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HUMANIZED ANTI-TN-MUC1 ANTIBODIES AND THEIR CONJUGATES

20170267778 · 2017-09-21

    Inventors

    Cpc classification

    International classification

    Abstract

    Humanized anti-Tn-MUC1 antibodies and conjugates thereof. Conjugates comprising pyrrolobenzodiazepines (PBDs) having a labile protecting group in the form of a linker to the antibody are described.

    Claims

    1. An isolated humanized antibody that binds to Tn-MUC1, wherein the isolated humanized antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, a light chain variable region having the amino acid sequence of SEQ ID NO: 31, 32, 33, or 34, and optionally comprises a constant region derived from one or more human antibodies.

    2. The isolated humanized antibody according to claim 1, wherein the isolated humanized antibody comprises: (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (ii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (iii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (iv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (v) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (vi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (vii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (viii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (ix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (x) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30; (xxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xl) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xli) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xlii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xliii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xliv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xlv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xlvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xlvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xlviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (xlix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (l) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (li) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (lii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (liii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (liv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (Iv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (lvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (lvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; (lviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (lxxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (lxxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (lxxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xc) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xci) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xciv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xciv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xciv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (xcix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (ci) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (cii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (ciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (civ) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (cv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; (cvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; (cxxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; or (cxxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34.

    3. The humanized antibody according to claim 1, wherein said antibody binds Tn-MUC1 with an affinity (Kd) of at least 10.sup.−6 M.

    4. The humanized antibody according to claim 3, wherein said antibody binds Tn-MUC1 with an affinity (Kd) of at least 10.sup.−9 M.

    5. The humanized antibody according to claim 1, wherein said antibody competitively inhibits the binding to Tn-MUC1 of an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30.

    6. The humanized antibody according to claim 1, wherein said antibody competitively inhibits the binding to Tn-MUC1 of the mouse 5E5 antibody.

    7. The humanized antibody according to claim 1, wherein said antibody or antibody fragment substantially neutralizes at least one activity of at least one Tn-MUC1.

    8. The humanized antibody according to claim 1, wherein said antibody or antibody fragment expresses at a level of at least 10 micrograms/ml in a static HEK293T transient expression system.

    9. The humanized antibody according to claim 1, wherein said antibody or antibody fragment expresses at a level of at least 20 micrograms/ml in a shaken HEK293T transient expression system.

    10. The humanized antibody according to claim 1, wherein said antibody has a melting temperature (T.sub.m) of at least 62° C.

    11. The humanized antibody according to claim 1, wherein said antibody or antibody fragment has a constant region of either isotype IgG1, IgG2, IgG3 or IgG4, or a mutated IgG constant region, and optionally a light chain constant region of isotype kappa or lambda.

    12. The humanized antibody according to claim 1, wherein the humanized antibody fragment is a scFv, Fab or F(ab′).sub.2.

    13.-123. (canceled)

    124. A polynucleotide encoding a humanized antibody according to claim 1.

    125.-128. (canceled)

    129. A conjugate comprising the humanized antibody according to claim 1 coupled to a functional moiety.

    130. The conjugate according to claim 129, wherein the functional moiety is selected from a drug, a reporter, a toxin, an organic moiety, and a binding member.

    131. The conjugate according to claim 130 wherein the reporter is a fluorescent compound, a radionuclide, or an enzyme.

    132. The conjugate according to claim 130 wherein the binding member is an antibody or antibody fragment.

    133. The conjugate according to claim 129, wherein the humanized antibody is covalently bonded to the functional moiety.

    134.-136. (canceled)

    137. The humanized antibody according to claim 1 or the conjugate according to claim 129, for use in therapy.

    138. A pharmaceutical composition comprising the humanized antibody according to claim 1, and a pharmaceutically acceptable diluent, carrier or excipient.

    139. The pharmaceutical composition of claim 138 further comprising a therapeutically effective amount of a chemotherapeutic agent.

    140. A method of treating cancer comprising administering to a patient the pharmaceutical composition according to claim 138.

    141. The method of claim 140 wherein the patient is administered a chemotherapeutic agent, in combination with the humanized antibody or conjugate.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [1094] FIG. 1: Tn-MUC1 binding of final humanised versions Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6.

    [1095] FIG. 2: Thermal denaturation of humanised 5E5 antibodies: unpurified antibodies

    [1096] FIG. 3 Thermal denaturation of humanised 5E5 antibodies: purified antibodies

    [1097] FIG. 4: In-vivo cytotoxicity of humanised 5E5 antibodies at 0.3 mg/kg

    [1098] FIG. 5: In-vivo cytotoxicity of humanised 5E5 antibodies at 1.0 mg/kg

    [1099] FIG. 6: In-vivo anti-tumour activity of humanised 5E5 antibodies at 0.3 mg/kg and 1.0 mg/kg. Arrows below x-axis indicate day of treatment (day 33, day 40, and day 47).

    MATERIALS AND METHODS

    [1100] Protocol 1: Subcloning from GeneArt Vector into Expression Vectors

    Materials

    [1101] Restriction enzymes (New England Biolabs): Agel (#R0552); BsiWI (#R0553); EcoRI: (#R0101); NheI: (#R0131) [1102] QIAprep Spin Miniprep Kit (Qiagen#27104 or 27105) [1103] QIAquick Gel Extraction (Qiagen#28704) [1104] Antarctic Phosphatase (NEB#M0289S) [1105] pFUSE2-CLIg-hk vector (Invivogen #pfuse2-hclk) [1106] pFUSE-CHIg-hG1 (Invivogen #pfuse-hchg1) [1107] Zeocin 100 mg/ml (Invivogen #ant-zn-1) [1108] Blasticidin 10 mg/ml (Invivogen #ant-bl-1) [1109] 5-alpha Competent E. coli (High Efficiency) (New England Biolabs #C29871) [1110] LigaFast™ Rapid DNA Ligation System (Promega #M8221) [1111] Agarose-LE0020 (Life Technologies #AM9040) [1112] TAE buffer [1113] Fushk-fwd; CTT TGC CTG ACC CTG CTT GC [1114] FushG1-bak; TCA CCG GTT CGG GGA AGT AG [1115] Fushk-bak: TCA GCA GGC ACA CAA CAG AGG [1116] Pfuse3′bak: TTC CAT ACC ACATTT GTA GAG GT Method [1117] 1) 5 μg of each GeneArt construct and each pfuse vector were restriction-digested in 10 μl: [1118] a) Light chain construct and vector pFUSE2-CHIg-hk were digested with Agel+BsiW1 in NEB buffer 1, incubating 1 hr at 37 C followed by 1 hr at 55 C. [1119] b) Heavy chain construct and vector pFUSE-CHIg-hG1 were digested with ECoRI+NheI in NEB buffer 1, incubating 1 hr at 37 C. [1120] 2) Digested heavy and light chain vectors were dephosphorylated using Antarctic Phosphatase: [1121] a) Add 1/10 volume of 10× Antarctic Phosphatase Reaction Buffer. [1122] b) Add 1 μl of Antarctic Phosphatase (NEB, 5,000 units/ml) and mix. [1123] c) Incubate for 15 minutes at 37° C. [1124] d) Heat inactivate for 5 minutes at 65° C. [1125] 3) Cut vectors were purified by electrophoresis in agarose gel (1.5% w/v in TAE buffer) [1126] 4) Cut inserts were purified in agarose gel (1.0% w/v in TAE buffer). [1127] 5) Gel fragments containing DNA were solubilised and the DNA extracted using the QIAquick Gel Extraction kit following manufacturer's instructions. [1128] 6) Cut insert and complementary cut vector were ligated with T4 DNA Ligase for 1 hr at room temperature, following the manufacturer's instructions and using a 1:2 molar ratio of vector:insert DNA. [1129] 7) Competent E. coli were transformed with the ligation mix according to the manufacturer's instructions: 42C heat shock; add SOC 1 nl and shake 1 hr 37 C, then and spread on warmed selection LB agar plates: [1130] a) For heavy chain constructs, Zeocin 25 μg/ml [1131] b) For light chain constructs, Blasticidin 100 μg/ml [1132] 8) Plates were incubated overnight at 37 C, then colonies (3) were picked from each plate and inoculated into 3 ml LB containing Zeocin 25 μg/ml or Blasticidin 50 μg/ml, and shaken overnight [1133] 9) DNA plasmids were isolated from each culture using the QIAprep Spin Miniprep Kit following the manufacturer's instructions [1134] 10) Heavy or light chain construct plasmids (10 μl) were restriction digested as before, and electrophoresed on agarose gel (1.5% w/v in TAE buffer). [1135] 11) Those plasmids producing an approx 450 bp fragment were validated by DNA sequencing using primers Fushk-fwd (for VH and VL); FushG1-bak (for VH); Fushk-bak (for VL)
    Protocol 2. Transient Transfection of HEK293T Cells with Expression Constructs

    Materials

    [1136] Cells: HEK293T cells [1137] Culture medium: DMEM high glucose 4.5 g/L (PAA) with 10% v/v FCS, penicillin and streptomycin [1138] Fugene HD transfection reagent (Promega # E2311) [1139] Opti-MEM (Life Technologies #11058-021) or [1140] FreeStyle 293 Expression Medium (Life Technologies #12338-018)

    Method

    [1141] Grow HEK293T cells in a T75 or T175 flask in a CO.sub.2-gassed cell culture incubator. Split cultures 1:3 every 2 days or 1:4 to 1:5 every 3-4 days. The cells adhere weakly to the flasks and only a light trypsinisation is necessary to detach cells during passaging.

    [1142] The day before transfection: [1143] 1. Trypsinise the cells, wash 1× in DMEM/10% FCS and count the cells. [1144] 2. Seed cells in a 6 well plate in 2 ml per well containing 2×10.sup.5 cells.

    [1145] Next day, check cells are at least 80% confluent and replace the medium (2 ml/well). [1146] 1. 1.2 μg of total DNA (0.6 ug of each high and light chain DNA) is needed for each transfection and better results are obtained if the DNA concentration is at or above 90 ng/μl. [1147] 2. Add 0.6 ug of VH and 0.6 ug VK expression plasmid DNAs into of Fugene HD (4.5 μl) and OptiMEM/Freestyle medium, in a total volume of 60 ul, avoiding touching the sides of the tube with the Fugene HD. [1148] 3. Mix and leave at RT for 15 minutes. [1149] 4. Add Fugene mixture drop-wise around the well of HEK293T cells. [1150] 5. Return the 6-well plate to the CO.sub.2-gassed cell culture incubator for 4 days. [1151] 6. Harvest each conditioned medium, centrifuge, and store at 4° C.

