, 1.18 mmol, 41%).
[00425]LC-MS TR 4.30min, 1076 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ 7.41 (s, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.35 (s, 1H), 7.34 (bs, 1H), 7.29 (s, 1H), 7.16 (t, J = 1.9 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 5.53 (d, J = 10.0 Hz, 2H), 4.79 (d, J = 10.0 Hz, 1H), 4.78 (d, J = 10.0 Hz, 1H), 4.66 - 4.60 ( m, 2H), 4.30 (t, J = 5.7Hz, 4H), 4.0 - 3.94 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H) ), 3.84 (s, 3H), 3.83 - 3.60 (m, 4H), 3.22 - 3.10 (m, 2H), 2.45 (t, J = 5.9 Hz, 2H), 1.05 - 0.94 (m, 4H), 0 (s, 18H). (b) (S)-2-(3-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)propyloxy)-10-((2 -(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5,11(10H,11aH)-dione (18)
Solid 3-aminobenzeneboronic acid (0.143 g, 0.92 mmol) was added to a solution of the mono triflate (17) (0.619 g, 0.58 mmol), sodium carbonate (195 mg, 1.84 mmol) ) and tetrakis triphenylphosphine palladium (26.6 mg, 0.023 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir at room temperature overnight at 30°C. 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. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 70/30^85/15). Removal of excess eluent from selected fractions provided the desired product (0.502 g, 0.49 mmol, 85%).
[00427]LC-MS TR 4.02 min, 1019 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ 7.38 - 7.35 (m, 4H), 7.33 (bs, 1H), 7.30 (bs, 1H), 7.25 (s, 2H), 7.10 (t, J = 7.8 Hz, 1H), 6.88 - 6.80 (m, 3H), 6.72 (bs, 1H), 6.57 (dd, J = 7.9, 1.8 Hz, 1H), 5.50 (d, J = 10.0 Hz, 2H), 4.75 (d, 10.0 Hz, 2H), 4.58 (dd, J = 10.6, 3.3Hz, 2H), 4.27 (t, J = 5.8Hz, 4H), 3.95 - 3.91 (m, 2H), 3.90 (s, 6H), 3.80 ( s, 3H), 3.77 - 3.60 (m, 6H), 3.15 - 3.05 (m, 2H), 2.41 (p, J = 5.8 Hz, 2H), 0.95 (t, = 8.25 Hz, 4H), 0 (s, 18H). (c) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro -1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5(11aH)-one (19 )
[00428] A solution of the superhydride (0.56 mL, 0.56 mmol, 1.0 M in THF) was added dropwise to a solution of the SEM dilactam (18) (0.271 g, 0.27 mmol) in dry THF (10 mL) at -78°C under a nitrogen atmosphere. After 1 h, an additional aliquot of superhydride solution (0.13 ml, 0.13 mmol) was added and the reaction mixture was allowed to stir for another 0.5 h, at which time LC-MS indicated that the reduction was complete. The reaction mixture was diluted with water and allowed to warm to room temperature. The reaction mixture was partitioned between chloroform and water, the layers were separated and the aqueous layer extracted with additional chloroform (emulsions). Finally, the combined organic phase was washed with brine and dried over magnesium sulfate. The reduced product was dissolved in methanol, chloroform and water and allowed to stir in the presence of silica gel for 72 hours. The crude product was subjected to flash column chromatography (methanol/chloroform gradient) to provide the desired imine product (150 mg, 0.21 mmol, 77%) after removal of excess eluent from selected fractions.
[00429]LC-MS RT 2.63 min, 97 area%, 726 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ 7.85 (d, J = 3.9 Hz, 1H), 7.84 (d, J = 3.9 Hz, 1H), 7.50 (s, 1H) , 7.49 (s, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 7.32 (d, J = 7.3 Hz, 2H), 7.11 (t, (d, J = 7.8 Hz, 1H), 6.906.80 (m, 4H), 6.77 (d, J = 7.9 Hz, 1H), 4.40-4.20 (m, 6H) , 3.92 (s, 6H), 3.80 (s, 3H), 3.60-3.27 (m, 6H), 2.48-2.29 (m, 2H)
(a)11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-( (2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy- 5-oxo-2-(trifluoromethylsulfonyloxy)-11,11a-dihydro-pyrrolo[2.1-c][1,4]benzodiazepine-10(5H)-(1S,11aS)-2,2,2-carboxylate trichloroethyl 21
Solid 4-methoxybenzeneboronic acid (59 mg, 0.39 mmol) was added to a solution of the Troc-protected bis triflate (Compound 44, WO 2006/111759) (600 mg, 0.41 mmol), carbonated carbonate sodium (65 mg, 0.61 mmol) and tetrakis triphenylphosphine palladium (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. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 20/80^30/70^40/60^60/40) ) to remove unreacted bis-triflate. Removal of excess eluent from selected fractions provided the product coupled with 4-methoxyphenyl (261 mg, 0.18 mmol, 46%).
[00431]LC-MS TR 4.17 min, 1427 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ □ 7.38 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.25 (s, 1H), 7.20 (bs, 1H), 6.92 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.7 Hz, 2H), 6.0 - 5.90 (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), 4.18-4.08 (m, 2H), 4.07 - 3.89 (m, 10H), 3.81 (s, 3H), 3.44 - 3.25 (m, 2H), 2.85 (d, J = 16.6 Hz, 2H), 2.05 - 1.90 (m, 4H) , 1.76 - 1.64 (m, 2H), 0.93 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.26 (s, 6H). (b) 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-10-( (2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy- 2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (11S,11aS)-2, 2,2-trichloroethyl 22
[00432] The Suzuki coupling procedure described in step (a) was applied to the synthesis of Compound 21. Compound 20 (62.5 mg, 0.044 mmol) was treated with 1 equivalent of 4-hydroxybenzeneboronic acid (10 mg), at 30°C overnight to provide the desired compound after filtration through a silica gel pad (40 mg, 0.029 mmol, 66% yield). The compound was used directly in the subsequent step.
[00433]LC-MS TR 4.27 min, 1371 (M + H). (c) (S)-2-(4-hydroxyphenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro - 1H-pyrrolo[2,1-c][1,4]benzodiazepindiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 23
The cadmium/lead pair (100 mg, Q Dong et al. Tetrahedron Letters vol 36, edition 32, 5681-5682, 1995) was added to a solution of 21 (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 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, 0 4% MeOH/CHClβ). Removal of excess eluent by rotary evaporation under reduced pressure provided the desired imine product (17 mg, 0.023 mmol, 79%).
[00435]LC-MS TR 2.20min, 755 (M + H); 1 H-NMR (400 MHZ, CDCl) δ □ 7.89 (d, J = 3.94 Hz, 1H), 7.89 (d, J = 4.00 Hz, 1H), 7.53 (s, 1H) ), 7.52 (s, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 7.28 (s, 1H ), 6.90 (d, J = 8.7 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.82 (s, 1H), 6.81 (s, 1H ), 5.68 (bs, 1H), 4.50 - 4.30 (m, 2H), 4.22 - 4.00 (m, 4H), 3.93 (s, 6H), 3.82 ( s, 3H), 3.69 - 3.45 (m, 2H), 3.44 - 3.28 (m, 2H), 2.64 - 1.88 (m, 4H), 1.77 - 1. 62 (m, 2H). Example 6
(a) 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-formylphenyl)-7-methoxy-5-oxo-10-( (2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy- 2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (11S,11aS)-2, 2,2-trichloroethyl 24
[00436] The Suzuki coupling procedure described in Example 5, step (a) was applied to the synthesis of Compound 24. Compound 21 (62.5 mg, 0.044 mmol) was treated with 1 equivalent of 4-formylbenzeneboronic acid ( 10.5 mg) at room temperature overnight to provide the desired compound after filtration through a silica gel pad (45 mg, 0.033 mmol, 75% yield). The compound was used directly in the subsequent step.
[00437]LC-MS TR 4.42 min, 1383 (M + H). (b)4-((S)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[ 2.1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2.1-c][1,4]benzodiazepine-2-yl ) benzaldehyde 25
Compound 24 was deprotected by the method described in Example 5, step (c) to yield the desired compound (18 mg, 0.023 mmol, 79%).
[00439]LC-MS TR 3.18 min, 768 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ □ 9.98 (s, 1H), 7.91 (d, J = 3.90 Hz, 1H), 7.90 - 7.80 (m, 3H), 7 .68 (s, 1H), 7.60 - 7.45 (m, 4H), 7.39 (s, 1H), 7.33 (d, J = 8.7 Hz, 1H), 6.90 ( d, J = 8.7 Hz, 2H), 6.83 (s, 1H), 6.82 (s, 1H), 4.55 - 4.44 (m, 1H), 4.43 - 4.36 (m, 1H), 4.23 - 4.00 (m, 4H), 3.95 (s, 3H), 3.94 (s, 3H), 3.82 (s, 3H), 3.66 - 3.51 (m, 2H), 3.50 - 3.34 (m, 2H), 2.05 - 1.87 (m, 4H), 1.76 - 164 (m, 2H).
(a) 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-10-( (2,2,2-trichloroethoxy)carbonyl)-2-(trifluoromethylsulfonyloxy)-5,10,11,11a-tetrahydro-1H-_pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy )-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2.1-c][1,4]benzodiazepine-10(5H)-carboxylate (11S,11aS)-2.2, 2-trichloroethyl 26
[00440] The Suzuki coupling procedure described in Example 5, step (a) was applied to the synthesis of Compound 26 using 3-aminobenzeneboronic acid to provide the desired compound in 41% yield (230 mg, 0.163 mmol) .
[00441]LC-MS TR 4.28 min, 1411 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δD 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). (b)2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-(3-(dimethylamino)) propoxy)phenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-_pyrrole [2,1-c][1 ,4] benzodiazepindiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)- (11S,11aS)-2,2,2-trichloroethyl carboxylate 27
Solid 4-[3-(dimethylamino)propoxybenzeneboronic acid pinacol ester (25 mg, 0.082 mmol) was added to a solution of 26 (73 mg, 0.052 mmol), sodium carbonate (18 mg, 0.17 mmol) mmol) and tetrakis triphenylphosphine palladium (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 sulfate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was eluted through a silica gel filling with chloroform/ethanol. Removal of excess eluent from selected fractions provided the product coupled with 4-methoxyphenyl (50 mg, 0.035 mmol, 67%).
[00443]LC-MS TR 4.12 min, 1440 (M + H). (c) (S)-2-(3-aminophenyl)-8-(5-((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5 ,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine- 5(11aH)-one 28
Compound 27 was deprotected by the method described in Example 5, step (c) to yield the desired compound. The reaction mixture was partitioned between DCM and aqueous sodium hydrogen carbonate (emulsion) and the crude product purified by silica gel gradient column chromatography (5% methanol chloroform 35% methanol/chloroform) to provide the Desired asymmetric PBD imine (50 mg, 0.018 mmol, 58%).
[00445]LC-MS TR 2.55 min, 826 (M + H); 1 H-NMR (400 MHZ, CDCl 3 ) δ □ 7.92 - 7.82 (m, 2H), 7.52 (bs, 2H), 7.45 (bs, 1H), 7.39 (bs, 1H) , 7.31 (d, J = 8.6 Hz, 2H), 7.14 (t, J = 7.8 Hz, 1H), 6.89 (d, J = 8.6 Hz, 2H), 6 .85 - 6.75 (m, 3H), 6.72 (bs, 1H), 6.60 (d, J = 8.0 Hz, 1H), 4.46 - 4.33 (m, 2H), 4.21 - 3.98 (m, 6H), 3.94 (s, 6H), 3.63 - 3.50 (m, 2H), 3.43 - 3.29 (m, 2H), 2. 64 - 2.48 (m, 2H), 2.34 (s, 6H), 2.10 - 1.89 (m, 6H), 1.57 (m, 2H).
(a)2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-(4) -methylpiperazin-1-yl)phenyl)-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrole [2,1-c][ 1,4] benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate of (11S,11aS)-2,2,2-trichloroethyl 29