    Protocol 3: IgG Quantitation by ELISA

    Materials

    [1152] Nunc-Immuno Plate MaxiSorp (Life Technologies, 43945A) [1153] Goat Anti-Human IgG(Fc)-AffiniPure: Stratech Scientific, 109-005-098-JIR; 1 mg: 1.3 mg/ml; [1154] Human IgG1/kappa antibody (Sigma, 1-3889-1 mg: 1 mg/ml) [1155] Goat anti-human kappa light chain peroxidase conjugate (Sigma, A-7164-1 ml) [1156] 1-Step Turbo TMB-ELISA, 250 mL (Thermo Scientific: #34022) [1157] Acid stop=0.6M HCL [1158] Sample enzyme conjugate (SEC) buffer Tween 20 (0.02% v/v), BSA 0.2% (w/v) in PBS [1159] Washing buffer: 1×PBS, Tween 20 (0.1% v/v)

    Method

    [1160] 1. Coat each well of a 96-well immunoplate with 100 μl aliquots of goat anti-human IgG antibody, (0.4 μg/ml in PBS: dilute stock ×3000), [1161] 2. Incubate overnight at 4° C. (Plates may be stored for 1 month at this stage). [1162] 3. Wash coated plate 3× with PBS [1163] 4. Block the anti-Ig-coated plate: add 200 ul BSA (3% in PBS): [1164] 5. Incubate 37 C 1 hr [1165] 6. Prepare 1 ug/ml solution of the human IgG1/kappa antibody in SEC buffer (×1000 diln) [1166] 7. Into a polypropylene (low binding) plate, dispense 200 μl SEC buffer into all wells excepting cols 1 & 7—dispense 300 ul SEC buffer. [1167] 8. Into this polypropylene plate, pipette 50 μl/well of IgG std (1 ug/ml) and 2 ul/well of unknowns into rows A-H, cols 1 & 7 [1168] 9. Serially transfer 100 μl across plate to achieve serial ×3 dilutions. [1169] 10. Transfer 100 ul from each well of polypropylene plate to the corresponding well of the blocked anti-IgG-coated plate. [1170] 11. Incubate at 37° C. for 1 hr. [1171] 12. Wash plate 3× with washing buffer. [1172] 13. Add 100 μl/well of goat anti-human kappa chain HRP conjugate (×1000 in SEC buffer). [1173] 14. Incubate at 37° C. for 1 hr. [1174] 15. Wash plate 3× with washing buffer [1175] 16. Wash plate 2× with PBS [1176] 17. Add 100 μl of TMB Turbo substrate to each well, [1177] 18. Incubate at room temp, 30 min. [1178] 19. Stop reaction by adding 100 μl of acid stop to each well. [1179] 20. Read the optical density at 450 nm (colour stable overnight at 4 C if you cannot read immediately). [1180] 21. Std curve plot in Excel: fit using polynomial to calculate unknowns

    Protocol 4:Tn-MUC1 Binding ELISA

    Materials

    [1181] 16TR-MUC1-Tn (Tn-MUC1) was from Guys Hospital and stored in aliquots at −20 C. [1182] Goat anti-human kappa light chain peroxidase conjugate (Sigma, A-7164-1 ml) [1183] Nunc-Immuno Plate MaxiSorp (Life Technologies, 43945A) [1184] Plate washer: Biotek LS405 [1185] 3% BSA: BSA 3% w/v in PBS [1186] PBS Tween: Tween 20 0.05% v/v in PBS [1187] PBS/Tween/BSA: BSA 0.5% w/v in PBS/Tween [1188] 1-Step Turbo TMB-ELISA (Thermo Scientific #3402)

    Protocol

    [1189] 1. Dispense 50 μl/well of 16TR-MUC1-Tn (2 ug/ml in PBS) [1190] 2. Cover with adhesive plate sealer and incubate at 4 C overnight. [1191] 3. Block: Dispense 50 μl/well of 3% BSA and incubate for 1 hr 37 C, [1192] 4. Wash plate with PBS/Tween 3× [1193] 5. Serially 3-fold dilute 5E5 antibodies (2 ml HEK293T culture supernatants) on non-binding polypropylene plate in PBS/Tween/BSA: serially transfer 50 ul onto 100 ul. [1194] 6. Transfer 50 ul from antibody dilution plate onto washed, blocked Tn-MUC1-coated plate [1195] 7. Incubate 37 C 1 hr [1196] 8. Wash plate with PBS/Tween 3× [1197] 9. Dispense anti-human IgG-HRP conjugate, diluted 1:1000 in PBS/Tween/BSA [1198] 10. Incubate 37 C 1 hr [1199] 11. Wash plate with PBS/Tween 3 [1200] 12. Wash plate with PBS 3× [1201] 13. Dispense 100 ul/well 1-Step Turbo TMB-ELISA substrate solution [1202] 14. Incubate 30 min at room temperature (or less if reaction is rapid) [1203] 15. Dispense 100 ul/well 0.6M HCl to stop the substrate reaction [1204] 16. Measure optical density at 450 nm

    Protocol 5: Thermal Denaturation Assay

    Materials

    [1205] Purified antibody or unpurified HEK293T transient transfection conditioned medium containing antibody [1206] PBS [1207] 0.5 ml PCR tubes [1208] PCR machine [1209] Antigen-binding ELISA protocol and materials [1210] Antigen-coated ELISA plate

    Method

    [1211] 1. Dispense 0.2 ml of recombinant antibody at or near EC50 i.e. 0.05 ug/ml diluted in HEK293F medium) in 8×PCR tubes [1212] 2. Take each tube individually and run in its individual 2-step PCR cycle (Table 1 below)—use a PCR machine with the unheated lid option selected [1213] 3. Hold at 4 C until the ELISA assay, to be run on the same day. [1214] 4. Apply each sample to 2 wells of a (BSA−) blocked antigen-coated ELISA plate at 50 ul/well [1215] 5. Run the antigen-binding ELISA [1216] 6. Plot OD.sub.450 nm vs temperature of heating [1217] 7. Compare the 50% inactivation temperature of each antibody

    TABLE-US-00001 PCR heating programs: Tube number 10 minutes at Hold at 1  4 4 2 50 4 3 55 4 4 60 4 5 65 4 6 70 4 7 75 4 8 80 4

    Protocol 6: ZR-75-1 Cell Binding Assay by Flow Cytometry

    Materials

    [1218] Wash solution: PBS 0.1% (w/v) sodium azide [1219] Binding buffer: PBS, 10% normal goat serum, 0.1% (w/v) sodium azide) [1220] 96 well U-well microplate

    Protocol

    [1221] Exponentially growing cells are suspended by incubation with cell dissociation buffer enzyme-free, Hanks-based (Gibco 13150-016) following the manufacturer's instructions. A cell count/viability is done on a dilution of 10 ul cells+Trypan Blue, 10 ul, with a haemocytomer. The suspension is centrifuged and the cell pellet re-suspended in binding buffer at 3×10̂6 per ml. If significant levels of aggregates are present in the cell count, the suspension is filtered to remove aggregates. The cell suspension is dispensed (50 ul/well) in 96 well U-bottom plates and cooled on ice for 10 minutes. Primary antibody (50 ul/well) diluted in binding buffer (typically 1 ug per ml or less) is added to each well and incubated at 4° C. for 1 hour. The microplate is centrifuged 5 min at 300-400 g at 4° C., flicked to remove the supernatant and then the cell pellet re-suspended in 200 uL of wash buffer (PBS, 0.1% sodium azide). Centrifugation and re-suspension are done 4 times in all. After the last centrifugation, cells are re-suspended in 50 uL of binding buffer containing the biotin labelled conjugate 1:250 (kept at −20 C: Biotin-SP-AffiniPure F(ab′)2 Fragment Goat antihuman IgG (H+L); Jackson 109-066-088). Cells are incubated for 1 hour at 4 C, then washed 4 times as before. Cells are resuspended in 50 ul of binding buffer containing Streptavidin, Alexa Fluor® 488 conjugate (Life Technologies s11223) diluted 1:250 and Fixable Viability Dye eFluor@780 (eBioscience 65-0865) diluted 1:2000, The cells are incubated at 4° C. for 1 hour and then washed 3 times, as above. Cells are then re-suspended in PBS if reading immediately or fixed in 100 ul of paraformaldehyde (2% (w/v) in PBS) and analysed with by flow cytometer (Accuri C6), gating on live cells which have a lower Fixable Viability Dye MFI compared to dead cells.

    Protocol 7: HEK293F Shake Culture

    [1222] FreeStyle™ 293-F cells were cultured according to the Life Technologies protocol (in 400 ml to 10̂6 cells/ml. FreeStyle™ 293 Expression Medium (40 ml) was added to a 50 ml tube followed by 133 g of VH plasmid DNA and 160 g of VH plasmid DNA together with DNA plasmids to enhance expression such as pAdvantage, p21, SV40LT and p27. The total DNA added was 467 μg. The solution was mixed twice by inversion and 800 μl of a 1 mg/ml linear PEI solution was added dropwise. The transfection solution was vortexed briefly and incubated at room temperature for 10 minutes. The transfection solution was then added dropwise directly to the cell culture whilst swirling the flask gently. The culture was then returned to shaken incubation conditions. Antibiotic was added after 8 hours. Medium was harvested after about 14 days.

    General Experimental Methods

    [1223] 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. .sup.1H and .sup.13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (b=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F.sub.254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the 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 a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C.

    General LC/MS Conditions:

    Method 1 (Default Method, Used Unless Stated Otherwise)

    [1224] The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B held over 1.0 min, then increase from 5% B to 95% B over a 3 min period. The composition was held for 0.1 min at 95% B, then returned to 5% B in 0.03 minutes and hold there for 0.87 min. Total gradient run time equals 5 minutes.

    Method 2

    [1225] The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B held over 1.0 minute, then increase from 5% B to 95% B over a 2.5 minute period. The composition was held for 0.5 minutes at 95% B, then returned to 5% B in 0.1 minutes and hold there for 0.9 min. Total gradient run time equals 5 minutes.

    For Both Methods

    [1226] Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex Onyx Monolithic C18 50×4.60 mm.

    [1227] The reverse phase flash purification conditions were as follows: The Flash purification system (Varian 971-Fp) was run using a mobile phase of water (A) and acetonitrile (B). Gradient: initial composition 5% B over 20 C.V. (Column Volume) then 5% B to 70% B within 60 C.V. The composition was held for 15 C.V. at 95% B, and then returned to 5% B in 5 C.V. and held at 5% B for 10 C.V. Total gradient run time equals 120 C.V. Flow rate 6.0 mL/min. Wavelength detection range: 254 nm. Column: Agilent AX1372-1 SF10-5.5gC8.

    [1228] Preparative HPLC: Reverse-phase ultra-high-performance liquid chromatography (UPLC) was carried out on Phenomenex Gemini NX 5μ C-18 columns of the following dimensions: 150×4.6 mm for analysis, and 150×21.20 mm for preparative work. All UPLC experiments were performed with gradient conditions. Eluents used were solvent A (H.sub.2O with 0.1% Formic acid) and solvent B (CH.sub.3CN with 0.1% Formic acid). Flow rates used were 1.0 ml/min for analytical, and 20.0 ml/min for preparative HPLC. Detection was at 254 and 280 nm.

    Synthesis of Intermediate 12

    [1229] ##STR00125##

    (a) 1′,3′-Bis[2-methoxy-4-(methoxycarbonyl)phenoxy]propane (3)

    [1230] Diisopropyl azodicarboxylate (71.3 mL, 73.2 g, 362 mmol) was added drop-wise over a period of 60 min to an overhead stirred solution of methyl vanillate 2 (60.0 g, 329 mmol) and Ph.sub.3P (129.4 g, 494 mmol) in anhydrous THF (800 mL) at 0-5° C. (ice/acetone) under a nitrogen atmosphere. The reaction mixture was allowed to stir at 0-5° C. for an additional 1 hour after which time a solution of 1,3-propanediol (11.4 mL, 12.0 g, 158 mmol) in THF (12 mL) was added drop-wise over a period of 20 min. The reaction mixture was allowed to warm to room temperature and stirred for 5 days. The resulting white precipitate 3 was collected by vacuum filtration, washed with THF and dried in a vacuum desiccator to constant weight. Yield=54.7 g (84% based on 1,3-propanediol). Purity satisfactory by LC/MS (3.20 min (ES+) m/z (relative intensity) 427 ([M+Na].sup.+., 10); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.64 (dd, 2H, J=1.8, 8.3 Hz), 7.54 (d, 2H, J=1.8 Hz), 6.93 (d, 2H, J=8.5 Hz), 4.30 (t, 4H, J=6.1 Hz), 3.90 (s, 6H), 3.89 (s, 6H), 2.40 (p, 2H, J=6.0 Hz).

    (b) 1′,3′-Bis[2-methoxy-4-(methoxycarbonyl)-5-nitrophenoxy]propane (4)

    [1231] Solid Cu(NO.sub.3).sub.2.3H.sub.2O (81.5 g, 337.5 mmol) was added slowly to an overhead stirred slurry of the bis-ester 3 (54.7 g, 135 mmol) in acetic anhydride (650 mL) at 0-5° C. (ice/acetone). The reaction mixture was allowed to stir for 1 hour at 0-5° C. and then allowed to warm to room temperature. A mild exotherm (ca. 40-50° C.), accompanied by thickening of the mixture and evolution of NO.sub.2 was observed at this stage. Additional acetic anhydride (300 mL) was added and the reaction mixture was allowed to stir for 16 hours at room temperature. The reaction mixture was poured on to ice (˜1.5 L), stirred and allowed to return to room temperature. The resulting yellow precipitate was collected by vacuum filtration and dried in a desiccator to afford the desired bis-nitro compound 4 as a yellow solid. Yield=66.7 g (100%). Purity satisfactory by LC/MS (3.25 min (ES+) m/z (relative intensity) 517 ([M+Na].sup.+., 40); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.49 (s, 2H), 7.06 (s, 2H), 4.32 (t, 4H, J=6.0 Hz), 3.95 (s, 6H), 3.90 (s, 6H), 2.45-2.40 (m, 2H).

    (c) 1′,3′-Bis(4-carboxy-2-methoxy-5-nitrophenoxy) propane (5)

    [1232] A slurry of the methyl ester 4 (66.7 g, 135 mmol) in THF (700 mL) was treated with 1N NaOH (700 mL) and the reaction mixture was allowed to stir vigorously at room temperature. After 4 days stirring, the slurry became a dark coloured solution which was subjected to rotary evaporation under reduced pressure to remove THF. The resulting aqueous residue was acidified to pH 1 with concentrated HCl and the colourless precipitate 5 was collected and dried thoroughly in a vacuum oven (50° C.). Yield=54.5 g (87%). Purity satisfactory by LC/MS (2.65 min (ES+) m/z (relative intensity) 489 ([M+Na].sup.+., 30)); .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.62 (s, 2H), 7.30 (s, 2H), 4.29 (t, 4H, J=6.0 Hz), 3.85 (s, 6H), 2.30-2.26 (m, 2H).

    (d) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (6)

    [1233] Oxalyl chloride (24.5 mL, 35.6 g, 281 mmol) was added to a stirred suspension of the nitrobenzoic acid 5 (43 g, 92.3 mmol) and DMF (6 mL) in anhydrous DCM (600 mL). Following 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 was observed by LC/MS. The majority of solvent was removed by evaporation under reduced pressure; the resulting concentrated solution was re-dissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40° C. The solid acid chloride was added portionwise over a period of 25 min to a stirred suspension of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (38.1 g, 210 mmol) and TEA (64.5 mL, g, 463 mmol) in DCM (400 mL) at −40° C. (dry ice/CH.sub.3CN). Immediately, the reaction was complete as judged by LC/MS (2.47 min (ES+) m/z (relative intensity) 721 ([M+H].sup.+., 100). The mixture was diluted with DCM (200 mL) and washed with 1N HCl (300 mL), saturated NaHCO.sub.3 (300 mL), brine (400 mL), dried (MgSO.sub.4), filtered and the solvent evaporated in vacuo to give the pure product 6 as an orange solid (66.7 g, 100%). [α].sup.22.sub.D=−46.1° (c=0.47, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.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), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); .sup.13C NMR (100 MHz, CDCl.sub.3) (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.sub.3) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211, 1177, 1072, 1050, 1008, 871 cm.sup.−1; MS (ES.sup.+) m/z (relative intensity) 721 ([M+H].sup.+., 47), 388 (80); HRMS [M+H].sup.+. theoretical C.sub.31H.sub.36N.sub.4O.sub.16 m/z 721.2199, found (ES.sup.+) m/z 721.2227.

    (e) 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] (7)

    Method A:

    [1234] A solution of the nitro-ester 6 (44 g, 61.1 mmol) in MeOH (2.8 L) was added to freshly purchased Raney® nickel (˜50 g of a ˜50% slurry in H.sub.2O) and anti-bumping granules in a 5 L 3-neck round bottomed flask. The mixture was heated at reflux and then treated dropwise with a solution of hydrazine hydrate (21.6 mL, 22.2 g, 693 mmol) in MeOH (200 mL) at which point vigorous effervescence was observed. When the addition was complete (˜45 min) additional Raney® nickel was added carefully until effervescence had ceased and the initial yellow colour of the reaction mixture was discharged. The mixture was heated at reflux for a further 5 min at which point the reaction was deemed complete by TLC (90:10 v/v CHCl.sub.3/MeOH) and LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H].sup.+., 100)). The reaction mixture was filtered hot immediately through a sinter funnel containing celite with vacuum suction. The filtrate was reduced in volume by evaporation in vacuo at which point a colourless precipitate formed which was collected by filtration and dried in a vacuum desiccator to provide 7 (31 g, 85%). [α].sup.27.sub.D=+404° (C=0.10, DMF); .sup.1H NMR (400 MHz, DMSO-d.sub.6) 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 (p, 2H, J=5.90 Hz), 1.99-1.89 (m, 2H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) 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, neat) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm.sup.−1; MS (ES.sup.+) m/z (relative intensity) 619 ([M+Na].sup.+., 10), 597 ([M+H].sup.+., 52), 445 (12), 326 (11); HRMS [M+H].sup.+. theoretical C.sub.29H.sub.32N.sub.4O.sub.10 m/z 597.2191, found (ES.sup.+) m/z 597.2205.

    Method B:

    [1235] 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 6 (75 g, 104 mmol) in DMF (360 mL). The suspension was hydrogenated in a Parr hydrogenation apparatus over 8 hours. Progress of the reaction was monitored by LC/MS after the hydrogen uptake had stopped. Solid Pd/C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 10 mbar) at 40° C. to afford a dark oil containing traces of DMF and residual charcoal. The residue was digested in EtOH (500 mL) at 40° C. on a water bath (rotary evaporator bath) and the resulting suspension was filtered through celite and washed with ethanol (500 mL) to give a clear filtrate. Hydrazine hydrate (10 mL, 321 mmol) was added to the solution and the reaction mixture was heated at reflux. After 20 minutes the formation of a white precipitate was observed and reflux was allowed to continue for a further 30 minutes. The mixture was allowed to cool down to room temperature and the precipitate was retrieved by filtration, washed with diethyl ether (2:1 volume of precipitate) and dried in a vacuum desiccator to provide 7 (50 g, 81%). Analytical data for method B: Identical to those obtained for Method A (optical rotation, .sup.1H NMR, LC/MS and TLC).

    (f) 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] (8)

    [1236] TBSCl (27.6 g, 182.9 mmol) and imidazole (29.9 g, 438.8 mmol) were added to a cloudy solution of the tetralactam 7 (21.8 g, 36.6 mmol) in anhydrous DMF (400 mL) at 0° C. (ice/acetone). The mixture was allowed to stir under a nitrogen atmosphere for 3 hours after which time the reaction was deemed complete as judged by LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M+H].sup.+., 100). The reaction mixture was poured onto ice (˜1.75 L) and allowed to warm to room temperature with stirring. The resulting white precipitate was collected by vacuum filtration, washed with H.sub.2O, diethyl ether and dried in the vacuum desicator to provide pure 8 (30.1 g, 99%). [α].sup.23.sub.D=+234° (c=0.41, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.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); .sup.13C NMR (100 MHz, CDCl.sub.3) 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.sub.3) 3235, 2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099, 1033 cm.sup.−1; MS (ES.sup.+) m/z (relative intensity) 825 ([M+H].sup.+., 62), 721 (14), 440 (38); HRMS [M+H].sup.+. theoretical C.sub.41H.sub.60N.sub.4O.sub.10Si.sub.2 m/z 825.3921, found (ES.sup.+) m/z 825.3948.

    (g) 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] (9)

    [1237] A solution of n-BuLi (68.3 mL of a 1.6 M solution in hexane, 109 mmol) was added dropwise to a stirred suspension of the tetralactam 8 (30.08 g, 36.4 mmol) in anhydrous THF (600 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 colour) at which point a solution of SEMCl (19.3 mL, 18.2 g, 109 mmol) in anhydrous THF (120 mL) was added dropwise. The reaction mixture was allowed to slowly warm to room temperature and was stirred for 16 hours under a nitrogen atmosphere. The reaction was deemed complete as judged by TLC (EtOAc) and LC/MS (4.77 min (ES+) m/z (relative intensity) 1085 ([M+H].sup.+., 100). The THF was removed by evaporation in vacuo and the resulting residue dissolved in EtOAc (750 mL), washed with H.sub.2O (250 mL), brine (250 mL), dried (MgSO.sub.4) filtered and evaporated in vacuo to provide the crude N10-SEM-protected tetralactam 9 as an oil (max.sup.m39.5 g, 100%). Product carried through to next step without purification. [α].sup.23.sub.D=+163° (c=0.41, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.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); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, −1.24, −4.73; IR (ATR, CHCl.sub.3) 2951, 1685, 1640, 1606, 1517, 1462, 1433, 1360, 1247, 1127, 1065 cm.sup.−1; MS (ES.sup.+) m/z (relative intensity) 1113 ([M+Na].sup.+., 48), 1085 ([M+H].sup.+., 100), 1009 (5), 813 (6); HRMS [M+H].sup.+. theoretical C.sub.53H.sub.88N.sub.4O.sub.12Si.sub.4 m/z 1085.5548, found (ES.sup.+) m/z 1085.5542.