[00446] The method of Example 7, step (b), was carried out to provide the desired product (58 mg, 0.040 mmol, 78%) after filtration through a silica gel filling (with methanol/chloroform 1/3) and removing excess solvent by rotary evaporation under reduced pressure.
[00447]LC-MS TR 4.08 min, 1439 (M + H). (b) (S)-2-(3-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)- 5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2.1-c][1,4]benzodiazepine -5(11aH)-one 30
The method of Example 7, step (c), was used to deprotect compound 29. The crude product was purified by silica gel gradient chromatography (2% methanol chloroform 35% methanol/chloroform) to provide the desired asymmetric imine PBD (18 mg, 0.022 mmol, 59%).
[00449]LC-MS TR 2.52 min, 823 (M + H); 1 H-NMR (400 MHZ, CDCl) δ □ 7.80 (d, J = 3.8Hz, 2H), 7.45 (s, 2H), 7.38 (s, 1H), 7.30 (s, 1H), 7.23 (d, J = 8.6Hz, 2H), 7.07 (t, J = 7.8Hz, 1H), 6.83 (d, J = 8.6Hz, 2H), 6.79-6.89 (m, 3H), 6.65 (s, 1H), 6.54 (d, J = 7.9 Hz, 1H), 4.40-4.24 (m, 2H) , 4.15-3.93 (m, 4H), 3.87 (s, 6H), 3.56-3.42 (m, 2H), 3.37-3.23 (m, 2H), 3 .22-3.08 (m, 4H), 2.61-2.41 (m, 4H), 2.29 (s, 3H), 1.981.80 (m, 4H), 1.67-1.54 (m, 2H). Example 9
(a) (S)-2-(4-(aminomethyl)phenyl)-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)propyloxy)-10- ((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione 31
Solid 4-aminomethylbenzeneboronic acid hydrochloride (0.111 g, 0.59 mmol) was added to a solution of 17 (0.394 g, 0.37 mmol), sodium carbonate (175 mg, 1.654 mmol) and tetrakis triphenylphosphine palladium (28.0 mg, 0.024 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir overnight at 30°C. The next day, the reaction mixture was heated for a further 3 hours at 70°C. 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. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 2/98 x 15/85). Removal of excess eluent from selected fractions provided the desired product (0.230 mg, 0.22 mmol, 61%).
[00451]LC-MS TR 3.63 min, 1034 (M + 2H); 1 H-NMR (400 MHz, DMSO d6) δ 11.7 (s, 2H), 7.52 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H ), 7.40 (s, 1H), 7.50 (d, J = 8.1 Hz, 2H), 7.38-7.19 (m, 5H) 6.93 (d, J = 8.7 Hz, 2H), 5.40 (d, J = 2.13 Hz, 1H), 5.38 (d, J = 2.12 Hz, 1H), 5.32 (d, J = 10.6 Hz, 2H), 5.25 (d, J = 10.6Hz, 2H), 4.874.72 (m, 2H), 4.35-4.15 (m, 4H), 3.85 (s, 6H), 3.79 (s, 3H), 3.73-3.56 (m, 2H), 3.553.39 (m, 4H), 3.22-3.02 (m, 2H), 2.39-2, 23 (m, 2H), 0.94-0.67 (m, 4H), -0.06 (s, 18H). (b) (S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5, 11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2.1-c][1,4]benzodiazepine-5(11aH)- ona 32
Compound 31 was deprotected following the method of Example 1, step (c). The crude product was purified by gradient column chromatography (MeOH/CHCl3 to 5/95·30/70) to provide the product as a mixture of imine and carbinolamine methyl ethers.
[00453]LC-MS TR 2.58 min, 740 (M + H). Example 10
(S)-2-(4-aminophenyl)-7-methoxy-11(S)-sulfo-8-(3-((S)-7-methoxy-11(S)-sulfo-2-(disodium salt 4-methoxyphenyl)-5-oxo-5,10,11,11a -tetrahydro-1H-pyrrolo[2.1-c] [1,4]benzodiazepin-8-yloxy)propyloxy)-1H-pyrrolo[2.1 -c] [1,4]benzodiazepine-5(11aH)-one 33
Sodium bisulfite (8.5 mg, 3.1 eq) was added to a stirred suspension of bisimine 11 (20 mg, 0.036 mmol) in isopropanol (4 mL) and water (2 mL). The reaction mixture was allowed to stir vigorously and finally became clear (about 1 hour). The reaction mixture was transferred to a funnel and filtered through a cotton plug (and then washed with 2 mL of water). The filtrate was snap frozen (liquid and to the bath) and lyophilized to provide the desired product 33 in quantitative yield.
[00455]LC-MS TR 11.77 min, 727.2 (M + H) (Mass of lead compound, unstable bisulfite adducts in mass spectrometer); 1 H-NMR (400 MHz, CDCl 3 ) δ 07.66-7.55 (m, 5H), 7.43 (s, 1H), 7.39 (d, J = 8.66 Hz, 2H), 7. 06 (m, 2H), 6.93 (d, J = 8.84 Hz, 2H), 6.54 (m, 2H), 5.29-5.21 (m, 2H), 4.32-4 .28 (m, 2H), 4.144.20 (m, 4H), 3.96-3.83 (m, 2H), 3.77 (s, 3H), 3.73 (m, 6H), 3. 52-3.43 (m, 2H), 3.303.08 (m, 2H), 2.24-2.21 (m, 2H). Example 11
(a) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10- ((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-pyrrolo[2.1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-10-(( 2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (103)
[00456] A catalytic amount of tetracistriphenylphosphine palladium (0) (11.2 mg) was added to a mixture of the mono triflate 17 (380 mg), the 2-aminophenylboronic acid ester pinacol (124 mg) and sodium carbonate (120 mg ) in ethanol 5m), toluene (5ml) and water (5ml). The reaction mixture was allowed to stir overnight at room temperature and at 40°C until the reaction was complete (about 2 hours). The reaction mixture was diluted with ethyl acetate and the organic layer was washed with water and brine. The ethyl acetate solution was dried over magnesium sulfate and vacuum filtered. Removal of ethyl acetate by rotary evaporation under reduced pressure provided the crude product which was subjected to flash chromatography (silica gel, ethyl acetate/hexane). Pure fractions were collected and combined. Removal of excess eluent by rotary evaporation under reduced pressure provided crude product 103 (330 mg, 86% yield). LC/MS RT: 4.17 min, ES+1018.48. (b) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a- dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-pyrrolo[2.1-c][1,4]benzodiazepin-5(11aH)-one (104)
[00457] A solution of the superhydride in dry tetrahydrofuran (1.0 M, 4.4 eq.) was added to a solution of the 2-analine compound 103 (300 mg) in dry tetrahydrofuran (5 ml) a - 78 °C under an inert atmosphere. As the reduction proceeded slowly, an aliquot of lithium borohydride (20 eq) was added and the reaction mixture was allowed to return to room temperature. Water/ice was added to the reaction mixture to quench unreacted hydrides and the reaction was diluted with dichloromethane. The organic layer was sequentially washed with water (twice), citric acid and brine. Excess dichloromethane was removed by rotary evaporation under reduced pressure and the residue was redissolved in ethanol and water and treated with silica gel for 96 hours. The reaction mixture was vacuum filtered and the filtrate evaporated to dryness. The residue was subjected to flash column chromatography (silica gel, chloroform/methanol gradient). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to yield pure product 104 (30 mg, 14% yield). LC/MS RT: 2.90 min, ES+726.09. Example 12: In Vitro Cytotoxicity Determination of Representative PBD Compounds Assay with K562
[00458]K562 human chronic myeloid leukemia cells were maintained in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine, at 37°C, in a humidified atmosphere containing 5% CO2, and were incubated with a specified dose of drug for 1 hour or 96 hours at 37°C in the dark. Incubation was terminated by centrifugation (5 min, 300 g) and cells were washed once with drug-free medium. After treatment with appropriate drug, cells were transferred to 96-well microtiter plates (104 cells per well, 8 wells per sample). The plates were then kept in the dark, at 37°C, in a humidified atmosphere containing 5% CO2. The assay is based on the ability of viable cells to reduce a yellow soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Aldrich-Sigma ), to an insoluble purple formazan precipitate. After incubating the plates for 4 days (to allow the control cells to increase in number by approximately 10-fold), 20 µL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well and the plates further incubated for 5 h. The plates were then centrifuged for 5 min at 300 g and most of the medium pipetted from the cell pellet, leaving 10-20 μL per well. DMSO (200 µL) was added to each well and the samples shaken to ensure complete mixing. Optical density was then read at a wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and a dose-response curve constructed. For each curve, an IC50 value was read as the dose required to reduce the final optical density to 50% of the control value.
Compound 13 has an IC50 of 30 pM in this assay. Test with A2780
Parental cell line A2780 was grown in Dulbecco's Modified Eagles Medium (DMEM) containing ~10% Fetal Calf Serum (FCS) and ~1% 200 mM L-Glutamine solution and grown in flasks. 75 cm2 Corning Cellbind.
A 190 µl cell suspension was added (at 1 x 104) to each well of columns 2 to 11 of a 96-well plate (Nunc 96F flat bottom TC plate). 190 µl of medium was added to each well of columns 1 and 12. The medium was Dulbecco's Modified Eagles Medium (DMEM) (which included ~10% Fetal Calf Serum (FCS) and ~1% L solution. -Glutamine 200 mM).
Plates were incubated overnight at 37°C before addition of drug if cells were adherent. 200 µM of test compound solutions (in 100% DMSO) were serially diluted through a 96-well plate. Each resulting dot was then further diluted 1/10 in sterile distilled water (SDW).
10% DMSO was added at 5% v/v to cell negative blanks and compound negative control wells. Assay plates were incubated during the following runs at 37°C in 5% CO2 in a humidified incubator for 72 hours. After incubation, MTT solution to a final concentration of 1.5 µM was added to each well. The plates were then incubated for an additional 4 hours at 37°C in 5% CO2 in a humidified incubator. The medium was then removed, and the dye was solubilized in 200 µl DMSO (99.99%).
Plates were read at absorbance at 540 nm using an Envision plate reader. Data was analyzed using Microsoft Excel and GraphPad Prism and IC50 values obtained.
Compound 11 has an IC50 of 11.7 pM in this assay. AML and Kidney Cell Lineage Assays
The cytotoxicity of several drug-free compounds was tested in one renal cancer cell line, 786-O a Hodgkin's lymphoma cell line, L428 and two AML cell lines, HL60 and HEL.
For a 96-hour assay, cells grown in log growth phase were seeded for 24 h in 96-well plates containing 150 µL of RPMI 1640 supplemented with 20% FBS. Serial dilutions of the test article (ie, drug free) into cell culture media were prepared at a working concentration of 4x; 50 uL of each dilution was added to 96-well plates. After addition of the test article, the cells were incubated with the test articles for 4 days at 37°C. Resazurin was then added to each well to obtain a final concentration of 50 µM and the plates were incubated for an additional 4 hours at 37°C. The plates were then read to measure dye reduction in a Fusion HT plate reader (Packard Instruments, Meridien, CT, USA), with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
[00468] Referring to the following Table 1, the para-aniline compound 11 shown significantly increased the activity in these cell lines compared to the meta-aniline compound 19 in this assay. Table 1: IC50 Summary for Over-the-Counter Drugs [nM]