    (h) 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] (10)

    [1238] A solution of TBAF (150 mL of a 1.0 M solution in THF, 150 mmol) was added to a stirred solution of the crude bis-silyl ether 9 [84.0 g (max.sup.m 56.8 g), 52.4 mmol] in THF (800 mL) at room temperature. After stirring for 1 hour, analysis of the reaction mixture by TLC (95:5 v/v CHCl.sub.3/MeOH) revealed completion of reaction. The THF was removed by evaporation under reduced pressure at room temperature and the resulting residue dissolved in EtOAc (500 mL) and washed with NH.sub.4Cl (300 mL). The combined organic layers were washed with brine (60 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCl.sub.3 to 96:4 v/v CHCl.sub.3/MeOH) gave the pure tetralactam 10 as a white foam (36.0 g, 79%). LC/MS 3.33 min (ES.sup.+) m/z (relative intensity) 879 ([M+Na].sup.+., 100), 857 ([M+H].sup.+., 40); [α].sup.23.sub.D=+202° (c=0.34, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.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); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 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.sub.3) 2956, 1684, 1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm.sup.−1; MS (ES.sup.+) m/z (relative intensity) 885 ([M+29].sup.+., 70), 857 ([M+H].sup.+., 100), 711 (8), 448 (17); HRMS [M+H].sup.+. theoretical C.sub.41H.sub.60N.sub.4O.sub.12Si.sub.2 m/z 857.3819, found (ES.sup.+) m/z 857.3826.

    (i) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-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] (11)

    [1239] Diol 10 (25.6 g, 30 mmol, 1 eq.), NaOAc (6.9 g, 84 mmol, 2.8 eq.) and TEMPO (188 mg, 1.2 mmol, 0.04 eq.) were dissolved in DCM (326 mL) under Ar. This was cooled to −8° C. (internal temperature) and TCCA (9.7 g, 42 mmol, 1.4 eq.) was added portionwise over 15 minutes. TLC (EtOAc) and LC/MS [3.60 min. (ES+) m/z (relative intensity) 854.21 ([M+H].sup.+., 40), (ES−) m/z (relative intensity) 887.07 ([M−H+Cl].sup.−., 10)] after 30 minutes indicated that reaction was complete. Cold DCM (200 mL) was added and the mixture was filtered through a pad of Celite before washing with a solution of saturated sodium hydrogen carbonate/sodium thiosulfate (1:1 v/v; 200 mL×2). The organic layer was dried with MgSO.sub.4, filtered and the solvent removed in vacuo to yield a yellow/orange sponge (25.4 g, 99%). LC/MS [3.60 min. (ES+) m/z (relative intensity) 854.21 ([M+H].sup.+., 40); [α].sup.20.sub.D=+291° (C=0.26, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.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); .sup.13C NMR (100 MHz, CDCl.sub.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.sub.3) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm.sup.−1; MS (ES.sup.+) m/z (relative intensity) 881 ([M+29].sup.+., 38), 853 ([M+H].sup.+., 100), 707 (8), 542 (12); HRMS [M+H].sup.+. theoretical C.sub.41H.sub.56N.sub.4O.sub.12Si.sub.2 m/z 853.3506, found (ES.sup.+) m/z 853.3502.

    (j) 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] (12)

    [1240] 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 11 (6.08 g, 7.1 mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile) under a nitrogen atmosphere. Anhydrous triflic anhydride, taken 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 (50/50 v/v n-hexane/EtOAc) revealed the complete consumption of starting material. The cold reaction mixture was immediately diluted with DCM (200 mL) and, with vigorous shaking, washed with water (1×100 mL), 5% citric acid solution (1×200 mL) saturated NaHCO.sub.3 (200 mL), brine (100 mL) and dried (MgSO.sub.4). Filtration and evaporation of the solvent under reduced pressure afforded the crude product which was purified by flash column chromatography (gradient elution: 90:10 v/v n-hexane/EtOAc to 70:30 v/v n-hexane/EtOAc) to afford bis-enol triflate 12 as a yellow foam (5.5 g, 70%). LC/MS 4.32 min (ES+) m/z (relative intensity) 1139 ([M+Na].sup.+., 20); [α].sup.24D=+271° (C=0.18, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.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); .sup.13C NMR (100 MHz, CDCl.sub.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.sub.3) 2958, 1690, 1646, 1605, 1517, 1456, 1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm.sup.−1; MS (ES.sup.+) m/z (relative intensity) 1144 ([M+28].sup.+., 100), 1117 ([M+H].sup.+., 48), 1041 (40), 578 (8); HRMS [M+H].sup.+. theoretical C.sub.43H.sub.54N.sub.4O.sub.16Si.sub.2S.sub.2F.sub.6 m/z 1117.2491, found (ES.sup.+) m/z 1117.2465.

    Example 1

    [1241] ##STR00126##

    (a) (S)-8-(3-(((S)-2-(4-aminophenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl trifluoromethanesulfonate (13)

    [1242] Pd(PPh.sub.3).sub.4 (116.9 mg, 0.101 mmol) was added to a stirred mixture of the bis-enol triflate 12 (5.65 g, 5.06 mmol), 4-Aminophenylboronic acid pinacol ester (1 g, 4.56 mmol), Na.sub.2CO.sub.3 (2.46 g, 23.2 mmol), MeOH (37 mL), toluene (74 mL) and water (37 mL). The reaction mixture was allowed to stir at 30° C. under a nitrogen atmosphere for 24 hours after which time all the boronic ester has consumed. The reaction mixture was then evaporated to dryness before the residue was taken up in EtOAc (150 mL) and washed with H.sub.2O (2×100 mL), brine (150 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 80:20 v/v Hexane/EtOAc to 60:40 v/v Hexane/EtOAc) afforded product 13 as a yellowish foam (2.4 g, 45%). LC/MS 4.02 min (ES+) m/z (relative intensity) 1060.21 ([M+H].sup.+., 100); .sup.1H-NMR: (CDCl.sub.3, 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), 4.00-3.85 (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). .sup.13C-NMR: (CDCl.sub.3, 100 MHz) 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, 127.5, 125.1, 123.21, 121.5, 120.5, 120.1, 116.4, 113.2, 108.7, 79.8, 79.6, 68.7, 68.5, 67.0, 66.8, 58.8, 58.0, 57.6, 32.8, 32.0, 30.3, 19.7, 0.25.

    (b) (S)-2-(4-Aminophenyl)-8-(3-(((S)-2-cyclopropyl-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (14)

    [1243] Triphenylarsine (0.24 g, 0.8 mmol), silver (I) oxide (1.02 g, 4.4 mmol), cyclopropylboronic acid (0.47 g, 5.5 mmol) and starting material 13 (1.15 g, 1.1 mmol) were dissolved in dioxane (30 mL) under an argon atmosphere. Potassium phosphate tribasic (2.8 g, 13.2 mmol) was ground-up with a pestle and mortar and quickly added to the reaction mixture. The reaction mixture was evacuated and flushed with argon 3 times and heated to 71° C. Palladium (11)bis (benzonitrile chloride) (84 mg, 0.22 mmol) was added and the reaction vessel was evacuated and flushed with argon 3 times. After 10 minutes a small sample was taken for analysis by TLC (80:20 v/v ethyl acetate/hexane) and LC/MS. After 30 minutes the reaction had gone to completion (LC/MS analysis indicated complete consumption of starting material) and the reaction was filtered through celite and the filter pad washed with ethyl acetate (400 mL). The filtrate was washed with water (2×200 mL) and brine (2×200 mL). The organic layer was dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (30:70 v/v Hexane/Ethyl acetate) afforded the product 14 as an orangey/yellow solid (0.66 g, 63%). Method 1, LC/MS (3.85 min (ES.sup.+) m/z (relative intensity) 952.17 ([M+H].sup.+., 100). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.36 (d, 2H, J=8.4 Hz), 7.30 (s, 1H), 7.25-7.19 (m, 4H), 6.68 (s, 1H), 6.62 (d, 2H, J=8.4 Hz), 5.49 (dd, 2H, J=5.6, 10.0 Hz), 4.73 (app. t, 2H, J=10.8 Hz), 4.54 (dd, 1H, J=3.2, 10.4 Hz), 4.40 (dd, 1H, J=3.2, 10.4 Hz), 4.29-4.23 (m, 4H), 3.91-3.85 (m, 7H), 3.80-3.71 (m, 2H), 3.70-3.61 (m, 2H), 3.38-3.32 (m, 1H), 3.12-3.01 (m, 1H), 2.50-2.69 (m, 1H), 2.40 (q, 2H, J=5.6 Hz), 1.50-1.43 (m, 1H), 0.99-0.71 (m, 6H), 0.54-0.59 (m, 2H), 0.00 (s, 18H) ppm.

    (c) (S)-2-(4-Aminophenyl)-8-(3-(((S)-2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (15)

    [1244] SEM dilactam 14 (0.66 g, 0.69 mmol) was dissolved in THF (23 mL) and cooled to −78° C. under an argon atmosphere. Super-Hydride® solution (1.7 mL, 1 M in THF) was added drop wise over 5 minutes while monitoring the temperature. After 20 minutes a small sample was taken and washed with water for LC/MS analysis. Water (50 mL) was added and the cold bath was removed. The organic layer was extracted and washed with brine (60 mL). The combined aqueous layers were washed with CH.sub.2Cl.sub.2/MeOH (90/10 v/v) (2×50 mL). The combined organic layers were dried with MgSO.sub.4, filtered and the solvent removed in vacuo. The crude product was dissolved in MeOH (48 mL), CH.sub.2Cl.sub.2 (18 mL) and water (6 mL) and sufficient silica gel was added to afford a thick suspension. After 5 days stirring, the suspension was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (˜200 mL) until product ceased to be eluted. The organic layer was washed with brine (2×70 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (100% CHCl.sub.3 to 96/4 v/v CHCl.sub.3/MeOH) afforded the product 15 as a yellow solid (302 mg, 66%). Method 1, LC/MS (2.42 min (ES.sup.+) m/z (relative intensity) 660.74 ([M+H].sup.+., 30). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.86 (d, 1H, J=3.6 Hz), 7.78 (d, 1H, J=3.6 Hz), 7.58-7.44 (m, 3H), 7.34-7.20 (m, 3H), 6.88-6.66 (m, 4H), 4.35-4.15 (m, 6H), 3.95-3.75 (m, 7H), 3.39-3.22 (m, 1H), 3.14-3.04 (m, 1H), 2.93-2.85 (m, 1H), 2.46-2.36 (m, 2H), 1.49-1.41 (m, 1H), 0.80-0.72 (m, 2H), 0.58-0.51 (app. s, 2H) ppm.

    (d) Allyl ((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (16)

    [1245] In a degassed round bottom flask filled with argon, HO-Ala-Val-alloc (149.6 mg, 0.549 mmol) and EEDQ (135.8 mg, 0.549 mmol) were dissolved in a 9:1 mixture of dry CH.sub.2Cl.sub.2/MeOH (5 mL). The flask was wrapped in aluminium foil and the reaction mixture was allowed to stir at room temperature for 1 hour before starting material 15 (302 mg, 0.457 mmol) was added. The reaction mixture was left to stir for a further 40 hours at room temperature before the volatiles were removed by rotary evaporation under reduced pressure (the reaction was followed by LC/MS, RT starting material 2.32 min, (ES.sup.+ 660.29 ([M+H].sup.+., 100)). The crude product was directly purified by silica gel chromatography column (100% CHCl.sub.3 to 90/10 v/v CHCl.sub.3/MeOH) to afford the pure product (16) in 42% yield (174 mg). Method 2 LC/MS (2.70 min (ES+) m/z (relative intensity) 914.73 ([M+H].sup.+., 60), 660.43 (60), 184.31 (100)).

    (e) (2S)-2-amino-N-((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (17)

    [1246] The starting material 16 (170 mg, 0.185 mmol) was dissolved in dry CH.sub.2Cl.sub.2 (5 mL) in a round bottom flask filled with argon, before pyrrolidine (41 μL, 0.21 mmol) was added. The flask was purged/refilled three times with argon before Pd(PPh.sub.3).sub.4 (14 mg, 0.084 mmol) was added and the flushing operation repeated. After 1 hour, complete consumption of starting material was observed (the reaction was followed by LC/MS) and Et.sub.2O (50 mL) was added to the reaction mixture which was allowed to stir until all the product had crashed out of solution. The solid was filtered through a sintered funnel and washed twice with Et.sub.2O (2×25 mL). The collecting flask was replaced and the isolated solid was dissolved in CHCl.sub.3 (100 mL or until all the product had passed through the sintered funnel). The volatiles were then removed by rotary evaporation under reduced pressure to afford the crude product 17 which was used directly in the next step (168 mg). LC/MS method 2 (2.70 min (ES+) m/z (relative intensity) 830.27 ([M+H].sup.+., 50), 660.13 (80), 171.15 (100)).

    (f) N—((R)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (18)

    [1247] Starting material 17 (154 mg, 0.185 mmol) and EDCl.HCl (110 mg, 0.185 mmol) were solubilised in dry CH.sub.2Cl.sub.2 (5 mL) in a round bottom flask purged and filled with argon. The mixture was left to stir at room temperature for 1 hour before PEG.sub.8-maleimide (35.6 mg, 0.185 mmol) was added and the reaction mixture stirred for a further 16 hours (or until the reaction is complete, monitored by LC/MS). The reaction solution was diluted with CH.sub.2Cl.sub.2 (50 mL) and the organics were washed with H.sub.2O (50 mL) and brine (50 mL) before being dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure to afford the crude product. Purification on silica gel column chromatography (100% CHCl.sub.3 to 85/15 v/v CHCl.sub.3/MeOH) gave the desired product (135 mg), however remaining traces of unreacted PEG.sub.8-maleimide were observed (by LC/MS, 2.21 min, method 2). Automated reverse phase silica gel chromatography (H.sub.2O/CH.sub.3CN) (see general information for conditions) successfully removed the impurity affording pure final product (18, 37 mg of pure product starting from 110 mg, 33%). Overall yield=17%. Method 2 LC/MS (2.58 min (ES+) m/z (relative intensity) 1404.03 ([M+H].sup.+., 20), 702.63 (100)). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91 (t, J=3.5 Hz, 1H), 7.80 (d, J=4.0 Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H), 7.39-7.31 (m, 2H), 6.87 (d, J=10.5 Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J=10.2, 5.2 Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J=16.1, 4.1 Hz, 1H), 2.62-2.49 (m, 4H), 2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J=9.2, 5.3 Hz, 2H), 0.57 (dd, J=9.2, 5.3 Hz, 2H), NH were not observed.

    Example 2

    [1248] ##STR00127## ##STR00128##

    (a) (R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido) propanoic acid (20b)

    [1249] HO-Ala-Val-H 20a (350 mg, 1.86 mmol) and Na.sub.2CO.sub.3 (493 mg, 4.65 mmol) were dissolved in distilled H.sub.2O (15 mL) and the mixture was cooled to 0° C. before dioxane (15 mL) was added (partial precipitation of the amino acid salt occurred). A solution of Fmoc-Cl (504 mg, 1.95 mmol) in dioxane (15 mL) was added dropwise with vigorous stirring over 10 minutes. The resulting mixture was stirred at 0° C. for 2 hours before the ice bath was removed and stirring was maintained for 16 hours. The solvent was removed by rotary evaporation under reduced pressure and the residue dissolved in water (150 mL). The pH was adjusted from 9 to 2 with 1N HCl and the aqueous layer was subsequently extracted with EtOAc (3×100 mL). The combined organics were washed with brine (100 mL), dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure to afford pure HO-Ala-Val-Fmoc 20b (746 mg, 97% yield). LC/MS 2.85 min (ES+) m/z (relative intensity) 410.60; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.79 (d, J=7.77 Hz, 2H), 7.60 (d, J=7.77 Hz, 2H), 7.43 (d, J=7.5 Hz, 2H), 7.34 (d, J=7.5 Hz, 2H), 6.30 (bs, 1H), 5.30 (bs, 1H), 4.71-7.56 (m, 1H), 4.54-4.36 (m, 2H), 4.08-3.91 (m, 1H), 2.21-2.07 (m, 1H), 1.50 (d, J=7.1 Hz, 3H), 1.06-0.90 (m, 6H).

    (b) (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-(4,4,5,5-tetramethyl-1,3, 2-dioxaborolan-2-yl)phenyl)amino)propan-2-yl)amino)butan-2-yl) carbamate (20)

    [1250] 4-Aminophenylboronic acid pinacol ester was added (146.9 mg, 0.67 mmol) was added to a solution of HO-Ala-Val-Fmoc 20b (330 mg, 0.8 mmol), DCC (166 mg, 0.8 mmol) and DMAP (5 mg, cat.) in dry DCM (8 mL) previously stirred for 30 minutes at room temperature in a flask flushed with argon. The reaction mixture was then allowed to stir at room temperature overnight. The reaction was followed by LCMS and TLC. The reaction mixture was diluted with CH.sub.2Cl.sub.2 and the organics were washed with H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dryloaded on a silicagel chromatography column (Hexane/EtOAc, 6:4) and pure product 20 was isolated as a white solid in 88% yield (360 mg).

    (c) 8-(3-((2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl trifluoromethanesulfonate (21)

    [1251] Bis-triflate 12 (2.03 g, 1.81 mmol), boronic pinacol ester (1 g, 1.63 mmol) and Na.sub.2CO.sub.3 (881 mg, 8.31 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (40 mL). The reaction flask was purged and filled with argon three times before tetrakis(triphenylphosphine)palladium(0) (41 mg, 0.035 mmol) was added and the reaction mixture heated to 30° C. overnight. The solvents were removed under reduce pressure and the residue was taken up in H.sub.2O (100 mL) and extracted with EtOAc (3×100 mL). The combined organics were washed with brine (100 mL), dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel chromatography column (Hexane/EtOAc, 8:2 to 25:75) to afford pure 21 in 33% yield (885 mg). LC/MS 3.85 min (ES+) m/z (relative intensity) 1452.90; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.78-7.16 (m, 17H), 7.13 (s, 1H), 6.51-6.24 (m, 1H), 5.51 (dd, J=10.0, 5.1 Hz, 2H), 5.36-5.11 (m, 1H), 4.74 (dd, J=10.1, 4.4 Hz, 2H), 4.70-4.53 (m, 2H), 4.47 (d, J=6.4 Hz, 1H), 4.37 (d, J=7.2 Hz, 1H), 4.27 (m, 4H), 4.20-4.14 (m, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.77 (ddd, J=16.7, 9.0, 6.4 Hz, 3H), 3.71-3.61 (m, 2H), 3.24-2.91 (m, 3H), 2.55-2.33 (m, 2H), 2.22-2.07 (m, 1H), 1.52-1.37 (m, 3H), 1.04-0.86 (m, 10H), 0.00 (s, 18H).

    (d) (9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate (22)

    [1252] Triphenylarsine (42 mg, 0.137 mmol) was added to a mixture of PBD-triflate 21 (250 mg, 0.172 mmol), cyclopropylboronic acid (73.9 mg, 0.86 mmol), silver oxide (159 mg, 0.688 mmol) and potassium phosphate tribasic (438 mg, 2.06 mmol) in dry dioxane (10 mL) under an argon atmosphere. The reaction was flushed with argon 3 times and bis(benzonitrile)palladium(II) chloride (13.2 mg, 0.034 mmol) was added. The reaction was flushed with Argon 3 more times before being warmed to 75° C. and stirred for 10 minutes. The reaction mixture was filtered through a pad of celite which was subsequently rinsed with ethyl acetate. The solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 1% methanol/chloroform). Pure fractions were collected and combined, and excess eluent was removed by rotary evaporation under reduced pressure to afford the desired product 22 (132 mg, 50% yield). LC/MS 3.83 min (ES+) m/z (relative intensity) 1345.91; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.88-7.14 (m, 17H), 6.69 (s, 1H), 6.45-6.25 (m, 1H), 5.57-5.41 (m, 2H), 5.34-5.14 (m, 1H), 4.78-4.67 (m, 2H), 4.62-4.55 (m, 1H), 4.50-4.45 (m, 2H), 4.51-4.44 (m, 1H), 4.31-4.21 (m, 4H), 4.16 (m, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.82-3.71 (m, 2H), 3.66 (m, 3H), 3.40-3.28 (m, 1H), 3.07 (m, 1H), 2.70-2.57 (m, 1H), 2.47-2.36 (m, 2H), 2.15 (m, 1H), 1.51-1.40 (m, 3H), 1.03-0.87 (m, 11H), 0.77-0.71 (m, 2H), 0.60-0.54 (m, 2H), 0.00 (t, J=3.0 Hz, 18H).