[00469] Referring to the following Table 2, the activity of compounds 28, 30 and 32 is shown in L428, 786-O, HEL, HL-60 and MCF-7 cells, as well as the activity for compound 19 in MCF cells -7. Table 2: IC50 Summary for Over-the-Counter Drugs [nM]

[00470] Referring to the following Table 3, the activities of compounds 23, 25 are compared to those of 1 in 786-O, Caki-1, MCF-7, HL-60, THP-1, HEL, and TF1 cells. Cells were placed in 150 μL of growth medium per well in light-bottomed, dark-walled 96-well plates (Costar, Corning) and allowed to settle for 1 hour in the biological cabin before placing in an incubator at 37°C, 5 % CO2. The next day, 4X drug stock concentration was prepared, and then titrated as 10-fold serial dilutions producing 8-point dose curves and added to 50 µl per well in duplicate. The cells were then incubated for 48 hours at 37oC, 5% CO2. Cytotoxicity was measured by incubation with 100 μL of Cell Titer Glo solution (Promega) for 1 hour, and then luminescence was measured in a Fusion HT plate reader (Perkin Elmer). Data were processed with Excel (Microsoft) and GraphPad (Prism) to produce dose response curves and IC50 values were generated and data collected. Table 3: IC50 Summary for Over-the-Counter Drugs [nM]

In Examples 13 to 16, the following compounds are referred to by compound numbers as shown below:

Example 13: Synthesis of PBD Drug Binding Compounds
[00472]General Information. In the Examples that follow, all commercially available anhydrous solvents were used without further purification. Analytical thin layer chromatography was performed on F254 aluminum sheets of silica gel 60 (EMD Chemicals, Gibbstown, NJ). Radial chromatography was performed on the Chromatotron apparatus (Harris Research, Palo Alto, CA). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Samples were eluted on a C12 Phenomenex Synergi 2.0 x 150 mm, 4 µm, 80 A reverse phase column. The acidic mobile phase consisted of acetonitrile and water both containing 0.05% trifluoroacetic acid or 0.1% formic acid (indicated for each compound). Compounds were eluted with a linear gradient of acidic acetonitrile from 5% in 1 min after injection, to 95% in 11 min, followed by isocratic 95% acetonitrile for 15 min (flow=1.0 mL/min). LC-MS was performed on a ZMD Micromass mass spectrometer interfaced with an HP Agilent 1100 HPLC instrument equipped with a C12 Phenomenex Synergi 2.0 x 150 mm, 4 µm, 80 A reverse phase column. linear gradient of acetonitrile from 5% to 95% in 0.1% aqueous formic acid for 10 min, followed by isocratic 95% acetonitrile for 5 min (flow=0.4 mL/min). Preparative HPLC was performed on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Products were purified on a C12 Phenomenex Synergi 10.0 x 250 mm, 4 µm, 80 reverse phase column. A eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The purification method consisted of the following gradient from solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min with monitoring at 254 nm. NMR spectral data were collected on a Varian Mercury 400 MHz spectrometer. Coupling constants (J) are reported in hertz. Scheme 1

[00473] (S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid ( 36): To a solution of Dipeptide Val-Alum 34 (200 mg, 1.06 mmol) dissolved in 10.6 mL of anhydrous DMF was added NHS ester of maleimidocaproyl 35 (327 mg, 1.06 mmol). Diisopropylethylamine (0.92 mL, 5.3 mmol) was then added and the reaction stirred under nitrogen at room temperature for 18 h, at which time TLC and analytical HPLC showed consumption of starting material. The reaction was diluted with 0.1 M HCL (100 mL), and the aqueous layer was extracted with ethyl acetate (100 mL, 3x). The combined organic layer was washed with water and brine, then dried over sodium sulfate, filtered and concentrated. The crude product was dissolved in minimal methylene chloride and purified by radial chromatography on a 2 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (95:5 to 90:10 CH2Cl2/MeOH) to provide 36 (158 mg, 39 %) as an oily residue. TLC: Rf = 0.26, 10% MeOH in CH2Cl2. 1H NMR (CDCl3) δ (ppm) 0.95 (d, J = 17 Hz, 3H), 0.98 (d, J = 17 Hz, 3H), 1.30 (m, 2H), 1.40 ( d, J = 17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1H), 2.25 (dt, J = 4.19 Hz, 2H), 3.35 (s, 1H), 3.49 (t, J = 17 Hz, 2H), 4.20 (d, J = 18 Hz, 1H), 4.38 (m, 1H), 6.80 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.05 min. LC-MS: tR 11.17 min, m/z (ES+) found 381.9 (M+H)+, m/z (ES-) found 379.9 (M-H)-.
[00474]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(( S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c] [1,4]benzodiazepin-8-yl)oxy)propoxy)-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 (38): A flame-dried 10 ml flask was charged with acid 36 (3.6 mg, 9 .5 µmoles), EEDQ (2.8 mg, 11.4 µmoles), and 0.33 mL of anhydrous CH2Cl2. Methanol (four drops, ~80 µL) was added to facilitate dissolution and the mixture was stirred under nitrogen for 1 h. PBD 37 dimer (5.7 mg, 7.9 µmoles) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS showed product conversion. The reaction was concentrated, dissolved in minimal CH2Cl2 and purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 90:10 CH2Cl2/MeOH) to provide drug binder 38 (3 .9mg, 45%). TLC: Rf = 0.06.5% MeOH in CH 2 Cl 2 , analytical HPLC (0.1% formic acid): tR 11.51 min. LC-MS: tR 12.73 min, m/z (ES+) found 1089.6 (M+H)+, m/z (ES-) found 1087.3 (MH)-. Scheme 2
EEDQ, pyridine, MeOH, CHCl3
[00475]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-((S)-7-methoxy-8-(3-(((S) )-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)- 5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (40): To a flame-dried 10 mL flask was added PBD 37 dimer (25 mg, 34.4 µmoles), which was dissolved in 1.4 mL of 10% MeOH in CHCl3 solvent mixture. Maleimidocaproic Acid (39) was added (7.3 mg, 34.4 μmoles), followed by EEDQ (10.2 mg, 41.3 μmoles) and pyridine (6 μL, 68.8 μmoles). The reaction was stirred at room temperature under a nitrogen atmosphere for 14 h, during which time LC-MS showed product conversion. The reaction was concentrated, dissolved in minimal CH2Cl2 and purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 90:10 CH2Cl2/MeOH) to provide drug binder 40 (14, 1mg, 45%). LC-MS: tR 12.81 min, m/z (ES+) found 918.9 (M+H)+, m/z (ES-) found 917.0 (MH)-. Scheme 3

[00476]2-bromo-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5, 11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11adihydro-1H-pyrrolo[2.1-c][1 ,4]benzodiazepin-2-yl)phenyl)acetamide (41): To a flame-dried 10 mL vial was added PBD 37 dimer (16.5 mg, 22.7 µmoles), which was dissolved in 0. 9 mL of 10% MeOH in CHCl3 solvent mixture. Bromoacetic acid was added (3.2 mg, 22.7 µmoles), followed by EEDQ (6.8 mg, 27.2 µmoles). The reaction was stirred at room temperature under a nitrogen atmosphere for 4 h, at which time LC-MS showed product conversion. The reaction was concentrated, dissolved in minimal CH2Cl2 and purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 95:5 CH2Cl2/MeOH) to provide drug binder 41 (9, 9mg, 52%). TLC: Rf = 0.09, 5% MeOH in CH2Cl2. LC-MS: tR 12.44min, m/z (ES+) found 848.1 (M+H)+, m/z (ES-) found 845.7 (MH)-. Scheme 4

[00477]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-(( S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c] [1,4]benzodiazepin-8-yl)oxy)propoxy)-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 (43): A flame dried 10 ml flask was charged with acid 36 (3.6 mg, 9 .4 μmoles), EEDQ (2.8 mg, 11.3 μmoles), and 0.38 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD 42 dimer (6.8 mg, 9.4 µmoles) was then added and the reaction was stirred at room temperature for 2 h, at which time LC-MS showed product conversion. The reaction was concentrated, dissolved in minimal CH2Cl2 and purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 90:10 CH2Cl2/MeOH) to provide drug binder 43 (3, 1mg, 30%). TLC: Rf = 0.31, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.49 min. LC-MS: tR 12.28 min, m/z (ES+) found 1089.5 (M+H)+, m/z (ES-) found 1087.3 (MH)-.

[00478]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-((S)-7-methoxy-8-(3-(((S) )-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)- 5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (44): To a flame-dried 10 mL flask was added PBD 42 dimer (8.0 mg, 11 µmoles), which was dissolved in 0.44 mL of 10% MeOH in CH2Cl2 solvent mixture. Maleimidocaproic acid (39) was added (2.3 mg, 11 μmoles), followed by EEDQ (3.3 mg, 13.2 μmoles) and pyridine (1.8 μL, 22 μmoles). The reaction was stirred at room temperature under a nitrogen atmosphere for 3 h, at which time LC-MS showed product conversion. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 90:10 CH2Cl2/MeOH) to provide drug binding compound 44 (1.2 mg, 12% ). TLC: Rf = 0.45, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.71 min. LC-MS: tR (ES+) found 919.1 (M+H)+, m/z (ES-) found 917.1 (MH)-. Scheme 6

(2S,3R,4S,5R,6R)-2-(2-(3-(((9H-fluoren-9-Oyl)methoxy)carbonyl)amino)propanamido)-4-( (((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl))-5-oxo-5,11a-dihydro-1H-pyrrole [2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin- 2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl (46): A flame dried flask was charged with glucuronide binder intermediate 45 (reference : Jeffrey et al., Bioconjugate Chemistry, 2006, 17, 831-840) (15 mg, 20 μmoles), 1.4 mL anhydrous CH2Cl2, pyridine (20 μL, 240 μmoles), and then cooled to -78° C under nitrogen. Diphosgene (3.0 μL, 24 μmoles) was then added and the reaction stirred for 2 h at -78 °C, after which time a small aliquot was quenched with methanol and analyzed by LC-MS for carbonate formation. methyl, which confirmed the formation of glucuronide chloroformate. PBD 42 dimer (15 mg, 20 µmoles) was then dissolved in 0.7 mL of anhydrous CH2Cl2 and added dropwise to a reaction vessel. The reaction was warmed to 0 °C for 2 h and then diluted with 50 mL of CH2Cl2. The organic layer was washed with water (50 ml), brine (50 ml), dried over sodium sulfate, filtered and concentrated. The crude reaction product was purified by radial chromatography on a 1 mm chromatotron plate eluted with 10% MeOH in CH 2 Cl 2 to provide 46 (5.7 mg, 19%). TLC: Rf = 0.47, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 12.09 min. LC-MS: tR 14.05 min, m/z (ES+) found 1500.3 (M+H)+.
[00480] (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((3-((S)-7-methoxy-8-(3-() acid ((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy) propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5 -trihydroxytetrahydro-2H-pyran-2-acid (47): One vial containing 46 (5.7 mg, 3.8 µmoles) dissolved in a solvent mixture of 0.2 mL each of MeOH, tetrahydrofuran, and water was cooled to 0 °C. To the stirred solution, lithium hydroxide monohydrate (0.8 mg, 19 µmoles) was added and the reaction was stirred at room temperature for 4 h at which time LC-MS indicated product conversion. Glacial acetic acid (1.1 µL, 19 µmol) was added and the reaction was concentrated to provide 47, which was carried on without further purification. LC-MS: tR 11.59 min, m/z (ES+) found 1138.4 (M+H)+.
[00481] Acid (2S,3S,4S,5R,6S)-6-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido) propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S))-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a -dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2.1-c][ 1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-acid (48): To a solution of 47 (4.3 mg , 3.8 µmoles) dissolved in 0.38 mL of anhydrous DMF, NHS ester of maleimidocaproyl 35 (1.2 mg, 3.8 µmoles) was added, followed by diisopropylethylamine (4.0 µL, 22.8 µmoles). ). The reaction was stirred at room temperature under nitrogen for 2 h, at which time LC-MS showed product conversion. The reaction was diluted with a mixture of acetonitrile (0.5 mL), DMSO (1 mL), water (0.5 mL), and then purified by preparative HPLC. The mobile phase consisted of A = water and B = acetonitrile, both containing 0.1% formic acid. A linear elution gradient from 90:10 A:B to 10:90 A:B over 75 minutes was employed and fractions containing the desired product were lyophilized to provide drug binding compound 48 (1.2 mg, 24% in two steps). Analytical HPLC (0.1% formic acid): tR 10.85 min. LC-MS: tR 12.12min, m/z (ES+) found 1331.4 (M+H)+, m/z (ES-) found 1329.5 (MH)-. Scheme 7

[00482]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-(( S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro- 1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-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 (51): One 10 ml flame-dried flask was loaded with acid 36 (2.7 mg, 7.1 µmoles), EEDQ (2.1 mg, 8.5 µmoles), and 0.28 mL of anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD 49 dimer (5.8 mg, 7.1 µmoles) was then added and the reaction was stirred at room temperature for 20 h, at which time LC-MS showed product conversion. The reaction was concentrated then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug binding compound 51 (2.7 mg, 32%). Analytical HPLC (0.1% formic acid): tR 9.17 min. LC-MS: tR 11.25min, m/z (ES+) found 1185.3 (M+H)+, m/z (ES-) found 1182.9 (M-H)-.
[00483]N-((S)-1-(((S)-1-((3-((S)-8-((5-(((S))-2-(4-(3- ( dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7 -methoxy-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)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (52): A 10 ml flame-dried vial was loaded with acid 36 (3.7 mg, 9.7 μmoles), EEDQ (2.9 mg, 11.6 μmoles), and 0.4 mL of anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD 50 dimer (8.0 mg, 9.7 µmoles) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS revealed the presence of product. The reaction was concentrated, then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug binding compound 52 (3.1 mg, 25%). Analytical HPLC (0.1% formic acid): tR 9.45 min. LC-MS: tR 11.75 min, m/z (ES+) found 1188.4 (M+H)+, m/z (ES-) found 1186.0 (MH)-.

[00484]4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(( S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c] [1,4]benzodiazepin-8-yl)oxy)propoxy)-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)benzamide (54): To a flame-dried 10 ml flask was added linker fragment 53 (7.7 mg, 20 µmoles), which was dissolved in 0.33 mL of 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.1 mg, 25 µmoles) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD 37 dimer (12 mg, 16.5 µmoles) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for a further 3 h, at which time LC-MS showed product conversion. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 90:10 CH2Cl2/MeOH) to provide 54 (2.4 mg, 13%). TLC: Rf = 0.44, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.53 min. LC-MS: tR 12.61 min, m/z (ES+) found 1095.4 (M+H)+, m/z (ES-) found 1093.9 (M-H)-.
[00485](S)-2-(2-iodoacetamido)-N-((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-) methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo- 5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (56): One vial Flame dried was charged with binder 55 (7.8 mg, 22 µmoles), which was dissolved in 0.37 mL of 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.8 mg, 27.5 µmoles) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD 37 dimer (13 mg, 18 µmoles) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h, at which time LC-MS showed product conversion. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 80:20 CH2Cl2/MeOH) to provide 56 (3.5 mg, 18%). Analytical HPLC (0.1% formic acid): tR 11.43 min. LC-MS: tR 12.49 min, m/z (ES+) found 1064.6 (M+H)+, m/z (ES-) found 1098.9 (M+2H2O-H)-.

[00486]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(( S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c) ][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-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 (58): To a flame-dried 10 ml flask was added ligand fragment 36 ( 19 mg, 50 μmoles), which was dissolved in 0.33 mL of 5% MeOH in CH2Cl2 solvent mixture. EEDQ (12.4 mg, 50 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD 57 dimer (12.5 mg, 16.6 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 5 h, at which time LC-MS showed product conversion. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 80:20 CH2Cl2/MeOH) to provide 58 (2.1 mg, 11%). Analytical HPLC (0.1% formic acid): tR 12.19 min. LC-MS: tR 12.58 min, m/z (ES+) found 1117.8 (M+H)+, m/z (ES-) found 1133.7 (M+H2O-H)-. Scheme 10

[00487](R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoic acid (60): A flame-dried flask was charged with Fmoc-D-Valine (200 mg, 0.59 mmol) and 5.9 mL of anhydrous THF. N-hydroxysuccinimide (75 mg, 0.65 mmol) was added, followed by diisopropylcarbodiimide (0.1 mL, 0.65 mmol), and the reaction was stirred at room temperature overnight, at which time LC-MS showed conversion. of product. The reaction mixture was diluted with CH2Cl2 and washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to dryness. The material was carried on without further purification. LC-MS: tR 13.89 min, m/z (ES+) found 437.0 (M+H)+. Crude Fmoc-D-Val-OSu (0.59 mmol) was dissolved in dimethoxyethane (1.5 mL) and THF (0.8 mL). D-alanine (73 mg, 0.89 mmol) was dissolved in 2.3 mL water and added to the reaction mixture, followed by sodium bicarbonate (99 mg, 1.2 mmol). The resulting slurry was stirred at room temperature overnight, at which time the reaction cleared and LC-MS showed completion. The reaction was poured into 50 mL CH2Cl2 and the organic layer was washed with 50 mL 0.1 M HCL and then brine, dried over sodium sulfate, and then concentrated to dryness. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2 to provide 60 (128 mg, 54%). TLC: Rf = 0.18, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 9.47 min. LC-MS: tR 13.09 min, m/z (ES+) found 411.1 (M+H)+, m/z (ES-) found 409.2 (M-H)-.
[00488](R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid ( 61): Protected Dipeptide 60 (70 mg, 0.37 mmol) was suspended in 6 mL of anhydrous CH 2 Cl 2 , cooled in ice under nitrogen, and 2 mL of diethylamine was added dropwise. The reaction was warmed to room temperature and stirred under nitrogen for 2 h, at which time HPLC showed consumption of starting material. The reaction was diluted with 6 mL of chloroform and concentrated. The crude reaction residue was re-dissolved in 6 mL chloroform and concentrated twice, followed by drying on a vacuum line for 2 h. The protected depeptide was then dissolved in 3.7 mL of anhydrous DMF. MC-OSu (138 mg, 0.44 mmol) was then added, followed by diisopropylethylamine (0.32 mL, 1.9 mmol). The reaction was stirred under an atmosphere of nitrogen at room temperature overnight. Workup was achieved by pouring the reaction into 50 mL of 0.1 M HCL and extracting with ethyl acetate (50 mL, 3x). The combined organic layer was washed with water (50 ml) and brine (50 ml), dried over sodium sulfate and concentrated. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (99:1 to 95:5 CH2Cl2/MeOH) to provide 61 (14 mg, 22%). 1H NMR (CD3OD) δ (ppm) 0.94 (d, J = 14 Hz, 3H), 0.98 (d, J = 14 Hz, 3H), 1.29 (m, 2H), 1.39 ( d, J = 7.4 Hz, 3H), 1.61 (m, 4H), 2.05 (m, 1H), 2.25 (dt, J = 1.2, 7.4 Hz, 2H), 3.48 (t, J = 7Hz, 2H), 4.19 (m, 1H), 4.37 (m, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 10.04 min. LC-MS: tR 11.22 min, m/z (ES+) found 382.1 (M+H)+, m/z (ES-) found 380.0 (M-H)-.
[00489]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((R)-1-(((R)-1-((4-(( S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c] [1,4]benzodiazepin-8-yl)oxy)propoxy)-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 (62): To a flame-dried 10 ml flask was added binder 61 (9.5 mg, 25 µmoles), which was dissolved in 0.33 mL of 5% MeOH in CH2Cl2 solvent mixture. EEDQ (7.3 mg, 30 µmoles) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD 37 dimer (12 mg, 16.5 µmoles) was added. The reaction was stirred at room temperature under an atmosphere of nitrogen for a further 3 h, at which time LC-MS showed product conversion. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 80:20 CH2Cl2/MeOH) to provide 62 (2.8 mg, 16%). TLC: Rf = 0.39, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.50 min. LC-MS: tR 12.50 min, m/z (ES+) found 1089.7 (M+H)+, m/z (ES-) found 1088.0 (MH)-.
(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanoic (64): L-alanine (58 mg, 0.65 mmol) was suspended in 6.5 mL of anhydrous DMF and MC-OSu 35 (100 mg, 0.324 mmol) was then added. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added and the reaction was stirred overnight at room temperature under nitrogen. The reaction was then diluted with 50 mL 0.1M HCL and the aqueous layer was then extracted with ethyl acetate (50 mL, 3x). The combined organic layer was then washed with water (50 ml) and brine (50 ml), dried over sodium sulfate, and then concentrated to dryness. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (97.5:2.5 to 90:10 CH2Cl2/MeOH) to provide 64 (25 mg, 27%). TLC: Rf = 0.25, 10% MeOH in CH2Cl2. 1H NMR (CD3OD) δ (ppm) 1.30 (m, 2H), 1.37 (d, J = 7.4 Hz, 3H), 1.60 (m, 4H), 2.21 (t, J = 7.4 Hz, 2H), 3.48 (t, J = 7 Hz, 2H), 4.35 (q, J = 7.4 Hz, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.06 min.
[00490] (S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanoic acid (64): L-alanine (58 mg, 0, 65 mmol) was suspended in 6.5 mL of anhydrous DMF and MC-OSu 35 (100 mg, 0.324 mmol) was then added. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added and the reaction was stirred overnight at room temperature under nitrogen. The reaction was then diluted with 50 mL 0.1M HCL and the aqueous layer was then extracted with ethyl acetate (50 mL, 3x). The combined organic layer was then washed with water (50 ml) and brine (50 ml), dried over sodium sulfate, and then concentrated to dryness. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (97.5:2.5 to 90:10 CH2Cl2/MeOH) to provide 64 (25 mg, 27%). TLC: Rf = 0.25, 10% MeOH in CH2Cl2. 1H NMR (CD3OD) δ (ppm) 1.30 (m, 2H), 1.37 (d, J = 7.4 Hz, 3H), 1.60 (m, 4H), 2.21 (t, J = 7.4 Hz, 2H), 3.48 (t, J = 7 Hz, 2H), 4.35 (q, J = 7.4 Hz, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.06 min.
[00491]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-((4-((S)-7-methoxy-8 -(3- (((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8 -yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2- yl)hexanamide (65): To a flame-dried 10 mL vial, binder 64 (14 mg, 50 μmoles) was added, which was dissolved in 0.66 mL of 5% MeOH in CH2Cl2 solvent mixture. EEDQ (15 mg, 60 μmoles) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD 37 dimer (24 mg, 33 μmoles) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with mixtures of CH2Cl2/MeOH (100:0 to 90:10 CH2Cl2/MeOH) to provide 65 (3.5 mg, 11%). Analytical HPLC (0.1% formic acid): tR 11.40 min. LC-MS: tR 12.39 min, m/z (ES+) found 990.6 (M+H)+, m/z (ES-) found 989.0 (MH)-. Scheme 12