    (e) (9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (23)

    [1253] A solution of Super-Hydride® (0.5 mL, 1M in THF) was added dropwise to a solution of SEM dilactam 22 (265 mg g, 0.19 mmol) in THF (10 mL) at −78° C. under an argon atmosphere.

    [1254] The addition was completed over 5 minutes in order to maintain the internal temperature of the reaction mixture constant. After 20 minutes, an aliquot was quenched with water for LC/MS analysis, which revealed that the reaction was complete. Water (20 mL) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with EtOAc (3×30 mL) and the combined organics were washed with brine (50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in MeOH (12 mL), CH.sub.2Cl.sub.2 (6 mL), water (2 mL) and enough silica gel to form a thick stirring suspension. After 5 days, the suspension was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (200 mL) until the elution of the product was complete. The organic layer was washed with brine (2×70 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCl.sub.3 to 96% CHCl.sub.3/4% MeOH) afforded the product 23 as a yellow solid (162 mg, 78%). LC/MS 3.02 min (ES+) m/z (relative intensity) 1052.37.

    (f) (2S)-2-amino-N-((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11α-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (17)

    [1255] Excess piperidine was added (0.2 mL, 2 mmol) to a solution of SEM-dilactam 23 (76 mg, 0.073 mmol) in DMF (1 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH.sub.2Cl.sub.2 (75 mL) and the organic phase was washed with H.sub.2O (3×75 mL) until complete piperidine removal. The organic phase was dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude product 17 which was used as such in the next step. LC/MS 2.32 min (ES+) m/z (relative intensity) 830.00.

    (g) N-((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (18)

    [1256] EDCl hydrochloride (14 mg, 0.0732 mmol) was added to a suspension of Maleimide-PEG.sub.8-acid (43.4 mg, 0.0732 mmol) in dry CH.sub.2Cl.sub.2 (5 mL) under argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 17 (60.7 mg, 0.0732 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH.sub.2Cl.sub.2 and the organic phase was washed with H.sub.2O and brine before being dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCl.sub.3 up to 9:1 CHCl.sub.3/MeOH) followed by reverse phase chromatography to remove unreacted maleimide-PEG.sub.8-acid. The product 18 was isolated in 17.6% (21.8 mg). LC/MS 2.57 min (ES+) m/z (relative intensity) 1405.30; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91 (t, J=3.5 Hz, 1H), 7.80 (d, J=4.0 Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H), 7.39-7.31 (m, 2H), 6.87 (d, J=10.5 Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J=10.2, 5.2 Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J=16.1, 4.1 Hz, 1H), 2.62-2.49 (m, 4H), 2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J=9.2, 5.3 Hz, 2H), 0.57 (dd, J=9.2, 5.3 Hz, 2H), NH were not observed.

    Example 3

    [1257] ##STR00129## ##STR00130##

    (a) (S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl trifluoromethanesulfonate (24)

    [1258] Pd(PPh.sub.3).sub.4 (20.6 mg, 0.018 mmol) was added to a stirred mixture of the bis-enol triflate 12 (500 mg, 0.44 mmol), N-methyl piperazine boronic ester (100 mg, 0.4 mmol), Na.sub.2CO.sub.3 (218 mg, 2.05 mmol), MeOH (2.5 mL), toluene (5 mL) and water (2.5 mL). The reaction mixture was allowed to stir at 30° C. under a nitrogen atmosphere for 24 hours after which time all the boronic ester has consumed. The reaction mixture was then evaporated to dryness before the residue was taken up in EtOAc (100 mL) and washed with H.sub.2O (2×50 mL), brine (50 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 80:20 v/v Hexane/EtOAc to 60:40 v/v Hexane/EtOAc) afforded product 24 as a yellowish foam (122.6 mg, 25%). LC/MS 3.15 min (ES+) m/z (relative intensity) 1144 ([M+H].sup.+., 20%).

    (b) (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate (25)

    [1259] PBD-triflate 24 (359 mg, 0.314 mmol), boronic pinacol ester 20 (250 mg, 0.408 mmol) and triethylamine (0.35 mL, 2.51 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (3 mL). The microwave vessel was purged and filled with argon three times before tetrakis(triphenylphosphine)palladium(0) (21.7 mg, 0.018 mmol) was added and the reaction mixture placed in the microwave at 80° C. for 10 minutes. Subsequently, CH.sub.2Cl.sub.2 (100 mL) was added and the organics were washed with water (2×50 mL) and brine (50 mL) before being dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel chromatography column (CHCl.sub.3/MeOH, 100% to 9:1) to afford pure 25 (200 mg, 43% yield). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478 ([M+H].sup.+., 100%).

    (c) (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-4-((S)-7-methoxy-8-(3-(((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)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 (26)

    [1260] A solution of Super-Hydride® (0.34 mL, 1M in THF) was added dropwise to a solution of SEM-dilactam 25 (200 mg, 0.135 mmol) in THF (5 mL) at −78° C. under an argon atmosphere. The addition was completed over 5 minutes in order to maintain the internal temperature of the reaction mixture constant. After 20 minutes, an aliquot was quenched with water for LC/MS analysis, which revealed that the reaction was complete. Water (20 mL) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with EtOAc (3×30 mL) and the combined organics were washed with brine (50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in MeOH (6 mL), CH.sub.2Cl.sub.2 (3 mL), water (1 mL) and enough silica gel to form a thick stirring suspension. After 5 days, the suspension was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (100 mL) until the elution of the product was complete. The organic layer was washed with brine (2×50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCl.sub.3 to 96% CHCl.sub.3/4% MeOH) afforded the product 26 as a yellow solid (100 mg, 63%). LC/MS 2.67 min (ES+) m/z (relative intensity) 1186 ([M+H].sup.+., 5%).

    (d) (S)-2-amino-N—((S)-1-((4-((R)-7-methoxy-8-(3-(((R)-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)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (27)

    [1261] Excess piperidine was added (0.1 mL, 1 mmol) to a solution of PBD 26 (36.4 mg, 0.03 mmol) in DMF (0.9 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL) and the organic phase was washed with H.sub.2O (3×50 mL) until complete piperidine removal. The organic phase was dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude product 27 which was used as such in the next step. LC/MS 2.20 min (ES+) m/z (relative intensity) 964 ([M+H].sup.+., 5%).

    (e) 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-(4-methylpiperazin-1-yl)phenyl)-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 (28)

    [1262] EDCl hydrochloride (4.7 mg, 0.03 mmol) was added to a suspension of 6-maleimidohexanoic acid (6.5 mg, 0.03 mmol) in dry CH.sub.2Cl.sub.2 (3 mL) under argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 27 (34 mg, crude) was added. Stirring was maintained until the reaction was complete (6 hours). The reaction was diluted with CH.sub.2Cl.sub.2 and the organic phase was washed with H.sub.2O and brine before being dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCl.sub.3 up to 9:1 CHCl.sub.3/MeOH) followed by reverse phase chromatography to remove unreacted maleimide-PEG.sub.8-acid. The product 28 was isolated in 41% over two steps (14.6 mg). LC/MS 2.40 min (ES+) m/z (relative intensity) 1157 ([M+H].sup.+., 5%)

    Example 4—Alternative Synthesis of Compound 25

    [1263] ##STR00131##

    [1264] PBD-triflate 21 (469 mg, 0.323 mmol), boronic pinacol ester (146.5 mg, 0.484 mmol) and Na.sub.2CO.sub.3 (157 mg, 1.48 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (10 mL). The reaction flask was purged with argon three times before tetrakis(triphenylphosphine)palladium(0) (7.41 mg, 0.0064 mmol) was added and the reaction mixture heated to 30° C. overnight. The solvents were removed under reduced pressure and the residue was taken up in H.sub.2O (50 mL) and extracted with EtOAc (3×50 mL). The combined organics were washed with brine (100 mL), dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography (CHCl.sub.3 100% to CHCl.sub.3/MeOH 95%:5%) to afford pure 25 in 33% yield (885 mg). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478 ([M+H].sup.+., 100%).

    Example 5

    [1265] ##STR00132##

    (a) (S)-2-(4-Aminophenyl)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (29)

    [1266] 3,4-(Methylenedioxy)phenyl boronic acid (356 mg, 2.1 mmol, 1.3 equiv.), TEA (1.8 mL, 12.9 mmol, 8 equiv.) and triflate/aniline 13 (1.75 g, 1.7 mmol, 1 equiv.) were dissolved in a mixture of ethanol (7 mL), toluene (13 mL) and water (2 mL) under an Ar atmosphere. The reaction mixture was evacuated and flushed with Ar 3 times, before addition of tetrakis(triphenylphosphine)palladium(0) (114 mg, 0.1 mmol, 0.06 equiv.). The flask was again evacuated and flushed with Ar 3 times and heated in a microwave at 80° C. for 8 minutes with 30 seconds pre-stirring time. Analysis by TLC (80:20 v/v ethyl acetate/hexane) indicated complete consumption of starting material. The reaction mixture was diluted with dichloromethane (50 mL) and washed with water (50 mL). The organic layer was dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (60:40 to 20:80 v/v hexane/ethyl acetate) afforded the product 29 as a yellow solid (1.21 g, 71%). LC/MS (3.92 min (ES.sup.+) m/z (relative intensity) 1032.44 ([M+H].sup.+., 100).

    (b) (S)-2-(4-Aminophenyl)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one (30)

    [1267] SEM dilactam 29 (0.25 g, 0.24 mmol, 1 equiv.) was dissolved in THF (8 mL) and cooled to −78° C. under an Ar atmosphere. Super-Hydride® (0.6 mL, 1 M in THF, 2.5 equiv.) was added drop wise over 5 minutes while monitoring the temperature. After 20 minutes a small sample was taken and worked-up for LCMS analysis. Water (50 mL) was added, the cold bath was removed and the solution washed with ethyl acetate (50 mL). The organic layer was extracted and washed with brine (60 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. The crude product was dissolved in EtOH (15 mL), CH.sub.2Cl.sub.2 (7.5 mL) and water (2.5 mL) and enough silica gel was added until it was a thick suspension. After 5 days stirring, it was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (100 mL) until product ceased to be eluted. The organic layer was washed with brine (2×50 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (CHCl.sub.3 with 1% to 4% MeOH gradient) afforded the product 30 as a yellow solid (94 mg, 53%). LC/MS (2.53 min (ES.sup.+) m/z (relative intensity) 739.64 ([M].sup.+., 70).