[00492] PBD 57 dimer bonded directly through maleimidocaproyl spacer (Scheme 14): PBD 57 dimer is coupled to maleimidocaproic acid 39 employing the chemistry described in Scheme 2.

[00493]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((S)-7-methoxy-8-(3-(((S) )-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)- 5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (68): To a mixture of 66 (10mg, 0.013mmol) in CH2Cl2 (300 □ L) were added DIPEA and MC-Cl (67) (3 mg, 0.013 mmol). After 1h, 3 more equiv. of DIPEA (7 µL) and 2 equiv. of the acid chloride (6 mg, 0.026 mmol) were added. After 1h, more amount of DIPEA (7 µL) and acid chloride (6 mg, 0.026 mmol) were added. After another 3h, the reaction mixture was aspirated directly onto a 1mm chromatotron plate and eluted with dichloromethane followed by a gradient of methanol (1% to 5%) in dichloromethane. Fractions containing product, as a mixture with the starting aniline, were concentrated to a residue and dissolved in a mixture of 0.5 ml DMSO, 0.5 ml acetonitrile and 0.5 ml deionized water and further purified by preparative HPLC. The highest peak was collected and the fractions were combined, frozen and lyophilized to give 2.1 mg (18%): MS (ES+) m/z 919.2 [M+H]+.
Note: Acid chloride 67 was prepared by dissolving 100 mg of 39 in oxalyl chloride (5 mL). One drop of DMF was added and the mixture was stirred at room temperature for several hours before being concentrated under reduced pressure. Dichloromethane was added and the mixture was concentrated a second time to give an off-white solid which was used directly: : 1H-NMR (400MHz, CDCl3) □ 6.70 (s, 2H), 3.46 (t, J = 7 Hz , 2H), 2.82 (t, J = 7.2 Hz, 2H), 1.72 (pent, J = 7.6 Hz, 2H), 1.61 (pent, J = 7.4 Hz, 2H ), 1.35 (pent, J = 7.6 Hz, 2H). Scheme 14

[00495] 2-(2-aminoacetamido)acetate tert-butyl (69): To a mixture of tert-butyl ester hydrogen chloride salt of glycine (70) (484 mg, 2.9 mmoles) in dichloromethane (25 mL) was added Fmoc-Gly-OH (71) (0.861 mg, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was stirred at room temperature for 16 h and then poured into ethyl acetate and washed with water (3X) and brine (1X). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified by radial chromatography on a 2 mm plate eluting with 5% methanol/dichloromethane. Fractions containing product were concentrated under reduced pressure and treated with 20% piperidine/dichloromethane (10 mL) for 1h, before being concentrated under reduced pressure and then purified twice by radial chromatography on a 2 mm plate eluting with a gradient of 5 to 10% methanol/dichloromethane to give (200 mg, 37%): 1 H-NMR (400MHz, CDCl 3 ) □ 7.62 (s, 1H), 4.00 (s, 2H), 3 .39 (s, 2H), 1.47 (s, 9H).
[00496] 2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetamido)acetic acid (72): To a solution of amine 69 (200) mg, 0.11 mmol) in DMF (1 mL) was added (350 mg, 0.11 mmol) and the reaction mixture was allowed to stir at room temperature for 2 h. The mixture was concentrated under reduced pressure and purified by radial chromatography on a 1 mm plate eluting with dichloromethane and a gradient of methanol (1 to 5%) in dichloromethane. Fractions containing product were concentrated under reduced pressure, dissolved in dichloromethane (4ml) and treated with trifluoroacetic acid (4ml). After 40 min, the mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated to give 22.5 mg (19%) of 72 as a white solid: 1H-NMR (400 MHz, CD3OD) □□ 6, 79 (s, 2H), 3.93 (s, 2H), 3.89 (s, 2H), 3.49 (t, J = 6.8 Hz, 2H), 2.26 (t, J = 6 .8 Hz, 2H), 1.61 (m, 4H), 1.34 (m, 2H); MS (ES+) m/z 326.21 [M+H]+.
[00497] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((4-((S)-7-methoxy-8) -(3- (((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8 -yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-2-oxoethyl)amino) -2-oxoethyl)hexanamide (73): To a mixture of 72 (15 mg, 0.046 mmol) in 5% methanol/dichloromethane (0.5 mL) was added EEDQ (11 mg, 0.046 mmol) and the mixture was stirred for 30 min at room temperature, at which time 37°C (16 mg, 0.023 mmol) was added. The reaction mixture was stirred for 3 h and purified directly on a 1 mm radial chromatotron plate eluting with 1% to 4% methanol/dichloromethane gradient to give 6.8 mg (29%) of 73 as a yellow solid. : MS (ES+) m/z 1033.57 [M+H]+.

[00498] 1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (S)-tert-butyl ester (74): To a hydrogen chloride salt mixture of L-proline-tert-butyl ester 75 (0.5 g, 2.9 mmol) in dichloromethane (50 mL) was added 76 (0.98 g, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g , 3.5 mmoles). The reaction mixture was allowed to stir at room temperature for 16 h. The mixture was poured into ethyl acetate (100 ml) and washed with 0.2 N HCl (50 ml), water (50 ml), brine (50 ml) and dried over MgSO4. Chromatography was conducted on a 2 mm radial chromatotron plate eluting with 10% ethyl acetate in hexanes. Fractions containing product were concentrated under reduced pressure, dissolved in dichloromethane (8ml) and treated with piperidine (2ml). The mixture was stirred for 1 h, concentrated under reduced pressure and purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane. This gave 200 mg (26%) of the dipeptide 74: 1H-NMR (400 MHz, CDCl 3 ) □ D4.41 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H) , 3.57 (m, 4H), 3.2 (m, 1H), 2.82 (m, 1H), 2.83-1.65 (m, 5H), 1.44 (m, 9H).
[00499]1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (S)-tert-butyl (77): To a mixture of amine 74 (200 mg, 0.75 mmol), 39 (190 mg, 0.9 mmol) and DIPEA (0.32 mL, 1.8 mmol) HATU (342 mg, 0.9 mmol) was added and the mixture was allowed to stir at room temperature for 5h. The mixture was poured into ethyl acetate (100ml) and washed with water (3X100ml) and brine (1X100ml). The organic phase was dried over magnesium sulfate, filtered and concentrated. The resulting residue was subjected to radial chromatography on a 2 mm radial chromatotron plate eluting with dichloromethane followed by an increasing gradient of 1 to 5% methanol in dichloromethane. Two additional purifications, both eluting with a gradient of 1 to 5% methanol in dichloromethane, first on a 2 mm plate and then on a 1 mm plate yielded 113 mg (33%) of 77 as a solid. white: 1H-NMR (400 MHz, CDCl3) □ □ 4.63 (m, 1H), 4.41 (m, 1H), 3.82 (m, 1H), 3.6 3 (m, 1H), 3.55 (m, 1H), 3.45 (m, 3H), 2.38-1.83 (m, 10H), 1.70-1.50 (m, 5H), 1.45 (m, 9H), 1.35 (m, 2H); MS (ES+) m/z 462.33 [M+H]+.
[00500] (S)-1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine acid -2-carboxylic (78): To a mixture of tert-butyl ester 77 in dichloromethane (4 ml) was added trifluoroacetic acid (4 ml). After 40 min, the reaction was determined to be complete by HPLC analysis. The mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated a second time to give 37 mg (100%) of 78 as a white solid: 1 H-NMR (400 MHz, CDCl 3 m 6.68 (s) , 2H), 4.62 (m, 2H), 3.81 (m, 1H), 3.70 (m, 1H), 3.57 (m, 2H), 3.45 (m, 2H), 2 .40-1.91 (m, 10H), 1.70-1.45 (m, 4H), 1.33 (m, 2H); MS (ES+) m/z 406.2 [M+H]+ .
[00501]1-(1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)-N-(4-((S) -7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1 ,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)pyrrolidine-2 -carboxamide (79): To a mixture of 78 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (0.4 mL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 15 min at room temperature and then 37 (15 mg, 0.021 mmol) was added. The mixture was stirred for 4 h, the reaction mixture was diluted with dichloromethane (2mL) and was aspirated directly onto a 1 mm radial chromatotron plate. The product was eluted with a gradient of 1 to 5% methanol in dichloromethane to provide 6.8 mg (29%) of 79 as a yellow solid: MS (ES+) m/z 1113.51 [M+H]+. Scheme 16

[00502](S)-5-(Allyloxy)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-oxopentanoic acid (80): To a resin mixture of 2-chlorotrityl (1.0 g, 1.01 mmol) suspended in dichloromethane (10 ml) was added Fmoc-Glu-(OAlyl)-OH (81) (409 mg, 1.0 mmol) and DIPEA (173 DL , 1.0 mmol). The reaction mixture was stirred for 5 min, and an additional portion of DIPEA (260 DL, 1.5 mmol) was added and the mixture was stirred for 1h. Methanol (0.8 ml) was added and the mixture was stirred for 5 min, before being filtered and washed with DMF (6X), dichloromethane (6X), diethyl ether (6X) and dried under reduced pressure. The resulting resin was subjected to 20% piperidine in dichloromethane (10ml) for 1h, before being filtered and washed with DMF (6X), dichloromethane (6X), diethyl ether (6X) and dried under reduced pressure.
To a mixture of Fmoc-Val-OH (82) (1.03 g, 3.30 mmol) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3 .03 mmoles). After complete mixing, the solution was aspirated into a 10 mL syringe containing the resin prepared above. The mixture was capped and stirred for 16 h. The resin was washed with DMF (6X), dichloromethane (6X) and ether (6X). A small portion (10 mg) was isolated and treated with 20% TFA/Dichloromethane and the resulting solution analyzed by LC-MS which revealed a high purity peak which had the correct mass (MS (ES+) m/z 509.28 [M+H]+). The remaining resin was then treated with 20% piperidine/DMF (8 mL) for 2 h, before being washed with DMF (6X), dichloromethane (6X), diethyl ether (6X) and dried under reduced pressure.
A mixture of allyl chloroformate (529 αL, 5.05 mmoles), DIPEA (1.7 mL, 10 mmol) in dichloromethane (10 mL) was prepared and aspirated into a syringe containing the above resin. The mixture was capped and stirred. After approximately 2 h, the reaction mixture was drained, and washed with dichloromethane (6X). A small portion of the resin (~10 mg) was cleaved with 20% TFA/dichloromethane and analyzed by LC-MS for masses of starting material and product. The main component was still the unreacted amine, so the resin was again subjected to the conditions described above. After 4 h, the resin was washed with dichloromethane (6X), and then repeatedly treated with 5% TFA in dichloromethane (4X7 mL). The resulting solution was concentrated under reduced pressure. The mixture was purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 107 mg of 80:1H-NMR (400 MHz, CDCl3) αα 7.05 (s, 1H), 5.90 (m, 2H), 5.57 (d, 1H), 5.29 (d, J = 14.7 Hz, 2H), 5.22 (t, J = 10.9 Hz, 2H), 4.59 (m, 5H), 4.02 (m, 1H), 2.60-2.40 (m, 2H), 2.37-2.18 (m, 1H), 2.17-2.02 (m) , 2H), 0.96 (d, J=6.4 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H); MS (ES+) m/z 371.12 [M+H]+.
[00505]4-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-((( S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy) (S)-allyl -5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoate (83): To a mixture of acid 80 (30, 0.04 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (20 mg, 0.082 mmol). The mixture was stirred for 30 min at room temperature and then 37°C (30 mg, 0.04 mmol) was added and the mixture was stirred for approximately 5 h. Partial purification by aspirating directly onto a 1 mm radial chromatotron plate and eluting with a gradient of 1% methanol/5% dichloromethane yielded a mixture of the desired product and 37 (26 mg; ~3:1 respectively) which was transported without further purification.
[00506] (S)-4-((S)-2-amino-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3- (((S)-) acid) 7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5- oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoic (84): To a mixture of 83 and 37 (26 mg ) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 mmol), pyrrolidine (4 DL, 0.048 mmol) and palladium tetrakis (0.7 mg, 0.6 nmol). After 2 h, a further amount (0.7 mg, 0.6 Dmol) of tetrakis palladium was added and the reaction was allowed to stir for a further 1 h before being concentrated under reduced pressure. The residue was dissolved in DMSO (1 ml), acetonitrile with 0.05% formic acid (1 ml) and water with 0.05% formic acid (1 ml) and purified by preparative reverse phase HPLC. A single product fraction was collected and lyophilized to give 6 mg (14% for two steps) of 84: MS (ES+) m/z 1078.5 [M+H]+.
[00507] (S)-4-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5 acid -((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrole) [2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin- 2-yl)phenyl)amino)-5-oxopentanoic (85): To a mixture of 84 (6 mg, 6 Dmoles), and 35 (2 mg, 6 Dmoles) in DMF (200 DL) was added DIPEA (3 DL) , 18 Dmoles) and the reaction mixture was stirred at room temperature. After 1h, another equivalent of 35 (2mg, 6Dmoles) was added and the reaction was allowed to continue stirring at room temperature for 3h. A third equivalent of 35 (2 mg, 6 Dmoles) was added and the mixture was stirred for approximately 1 h, concentrated under reduced pressure, dissolved in dichloromethane and aspirated directly onto a 1 mm radial chromatotron plate and eluted with 5% methanol in dichloromethane. This gave 2.5 mg (36%) of high purity 85: MS (ES+) m/z 1147.49 [M+H]+.
[00508] Acid (21S,24S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-isopropyl-24- ((4-((S)-7 -methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4 ]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)-3, 19,22-trioxo-7,10,13,16-tetraoxa-4,20,23-triazaheptacosan-27-oic (86): To a mixture of 84 (8mg, 8.4 nmoles) and Mal-PEG4-NHS (87) (6.5 mg, 12.6 moles) in DMF (200 DL) was added DIPEA (4.3 DL, 25 nmoles). The reaction mixture was stirred at room temperature for 2 h, and was concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane and aspirated onto a 1 mm radial chromatotron plate. The material was polar and did not chromatograph on the silica gel-based chromatotron plate. The plate was eluted with methanol to recover the mixture which was isolated under reduced pressure. The residual material was purified by preparative reverse phase HPLC. A single major peak eluted and fractions were combined, frozen and lyophilized to a 0.9 mg (8%) residue of 86: MS (ES+) m/z 1353.04 [M+H]+. Scheme 17

[00509] (S)-6-(Dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)hexanoic acid (88): To a mixture of the resin of 2-chlorotrityl (1 g, 1.01 mmol) in CH 2 Cl 2 (10 mL) was added Fmoc-Lys(Me) 2-OH (89) (432 mg, 1.0 mmol) and DIPEA (433 DL, 2.5 mmoles). The reaction mixture was stirred for 1 h. Methanol (0.8 ml) was added and the mixture stirred for a further 5 min, before being filtered and washed with DMF (6x), dichloromethane (6x), diethyl ether (6x) and dried under reduced pressure. The dried resin was subjected to 20% piperidine in DMF (10 mL) for 1h, before being filtered and washed with DMF (6X), dichloromethane (6X), diethyl ether (6X).
To a mixture of 39 (3.0 mmol, 633 mg) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After complete mixing, the solution was aspirated into a 10 mL syringe containing the above resin. The mixture was capped, stirred for 16 h, filtered and the resin washed with DMF (6X), dichloromethane (6X), and ethyl ether (6X). The resin was repeatedly treated with 5% TFA/dichloromethane (6 mLX5), shaken for 1 min, and then filtered. The resulting solution was concentrated under reduced pressure and under high vacuum. The material was purified by preparative reverse phase HPLC to give 208 mg of 88:1H-NMR (400 MHz, CD3OH/CDCl3 1:1 mixture)DDD 6.73 (s, 2H), 4.41 (m, 1H) , 3.48 (t, 2H), 3.31 (s, 1H), 3.03 (m, 2H), 2.84 (s, 6H), 2.22 (m, 2H), 1.87 ( m, 2H), 1.78-1.52 (m, 6H), 1.43 (m, 2H), 1.31 (pent, 2H); MS (ES+) m/z 386.28 [M+H]+.
[00511](S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-(4-((S) )-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][ 1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide ( 90): To a mixture of 88 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (400 DL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 30 min at room temperature and then 37 (15 mg, 0.021 mmol) was added. After 4 h, the mixture was concentrated under reduced pressure, dissolved in a mixture of DMSO (1 ml), acetonitrile (2 ml containing 0.05% foric acid) and water (1 ml containing 0.05% foric acid) and purified by reverse phase HPLC (method A). Fractions containing product were contaminated with 37, then fractions were lyophilized to a residue and repurified as described above to give 0.5 mg (2%) of pure 90: MS (ES+) m/z 537.46 [M +H]/2+. Scheme 18

[00512] ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4) -methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-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)carbamate of Alila (91): To a mixture of 92 (45 mg, 0.123 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (30.4 mg, 0.123 mmol). The mixture was stirred for 30 min at room temperature and then 37 (30 mg, 0.041 mmol) was added. The reaction mixture was stirred for approximately 5 h and then purified on a 1 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 22mg (55%) of 91 which was not characterized but transported directly .
[00513]1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((S)-1-(((S)-1-( (4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2 ,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-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)-3,6,9,12-tetraoxapentadecan-15-amide (93): To a To a solution of 91 (22 mg, 0.022 mmol) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 mmol), pyrrolidine (4 DL, 0.048 mmol) and palladium tetrakis (0.7 mg, 6 nmoles). After approximately 2h, the reaction mixture was purified on a 1mm radial chromatotron plate eluting with 5% to 10% methanol/dichloromethane. Most of it was collected and concentrated to a residue which was dissolved in DMF (0.2 mL) and reacted with NHS ester 87 (10 mg, 0.19 mmol). The reaction was allowed to stir for 30 min, concentrated and purified by radial chromatography on a 1 mm plate eluting with 5% methanol/dichloromethane to give 3.2 mg (11%) of 93: MS (ES+) m/ z 1294.7 [M+H]+. Scheme 19

[00514](E)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N'-(4-((S)-7-methoxy-8-(( 5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl )oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)benzylidene)hexanehydrazide (94): To a mixture of aldehyde 95 (5.4 mg, 7 nmoles) in 5% methanol/dichloromethane at 0 °C was added the hydrazide-TFA salt 96 (4.5 mg, 14 nmoles). The reaction mixture was allowed to warm to room temperature and stir for 5 h before being concentrated under reduced pressure and purified on a silica gel column eluting with 3% methanol/dichloromethane to give 2.2 mg (32% ) of 94: MS (ES+) m/z 974.49 [M+H]+. Scheme 20