    (c) Allyl ((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-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)carbamate (31)

    [1268] Under an Ar atmosphere, Alanine-Valine-Alloc (180 mg, 0.66 mmol, 1.2 equiv.) was stirred with EEDQ (163 mg, 0.66 mmol, 1.2 equiv.) in anhydrous CH.sub.2Cl.sub.2 (21 mL) and methanol (1 mL) for 1 hour. The PBD 30 (407 mg, 0.55 mmol, 1 equiv.) was dissolved in anhydrous CH.sub.2Cl.sub.2 (21 mL) and methanol (1 mL) and added to the reaction. LC/MS after 5 days stirring at room temperature showed majority product formation. The solvent was removed in vacuo before purification by column chromatography (CH.sub.2Cl.sub.2 with 1% to 6% MeOH gradient) to yield the product 31 as a yellow solid (184 mg, 34%). LC/MS (2.95 min (ES.sup.+) m/z (relative intensity) 994.95 ([M+H].sup.+., 60).

    (d) (S)-2-Amino-N—((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (32)

    [1269] The imine 31 (100 mg, 0.1 mmol, 1 equiv.) was dissolved in anhydrous DCM (10 mL) (with the aid of one drop of methanol to aid dissolution) under an Ar atmosphere. Pyrrolidine (30 μL, 0.15 mmol, 1.5 equiv.) was added drop wise before the flask was evacuated and flushed with Ar three times. Pd(PPh.sub.3).sub.4 (7 mg, 6 μmol, 0.06 equiv.) was added and the flask was evacuated and flushed with Ar three times. LC/MS analysis after 1 hour indicated product formation and complete loss of starting material. Et.sub.2O (60 mL) was added to the reaction mixture and it was left to stir until all the product had crashed out of solution. The precipitate was filtered through a sintered funnel and washed twice with Et.sub.2O (2×20 mL). The collection flask was replaced and the isolated solid was dissolved and washed through the sinter with CHCl.sub.3 (100 mL). The solvent was removed in vacuo to afford the crude product 32 as a yellow solid which was used directly in the next step. LC/MS (1.14 min (ES.sup.+) m/z (relative intensity) 910.40 ([M+H].sup.+., 67).

    (e) N—((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(Benzo[d][1, 3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-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)-1-(3-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (33)

    [1270] The imine 32 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHCl.sub.3 (6 mL) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG.sub.8-acid (53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14 mmol, 1.5 equiv.). This was left to stir vigorously at room temperature under Ar for 4 days until LC/MS analysis showed majority product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHCl3 with 1% to 10% MeOH gradient) yielding 33 (81 mg). The material was purified further by preparative HPLC to give 33 as a yellow solid (26.3 mg, 18%). Fast Formic run: LC/MS (1.39 min (ES+) m/z (relative intensity) 1485.00 ([M+H]+., 64).

    Example 6

    [1271] ##STR00133##

    (a) 9H-Flouren-9-yl)methyl ((S)-1-((S)-1-((4-((S)-8-3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate (34)

    [1272] The triflate 21 (0.5 g, 0.35 mmol, 1 equiv.), 3, 4-(methylenedioxy)phenyl boronic acid (75 mg, 0.45 mmol, 1.3 equiv.) and Na.sub.2CO.sub.3 (0.17 g, 1.6 mmol, 4.5 equiv.) were dissolved in toluene (11 mL), EtOH (5.5 mL) and water (5.5 mL) under an Ar atmosphere. The flask was evacuated and flushed with Ar three times. Pd(PPh.sub.3).sub.4 (24 mg, 0.02 mmol, 0.06 equiv.) was added and again the flask was evacuated and flushed with Ar three times. This was heated to 30° C. and left stirring overnight. Analysis by LC/MS showed complete loss of starting material. The solvent was removed in vacuo and the residue dissolved in water (60 mL) before washing with ethyl acetate (60 mL×3). The combined organic layers were washed with brine (50 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by column chromatography (50:50 to 25:75 v/v hexane/ethyl acetate) afforded the product 34 as a yellow solid (310 mg, 64%). LC/MS (1.44 min (ES.sup.−) m/z (relative intensity) 1423.35 ([M−H].sup.−., 79).

    (b) (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-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)carbamate (35)

    [1273] SEM dilactam 34 (0.31 g, 0.22 mmol, 1 equiv.) was dissolved in THF (10 mL) and cooled to −78° C. under an Ar atmosphere. Super-Hydride® (0.5 mL, 1 M in THF, 2.5 equiv.) was added drop wise over 5 minutes while monitoring the temperature. After 30 minutes a small sample was taken and worked-up for LC/MS analysis. Water (50 mL) was added, the cold bath was removed and the solution washed with ethyl acetate (50 mL). The organic layer was extracted and washed with brine (60 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. The crude product was dissolved in EtOH (13.2 mL), CH.sub.2Cl.sub.2 (6.6 mL) and water (2.2 mL) and enough silica gel was added until it was a thick suspension. After 5 days stirring, it was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (100 mL) until product ceased to be eluted. The organic layer was washed with brine (2×50 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (CHCl.sub.3 with 1% to 4% MeOH gradient) afforded the pure product 35 as a yellow solid (185 mg, 75%). LC/MS (1.70 min (ES.sup.+) m/z (relative intensity) 1132.85 ([M+H].sup.+., 60).

    (c) (S)-2-Amino-N—((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (32)

    [1274] The imine 35 (82 mg, 0.07 mmol, 1 equiv.) was dissolved in DMF (1 mL) before piperidine (0.2 mL, 2 mmol, excess) was added slowly. This solution was left to stir at room temperature for 20 minutes until LC/MS analysis showed complete consumption of starting material. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL), washed with water (50 mL×4), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. The product 33 was used without further purification in the next step. LC/MS (1.15 min (ES.sup.+) m/z (relative intensity) 910.60 ([M+H].sup.+., 58).

    Example 7

    (i) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone (49)

    [1275] ##STR00134## ##STR00135##

    (a) 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (42)

    [1276] Neat triisopropylsilylchloride (56.4 mL, 262 mmol) was added to a mixture of imidazole (48.7 g, 715.23 mmol) and 4-hydroxy-5-methoxy-2-nitrobenzaldehyde 41 (47 g, 238 mmol) (ground together). The mixture was heated until the phenol and imidazole melted and went into solution (100° C.). The reaction mixture was allowed to stir for 15 minutes and was then allowed to cool, whereupon a solid was observed to form at the bottom of the flask (imidazole chloride). The reaction mixture was diluted with 5% EtOAc/hexanes and loaded directly onto silica gel and the pad was eluted with 5% EtOAc/hexanes, followed by 10% EtOAc/hexanes (due to the low excess, very little unreacted TIPSCl was found in the product). The desired product was eluted with 5% ethyl acetate in hexane. Excess eluent was removed by rotary evaporation under reduced pressure, followed by drying under high vacuum to afford a crystalline light sensitive solid (74.4 g, 88%). Purity satisfactory by LC/MS (4.22 min (ES+) m/z (relative intensity) 353.88 ([M+H].sup.+., 100)); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.43 (s, 1H), 7.60 (s, 1H), 7.40 (s, 1H), 3.96 (s, 3H), 1.35-1.24 (m, 3H), 1.10 (m, 18H).

    (b) 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (43)

    [1277] A solution of sodium chlorite (47.3 g, 523 mmol, 80% technical grade) and sodium dihydrogenphosphate monobasic (35.2 g, 293 mmol) (NaH.sub.2PO.sub.4) in water (800 mL) was added to a solution of compound 2 (74 g, 209 mmol) in tetrahydrofuran (500 mL) at room temperature. Hydrogen peroxide (60% w/w, 140 mL, 2.93 mol) was immediately added to the vigorously stirred biphasic mixture. The reaction mixture evolved gas (oxygen), the starting material dissolved and the temperature of the reaction mixture rose to 45° C. After 30 minutes LC/MS revealed that the reaction was complete. The reaction mixture was cooled in an ice bath and hydrochloric acid (1 M) was added to lower the pH to 3 (this step was found unnecessary in many instances, as the pH at the end of the reaction is already acidic; please check the pH before extraction). The reaction mixture was then extracted with ethyl acetate (1 L) and the organic phases washed with brine (2×100 mL) and dried over magnesium sulphate. The organic phase was filtered and excess solvent removed by rotary evaporation under reduced pressure to afford the product 43 in quantitative yield as a yellow solid. LC/MS (3.93 min (ES−) m/z (relative intensity) 367.74 ([M−H].sup.−., 100)); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.36 (s, 1H), 7.24 (s, 1H), 3.93 (s, 3H), 1.34-1.22 (m, 3H), 1.10 (m, 18H).

    (c) ((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidin-1-yl) (5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (45)

    [1278] DCC (29.2 g, 141 mmol, 1.2 eq) was added to a solution of acid 3 (43.5 g, 117.8 mmol, 1 eq), and hydroxybenzotriazole hydrate (19.8 g, 129.6 mmol, 1.1 eq) in dichloromethane (200 mL) at 0° C. The cold bath was removed and the reaction was allowed to proceed for 30 mins at room temperature, at which time a solution of (2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine 44 (30 g, 129.6 mmol, 1.1 eq) and triethylamine (24.66 mL, 176 mmol, 1.5 eq) in dichloromethane (100 mL) was added rapidly at −10° C. under argon (on large scale, the addition time could be shortened by cooling the reaction mixture even further. The reaction mixture was allowed to stir at room temperature for 40 minutes to 1 hour and monitored by LC/MS and TLC (EtOAc). The solids were removed by filtration over celite and the organic phase was washed with cold aqueous 0.1 M HCl until the pH was measured at 4 or 5. The organic phase was then washed with water, followed by saturated aqueous sodium bicarbonate and brine. The organic layer was dried over magnesium sulphate, filtered and excess solvent removed by rotary evaporation under reduced pressure. The residue was subjected to column flash chromatography (silica gel; gradient 40/60 ethyl acetate/hexane to 80/20 ethyl acetate/hexane). Excess solvent was removed by rotary evaporation under reduced pressure afforded the pure product 45, (45.5 g of pure product 66%, and 17 g of slightly impure product, 90% in total). LC/MS 4.43 min (ES+) m/z (relative intensity) 582.92 ([M+H].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.66 (s, 1H), 6.74 (s, 1H), 4.54 (s, 1H), 4.40 (s, 1H), 4.13 (s, 1H), 3.86 (s, 3H), 3.77 (d, J=9.2 Hz, 1H), 3.36 (dd, J=11.3, 4.5 Hz, 1H), 3.14-3.02 (m, 1H), 2.38-2.28 (m, 1H), 2.10 (ddd, J=13.3, 8.4, 2.2 Hz, 1H), 1.36-1.19 (m, 3H), 1.15-1.05 (m, 18H), 0.91 (s, 9H), 0.17-0.05 (m, 6H), (presence of rotamers).