[00515](S)-tert-butyl 2-((S)-2-amino-3-methylbutanamido)propanoate (97): To a mixture of alanine-O-tert-butyl ester hydrogen chloride salt (98) ( 500mg, 2.76mmol) in dichloromethane (5ml) was added Fmoc-val-OSu (99) (1.09g, 2.51mmol). DIPEA (0.96 ml, 5.5 mmol) was added and the reaction mixture was allowed to stir at room temperature for 16 h. The mixture was poured into dichloromethane (100ml) and washed with 1N HCl (50ml) and water (50ml) before being dried over magnesium sulphate. The material was chromatographed on a 2 mm radial chromatotron plate eluting with a 1 to 5% methanol/dichloromethane gradient and fractions containing product were combined and concentrated. The resulting residue was dissolved in dichloromethane (16 ml) and piperidine (4 ml) was added. The mixture was stirred for 10 min before being concentrated under reduced pressure. The resulting residue was chromatographed on a 2 mm plate eluting first with ammonia saturated dichloromethane followed by 5% methanol in ammonia saturated dichloromethane to give 494 mg (2.02 mmoles, 81% for two steps) of 97: 1H- NMR (400 MHz, CDCl 3 ) □ □□ 7.78 (bs, 1H), 4.47 (m, 1H), 3.30 (d, 1H), 2.30 (m, 1H), 1.38 ( d, 3H), 1.47 (s, 9H), 1.00 (d, J = 7.0 Hz, 3H), 0.84 (d, J = 6.9 Hz, 3H).
[00516] 2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoate (S)-tert -butyl (100): To a mixture of 97 (100 mg, 0.41 mmol) and 4-maleimidobenzoic acid (101) (98 mg, 0.45 mmol) was added dichloromethane (5 mL), followed by TBTU (157 mg, 0.49 mmol) and DIPEA (212 µL, 1.23 mmol). The mixture was stirred at room temperature for 16 h and then purified on a 2 mm radial chromatotron plate eluting with 50% ethyl acetate in hexanes to give 95 mg (51%) of 100:1H-NMR (400 MHz, CDCl3) □ □ 7.85 (d, J = 6.6 Hz, 2H), 7.42 (d, J = 6.6 Hz, 2H), 6.81 (s, 2H), 6.38 (bs, 1H), 4.43 (m, 2H), 2.14 (sept, J = 6.6 Hz, 1H), 1.41 (s, 9H), 1.31 (d, J = 7. 0 Hz, 3H), 0.98 (m, 6H); MS (ES-) m/z 441.90 [M-H]-.
[00517](R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic acid ( 53): To a mixture of 100 (47 mg, 0.11 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was monitored by TLC (50% ethyl acetate in hexane, after pumping the TLC plate under high vacuum for 5 min). After 75 min, no starting material could be detected by TLC. The reaction was carried out a second time using the same conditions and material from both reactions was combined and purified on a 2 mm radial chromatotron plate eluting with a gradient of 510% methanol in dichloromethane. Yield was 42 mg (49%) of 53: 1 H-NMR (400 MHz, CDCl 3 ) □ □7.92 (d, J = 6.6 Hz, 2H), 7.51 (d, J = 6.6 Hz, 2H), 7.0 (m, 1H), 6.89 (s, 2H), 6.70 (s, 1H), 4.60 M, 1H), 2.22 (m, 1H), 1 .18 (d, J = 6.6 Hz, 3H), 1.04 (m, 6H); MS (ES+) m/z 388.02 [M+H]+.
[00518] (S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (102): To a mixture of 97 (100 mg, 0.41 mmol) in dichloromethane was added iodoacetamide-NHS ester (103) (115 mg, 0.41 mmol) and the mixture was stirred at room temperature. After 30 min, the mixture was aspirated onto a 1 mm chromatotron plate and eluted with ethyl acetate in hexanes (1:1). A single part was collected and the structure was confirmed: 1H-NMR (400 MHz, CDCl3) □□ 6.70 (d, J = 7.8 Hz, 1H), 6.27 (d, J = 7.0 Hz) , 1H), 4.45 (m, 1H), 4.26 (dd, J = 8.5, 6.3 Hz, 1H), 3.72 (quart, J = 11.3 Hz, 2H), 2 1.13 (sept, J = 6.5 Hz, 1H), 1.47 (s, 9H), 1.38 (d, J = 7.1 Hz, 3H), 0.99 (m, 6H); MS (ES+) m/z 412.87 [M+H]+.
[00519](S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (55): See procedure for the synthesis of (R)-2-((S)- 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic (53). This gave 22 mg (15% for two steps): 1H-NMR (400 MHz, D6-DMSO) □ □ 8.27 (d, J = 9.4 Hz, 1H), 4.24 (m, 2H), 3.97 (bs, 2H), 3.83 (d, J = 9.4 Hz, 1H), 3.71 (d, J = 9.6 Hz, 1H), 2.07 (m, 1H), 1.33 (d, J = 7.3 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H); MS (ES-) m/z 354.84 [MH]-.

[00520]Dimers of PBD linked via aliphatic amines (Scheme 21). PBD dimers containing aliphatic amines, such as the benzyl amine (Example 9), are synthesized as peptide linkers, the glucuronide linker, and/or mAb degradation-dependent linkers for release (ie, non-cleavable linkers). Drug ligands conjugated through a benzyl amine will include: (1) a cleavable peptide employing chemistry similar to Scheme 1; (2) direct connection

Generic peptide-linked 2-, 3-, and 4-aniline PBD dimers (Scheme 22). PBD dimers with anilines at the 2-, 3-, and 4-positions will be conjugated to peptide-based linkers, employing the chemistry described in Scheme 1, or directly linked with maleimidocaproic acid, as exemplified in Scheme 2. Example 14: Preparation of PDB Dimer Conjugates
Antibody-drug conjugates were prepared as previously described (see Doronina et al., Nature Biotechnology, 21, 778-784 (2003)) or as described below. Briefly, for maleimide drug binder mAbs (4-5 mg/ml) in PBS containing 50 mM sodium borate at pH 7.4 were reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37°C. The progress of the reaction, which reduces interchain disulfides, was monitored by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb was reached. The reduced antibody was then cooled to 0°C and alkylated with 1.5 equivalents of maleimide drug binder per thiol antibody. After 1 h, the reaction was quenched by the addition of 5 equivalents of N-acetyl cysteine. The quenched drug binder was removed by gel filtration on a PD-10 column. The ADC is then sterile filtered through a 0.22 µm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size Exclusion Chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the levels of quench-cold ligand-drug - NAC remaining.
[00523] For halo acetamide-based drug binders, conjugation was generally performed as follows: To 10 mg/ml of reduced and reoxidized antibody solution (having cysteines introduced by substitution of S239C in heavy chains (see below)) in 10 mM Tris (pH 7.4), 50 mM NaCl and 2 mM DTPA was added 0.5 volume of propylene glycol. A 10 mM solution of acetamide-based drug binder in dimethylacetamide was prepared just prior to conjugation. An equivalent amount of propylene glycol as added to the antibody solution was added to a 6-fold molar excess of the drug binder. The diluted drug binding solution was added to the antibody solution and the pH was adjusted to 8.0-8.5 using 1M Tris (pH 9). The conjugation reaction was allowed to proceed for 45 min at 37 °C. Conjugation was verified by PLRP-S reverse phase chromatography of reduction and denaturation. Excess drug binding was removed with Quadrasil MP resin (Sigma Aldrich; Product # 679526) and the buffer was exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl, and 5% propylene glycol using a PD-10 desalting column (GE Heathcare; Product # 17-0851-01).
[00524] hIgG1 antibodies grown with introduced cysteines: CD70 antibodies containing a cysteine residue at position 239 of the heavy chain (h1F6d) were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA 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 reoxidize the native disulfides, while leaving cysteine 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 to a final concentration of 33% (eg, if the antibody solution was in a reaction volume of 60 mL, 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 drug-binding solution of PBD in propylene glycol was added to the antibody solution to effect conjugation; the final concentration of propylene glycol is 50%. The reaction was allowed to proceed for 30 minutes and then quenched by the addition of 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 as its sole purpose is to maintain the solubility of the drug binder in the aqueous medium). Example 15: Determination of In Vitro Activity of Selected Conjugates
The in vitro cytotoxic activity of selected antibody drug conjugates was evaluated using a resazurin reduction assay (Sigma, St. Louis, MO, USA) (reference: Doronina et al., Nature Biotechnology, 2003, 2003, 21, 778-784). antibody drug conjugates were prepared as described above in Example 13.
For a 96-hour assay, cells grown in log growth phase were seeded for 24 h in 96-well plates containing 150 µL of RPMI 1640 supplemented with 20% FBS. Serial dilutions of ADC in cell culture media were prepared at a working concentration of 4x; 50 uL of each dilution was added to 96-well plates. After the addition of the ADC, the cells were incubated with the test articles for 4 days at 37°C. Resazurin was then added to each well to obtain a final concentration of 50 µM and the plates were incubated for an additional 4 hours at 37°C. The plates were then read to measure dye reduction in a Fusion HT plate reader (Packard Instruments, Meridien, CT, USA), with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
[00527] Referring to Table 4 (infra), the in vitro cytotoxicity of ADCs having para-aniline PBD dimers using the 96 hour assay is shown. ADCs were tested against CD70+ CD30- cell lines and a CD70-CD30 control cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see United States Published Application No. 2009-148942), a CD30 antibody, chimeric AC10 (see United States Published Application No. 2008-0213289) and a CD70 antibody ( humanized 1F6) having introduced cysteine residues at position 239 of the heavy amino acid chain (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) had a lower IC50 than conjugates having a maleimidyl- or acetamide-based linker (compounds 40 and 41, respectively). In Vitro Cytotoxic Activity of ADCs with Drug Ligands Derived from Para-aniline PBD 37 Dimer: Table 4. In vitro Cytotoxic Activity on CD70+ Cell Lines (ng/mL), All ADCs 2 Drugs/mAb

[00528] Referring to Table 5, the in vitro cytotoxicity of ADCs conjugated to PBD dimers in CD30+ cell lines using the 96 hour assay is shown. ADCs were tested against CD70+ CD30- cell lines and one CD70-CD30 cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see United States Published Application No. 2009148942), a CD30 antibody, chimeric AC10 (see United States Published Application No. 2008-0213289). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) generally had a lower IC50 than conjugates with a maleimidyl- or acetamide-based linker (compounds 40 and 41, respectively). Table 5. In Vitro cytotoxic activity in CD30+ (ng/mL) cell lines, all ACDs 2 drugs/mAb
In Vitro Cytotoxic Activity of ADCs with drug ligands derived from meta-aniline PBD 42 dimer:
[00529] Referring to Table 6, the in vitro cytotoxicity of ADCs containing PBD dimers on CD30+ cell lines using the 96 hour assay is shown. Activity was tested against CD70-CD30 cell lines. The antibodies used were a CD70 antibody, humanized 1F6 (see U.S. Published Application No. 2009-148942), and a CD70 antibody (1F6 humanized) having introduced cysteine residues at position 239 of the heavy amino acid chain (according to with the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 43) and a glucuronide linker (48) had a lower IC50 than conjugates with a maleimidyl- or acetamide-based linker (compound 44). Table 6. In Vitro cytotoxic activity in CD70+ (ng/mL) cell lines
In Vitro Cytotoxic Activity of ADCs with drug ligands derived from PBD 38 and 42 dimer of para- and meta-aniline:
[00530] Referring to Table 7, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. Activity was tested against CD70+ L428 and 786O cell lines and CD70-AML cell lines. The antibodies used were a CD70 antibody, humanized 1F6 (see U.S. Published Application No. 2009-148942), and a CD70 antibody (1F6 humanized) having introduced cysteine residues at position 239 of the heavy amino acid chain (according to EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker with the meta-aniline (drug-binding compound 43) were less active than those having a maleimidyl-peptide linker with the para-aniline (drug-binding compound 38). Reducing the drug load of the meta-aniline compound to 2 per antibody reduced activity. Conjugates with a glucuronide linker from the compound para-aniline (48) generally had a lower IC50 than conjugates with a linker based on maleimidyl (compound 39). Additionally, an aryl maleimide of the compound para-aniline (54) has no activity in these cell lines. Additionally, a conjugate having a maleimide linker conjugated directly to compound 42 has reduced activity compared to the h1F6-43 conjugate (data not shown). Table 7. In Vitro Cytotoxic Activity in CD70+ (ng/mL) Cell Lines
In Vitro Cytotoxic Activity of ADCs with Drug Ligands Derived from Aniline-Linked PBD Dimers:
[00531] Referring to Table 8, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. Activity was tested against CD70+ cell lines Caki-1 and L428 and a CD70- cell line. The antibody used was a CD70 antibody (1F6 humanized) having cysteine residues introduced at position 239 of the heavy amino acid chain (according to the EU numbering system) (indicated as h1F6d). Binding of a PBD through an amine in the ortho position through a non-cleavable linker (compound 68) significantly reduced activity compared to an ADC linked through a cleavable linker linked to para-aniline (compound 54). Compounds 73 and 85, having a cleavable linker, showed comparable activity to compound 54; both of these compounds are linked via a para-aniline. Compounds with cleavable linkers need stronger cleavage, compounds 79 and 90, showed somewhat reduced activity compared to compound 54. Table 8. In vitro cytotoxic activity in CD70+(ng/mL) cell lines
In Vitro Cytotoxic Activity of ADCs with Drug Ligands Derived from Aniline-Linked PBD Dimers:
[00532] Referring to Table 9, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines Caki-1 and L428 and a leukemia cell line CD70-. The antibodies used were a CD70 antibody, humanized 1F6 (see U.S. Published Application No. 2009148942), and a CD70 antibody (1F6 humanized) having introduced cysteine residues at position 239 of the heavy amino acid chain (according to the system of EU numbering) (indicated as h1F6d). Compound 56, having a cleavable linker attached to the antibody through an acetamide, showed comparable activity to compound 38. A glucuronide-linked version of meta-aniline-linked PBD Dimer, compound 48, demonstrated little activity in this assay. Compound 58, having five methylene groups on the PBD bridge, demonstrated comparable activity to compound 38, having three methylene groups on the PBD bridge. Table 9: In vitro cytotoxic activity in cell lines CD70+ cell lines (ng/mL)
Example 16: In Vivo Cytotoxicity Determination of Selected Conjugates
[00533] All studies were conducted in accordance with the Animal Care and Use Committee in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. In vivo tolerance was first evaluated to ensure that the conjugates were tolerated at clinically relevant doses. BALB/c mice were treated with staggered doses of ADC formulated in PBS with 0.01% Tween 20. The mice were monitored for weight loss after drug treatment; those who experienced 20% weight loss or other signs of morbidity were euthanized. The antibodies used were a CD70 antibody, humanized 1F6 (see United States Published Application No. 2009-148942) and a CD30 antibody, chimeric AC10 (see United States Published Application No. 2008-0213289).
[00534] Referring to Figure 1, the results of a weight loss study are shown using cAC10-val-ala-SG3132(2) (cAC10-compound 38). A single dose of Conjugate given as 5 mg given IP or IV resulted in little weight loss. A higher dose of Conjugate (15 mg/kg) caused significant weight loss in mice.
[00535]Referring to Figure 2, the results of a weight loss study are shown using h1F6-val-ala-SG3132(2) (h1F6-compound 38). A single dose of Conjugate given as 5 mg given IP resulted in some weight loss. A higher dose of Conjugate (10 mg/kg) caused significant weight loss in mice.
[00536] Treatment studies were performed in two xenograft models of CD70+ renal cell carcinoma. Tumor fragments (786-O and Caki-1) were implanted in the right flank of Nude mice. Mice were randomized to study groups (n=5) on day eight (786-O) or day nine (Caki-1) with each group ranging around 100 mm3. ADCs or controls were dosed according to a q4dx4 schedule. Tumor volume as a function of time was determined using the formula (L x W2)/2. Animals were euthanized when tumor volume reached 1000 mm3. Mice that showed durable regressions were finished around day 100 post-implantation.
Referring to Figure 3, the results of a treatment study using a conjugate of h1F6-val-ala-SG3132(2) (h1F6-compound 38) are shown. A control conjugate, cAC10-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice given doses of the h1F6 conjugate at 0.1 mg/kg showed some tumor shrinkage, whereas higher doses at 0.3 mg/kg and 1 mg/kg appeared to show complete tumor shrinkage. The control (non-binding) conjugate was less active than the h1F6 conjugates.
Referring to Figure 4, the results of a treatment study using a conjugate of h1F6-mc-val-ala-SG3132(2) (h1F6-compound 38) are shown. A control conjugate, cAC10-mc-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice given doses of the h1F6 conjugate at 1 mg/kg appeared to show complete tumor reduction. Mice administered at doses below 0.3 mg/kg and 0.1 mg/kg showed less tumor reduction, respectively. The control (non-binding) conjugate was less active than the h1F6 conjugate given at a similar dose, although it had more activity than the h1F6 conjugate given at lower doses. The h1F6 conjugate was also more active than the h1F6-vc-MMAE conjugate (United States Application Published No. 2009-0148942) given in higher doses.
[00539] Referring to Figure 5, the results of a treatment study using a bicharged antibody h1F6d bound to compound 38 (h1F6d-38) compared to a bicharged non-binding control, H00d conjugated to the same compound (h00d- 38). The model was a subcutaneous Caki model in Nude mice. The doses were 0.1, 0.3 and 1 mg/kg q7dX2. The two highest doses of the h1F6 conjugate demonstrated complete regressions at 1 mg/kg and substantial tumor delay at 0.3 mg/kg. The non-binding control demonstrated tumor delay at a dose of 1 mg/kg.
[00540] Referring to Figure 6, the results of a treatment study using a bicharged antibody h1F6d bound to compound 38 (h1F6d-38) compared to a bicharged non-binding control, H00d conjugated to the same compound (h00d- 38). The model was a 786-O subcutaneous model in Nude mice. The doses were 0.1, 0.3 and 1 mg/kg q7dX2. All three doses of the h1F6 conjugate demonstrated complete regressions or tumor retardation, while non-binding controls demonstrated tumor retardation.

权利要求:
Claims (17)
[0001]
1. Conjugate CHARACTERIZED by the fact that it has the formula I: L - (LU-D)p (I), or a pharmaceutically acceptable salt or solvate thereof; where L is a Linker unit, where the Linker unit is an antibody or antigen-binding fragment of a full-length antibody thereof that specifically binds to target cancer cells, where the antibody is selected from a monoclonal antibody, chimeric antibody, humanized antibody, fully human antibody, or a single chain antibody, LU is a Linking unit, where the Linking unit (LU) has the formula Ia: -A1-L1- (Ia), where: A1 is selected from:
[0002]
2. Conjugate according to claim 1, CHARACTERIZED by the fact that Y is O, and R" is C3-7 alkylene.
[0003]
3. Conjugate according to claim 1 or 2, CHARACTERIZED by the fact that Q1 is a single bond and Q2 is a single bond.
[0004]
4. Conjugate according to any one of claims 1 to 3, CHARACTERIZED by the fact that R12 is phenyl optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl , C3-7 heterocyclyl, dimethyl-aminopropyloxy, piperazinyl and bis-oxy C1-3 alkylene.
[0005]
5. Conjugate 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. Conjugate, 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, R9, R10, R11 and Y, respectively.
[0007]
7. Conjugate, according to any one of claims 1 to 6, CHARACTERIZED by the fact that A1 is selected from:
[0008]
8. Conjugate, according to claim 1, CHARACTERIZED by the fact that L1 is selected from: - Phe-Lys-, - Val-Ala-, - Val-Lys-, - Ala-Lys-, and - Val -Cit, where Cit is citrulline.
[0009]
9. Conjugate, according to claim 8, CHARACTERIZED by the fact that L1 is selected from the group consisting of valine-alanine, valine-citrulline and phenylalanine-lysine.
[0010]
10. Conjugated, according to claim 1, CHARACTERIZED by the fact that D is:
[0011]
11. Conjugate, according to claim 1, CHARACTERIZED by the fact that LU-D is:
[0012]
12. Conjugate, according to claim 1, CHARACTERIZED by the fact that LU-D is
[0013]
13. Conjugated, according to any one of claims 1 to 12, CHARACTERIZED by the fact that the subscript p is 2.
[0014]
14. Pharmaceutical composition CHARACTERIZED by the fact that it includes a conjugate as defined in any one of claims 1 to 13.
[0015]
15. Use of a conjugate, or a pharmaceutically acceptable salt or solvate thereof, as defined in any one of claims 1 to 13, CHARACTERIZED by the fact that it is for the manufacture of a medicine to treat a proliferative disease or a autoimmune disease.
[0016]
16. Drug binding agent CHARACTERIZED by the fact that it has the formula: G1-L1-D, or a pharmaceutically acceptable salt or solvate thereof; where G1 is selected from:
[0017]
17. Drug linker, according to claim 16, CHARACTERIZED by the fact that it has the formula: or a pharmaceutically acceptable salt or solvate thereof.

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法律状态:
2017-10-24| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2017-11-28| B25A| Requested transfer of rights approved|Owner name: SEATTLE GENETICS, INC. (US) , SPIROGEN SARL (CH) |

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2017-12-19| B25A| Requested transfer of rights approved|Owner name: SEATTLE GENETICS, INC. (US) , MEDIMMUNE LIMITED (G |

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2018-03-20| B65X| Notification of requirement for priority examination of patent application|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 31/5517 (2006.01), A61K 47/68 (2017.01), C07K |

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-05-02| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

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2018-11-21| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-01-02| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2019-04-16| B09B| Patent application refused [chapter 9.2 patent gazette]|

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2019-07-02| B12B| Appeal against refusal [chapter 12.2 patent gazette]|

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2021-05-04| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/04/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME MEDIDA CAUTELAR DE 07/04/2021 - ADI 5.529/DF |

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2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/04/2011 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US32462310P| true| 2010-04-15|2010-04-15|

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US61/324,623|2010-04-15|

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PCT/US2011/032664|WO2011130613A1|2010-04-15|2011-04-15|Targeted pyrrolobenzodiazapine conjugates|

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