    (d) (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)pyrrolidin-3-one (46)

    [1279] TCCA (8.82 g, 40 mmol, 0.7 eq) was added to a stirred solution of 45 (31.7 g, 54 mmol, 1 eq) and TEMPO (0.85 g, 5.4 mmol, 0.1 eq) in dry dichloromethane (250 mL) at 0° C. The reaction mixture was vigorously stirred for 20 minutes, at which point TLC (50/50 ethyl acetate/hexane) revealed complete consumption of the starting material. The reaction mixture was filtered through celite and the filtrate washed with aqueous saturated sodium bicarbonate (100 mL), sodium thiosulphate (9 g in 300 mL), brine (100 mL) and dried over magnesium sulphate. Rotary evaporation under reduced pressure afforded product 46 in quantitative yield. LC/MS 4.52 min (ES+) m/z (relative intensity) 581.08 ([M+H].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.78-7.60 (m, 1H), 6.85-6.62 (m, 1H), 4.94 (dd, J=30.8, 7.8 Hz, 1H), 4.50-4.16 (m, 1H), 3.99-3.82 (m, 3H), 3.80-3.34 (m, 3H), 2.92-2.17 (m, 2H), 1.40-1.18 (m, 3H), 1.11 (t, J=6.2 Hz, 18H), 0.97-0.75 (m, 9H), 0.15-−0.06 (m, 6H), (presence of rotamers).

    (e) (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4, 5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate (47)

    [1280] Triflic anhydride (27.7 mL, 46.4 g, 165 mmol, 3 eq) was injected (temperature controlled) to a vigorously stirred suspension of ketone 46 (31.9 g, 55 mmol, 1 eq) in dry dichloromethane (900 mL) in the presence of 2,6-lutidine (25.6 mL, 23.5 g, 220 mmol, 4 eq, dried over sieves) at −50° C. (acetone/dry ice bath). The reaction mixture was allowed to stir for 1.5 hours when LC/MS, following a mini work-up (water/dichloromethane), revealed the reaction to be complete. Water was added to the still cold reaction mixture and the organic layer was separated and washed with saturated sodium bicarbonate, brine and magnesium sulphate. The organic phase was filtered and excess solvent was removed by rotary evaporation under reduced pressure. The residue was subjected to column flash chromatography (silica gel; 10/90 v/v ethyl acetate/hexane), removal of excess eluent afforded the product 47 (37.6 g, 96%) LC/MS, method 2, 4.32 min (ES+) m/z (relative intensity) 712.89 ([M+H].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.71 (s, 1H), 6.75 (s, 1H), 6.05 (d, J=1.8 Hz, 1H), 4.78 (dd, J=9.8, 5.5 Hz, 1H), 4.15-3.75 (m, 5H), 3.17 (ddd, J=16.2, 10.4, 2.3 Hz, 1H), 2.99 (ddd, J=16.3, 4.0, 1.6 Hz, 1H), 1.45-1.19 (m, 3H), 1.15-1.08 (m, 18H), 1.05 (s, 6H), 0.95-0.87 (m, 9H), 0.15-0.08 (m, 6H).

    (f) (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrol-1-yl) (5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (48)

    [1281] Triphenylarsine (1.71 g, 5.60 mmol, 0.4 eq) was added to a mixture of triflate 47 (10.00 g, 14 mmol, 1 eq), methylboronic acid (2.94 g, 49.1 mmol, 3.5 eq), silver oxide (13 g, 56 mmol, 4 eq) and potassium phosphate tribasic (17.8 g, 84 mmol, 6 eq) in dry dioxane (80 mL) under an argon atmosphere. The reaction was flushed with argon 3 times and bis(benzonitrile)palladium(II) chloride (540 mg, 1.40 mmol, 0.1 eq) was added. The reaction was flushed with argon 3 more times before being warmed instantaneously to 110° C. (the drysyn heating block was previously warmed to 110° C. prior addition of the flask). After 10 mins the reaction was cooled to room temperature and filtered through a pad celite. The solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to column flash chromatography (silica gel; 10% ethyl acetate/hexane). Pure fractions were collected and combined, and excess eluent was removed by rotary evaporation under reduced pressure afforded the product 48 (4.5 g, 55%). LC/MS, 4.27 min (ES+) m/z (relative intensity) 579.18 ([M+H].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.70 (s, 1H), 6.77 (s, 1H), 5.51 (d, J=1.7 Hz, 1H), 4.77-4.59 (m, 1H), 3.89 (s, 3H), 2.92-2.65 (m, 1H), 2.55 (d, J=14.8 Hz, 1H), 1.62 (d, J=1.1 Hz, 3H), 1.40-1.18 (m, 3H), 1.11 (s, 9H), 1.10 (s, 9H), 0.90 (s, 9H), 0.11 (d, J=2.3 Hz, 6H).

    (g) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl) (2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrol-1-yl)methanone (49)

    [1282] Zinc powder (28 g, 430 mmol, 37 eq) was added to a solution of compound 48 (6.7 g, 11.58 mmol) in 5% formic acid in ethanol v/v (70 mL) at around 15° C. The resulting exotherm was controlled using an ice bath to maintain the temperature of the reaction mixture below 30° C. After 30 minutes the reaction mixture was filtered through a pad of celite. The filtrate was diluted with ethyl acetate and the organic phase was washed with water, saturated aqueous sodium bicarbonate and brine. The organic phase was dried over magnesium sulphate, filtered and excess solvent removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 10% ethyl acetate in hexane). The pure fractions were collected and combined and excess solvent was removed by rotary evaporation under reduced pressure to afford the product 49 (5.1 g, 80%). LC/MS, 4.23 min (ES+) m/z (relative intensity) 550.21 ([M+H].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.28 (s, 1H), 6.67 (s, 1H), 6.19 (s, 1H), 4.64-4.53 (m, J=4.1 Hz, 1H), 4.17 (s, 1H), 3.87 (s, 1H), 3.77-3.69 (m, 1H), 3.66 (s, 3H), 2.71-2.60 (m, 1H), 2.53-2.43 (m, 1H), 2.04-1.97 (m, J=11.9 Hz, 1H), 1.62 (s, 3H), 1.26-1.13 (m, 3H), 1.08-0.99 (m, 18H), 0.82 (s, 9H), 0.03-−0.03 (m, J=6.2 Hz, 6H).

    (ii) (11S,11aS)-allyl 11-((tert-butyldimethylsilyl)oxy)-8-((5-iodopentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate

    [1283] ##STR00136## ##STR00137##

    (a) (S)-allyl (2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (50)

    [1284] Allyl chloroformate (0.30 mL, 3.00 mmol, 1.1 eq) was added to a solution of amine 49 (1.5 g, 2.73 mmol) in the presence of dry pyridine (0.48 mL, 6.00 mmol, 2.2 eq) in dry dichloromethane (20 mL) at −78° C. (acetone/dry ice bath). After 30 minutes, the bath was removed and the reaction mixture was allowed to warm to room temperature. The reaction mixture was diluted with dichloromethane and saturated aqueous copper sulphate was added. The organic layer was then washed sequentially with saturated aqueous sodium bicarbonate and brine. The organic phase was dried over magnesium sulphate, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford the product 50 which was used directly in the next reaction. LC/MS, 4.45 min (ES+) m/z (relative intensity) 632.91 ([M+H].sup.+., 100)

    (b) (S)-allyl (2-(2-(hydroxymethyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl) carbamate (51)

    [1285] The crude 50 was dissolved in a 7:1:1:2 mixture of acetic acid/methanol/tetrahydrofuran/water (28:4:4:8 mL) and allowed to stir at room temperature. After 3 hours, complete disappearance of starting material was observed by LC/MS. The reaction mixture was diluted with ethyl acetate and washed sequentially with water (2×500 mL), saturated aqueous sodium bicarbonate (200 mL) and brine. The organic phase was dried over magnesium sulphate filtered and excess ethyl acetate removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel, 25% ethyl acetate in hexane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to afford the desired product 51 (1 g, 71%). LC/MS, 3.70 min (ES+) m/z (relative intensity) 519.13 ([M+H].sup.+., 95); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.34 (s, 1H), 7.69 (s, 1H), 6.78 (s, 1H), 6.15 (s, 1H), 5.95 (ddt, J=17.2, 10.5, 5.7 Hz, 1H), 5.33 (dq, J=17.2, 1.5 Hz, 1H), 5.23 (ddd, J=10.4, 2.6, 1.3 Hz, 1H), 4.73 (tt, J=7.8, 4.8 Hz, 1H), 4.63 (dt, J=5.7, 1.4 Hz, 2H), 4.54 (s, 1H), 3.89-3.70 (m, 5H), 2.87 (dd, J=16.5, 10.5 Hz, 1H), 2.19 (dd, J=16.8, 4.6 Hz, 1H), 1.70 (d, J=1.3 Hz, 3H), 1.38-1.23 (m, 3H), 1.12 (s, 10H), 1.10 (s, 8H).

    (c) (11S,11aS)-allyl 11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (52)

    [1286] Dimethyl sulphoxide (0.35 mL, 4.83 mmol, 2.5 eq) was added dropwise to a solution of oxalyl chloride (0.2 mL, 2.32 mmol, 1.2 eq) in dry dichloromethane (10 mL) at −78° C. (dry ice/acetone bath) under an atmosphere of argon. After 10 minutes a solution of 51 (1 g, 1.93 mmol) in dry dichloromethane (8 mL) was added slowly with the temperature still at −78° C. After 15 min triethylamine (1.35 mL, dried over 4 Å molecular sieves, 9.65 mmol, 5 eq) was added dropwise and the dry ice/acetone bath was removed. The reaction mixture was allowed to reach room temperature and was extracted with cold hydrochloric acid (0.1 M), saturated aqueous sodium bicarbonate and brine. The organic phase was dried over magnesium sulphate, filtered and excess dichloromethane was removed by rotary evaporation under reduced pressure to afford product 52 (658 mg, 66%). LC/MS, 3.52 min (ES+) m/z (relative intensity) 517.14 ([M+H].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.20 (s, 1H), 6.75-6.63 (m, J=8.8, 4.0 Hz, 2H), 5.89-5.64 (m, J=9.6, 4.1 Hz, 2H), 5.23-5.03 (m, 2H), 4.68-4.38 (m, 2H), 3.84 (s, 3H), 3.83-3.77 (m, 1H), 3.40 (s, 1H), 3.05-2.83 (m, 1H), 2.59 (d, J=17.1 Hz, 1H), 1.78 (d, J=1.3 Hz, 3H), 1.33-1.16 (m, 3H), 1.09 (d, J=2.2 Hz, 9H), 1.07 (d, J=2.1 Hz, 9H).

    (d) (11S,11aS)-allyl 11-((tert-butyldimethylsilyl)oxy)7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (53)

    [1287] Tert-butyldimethylsilyltriflate (0.70 mL, 3.00 mmol, 3 eq) was added to a solution of compound 52 (520 mg, 1.00 mmol) and 2,6-lutidine (0.46 mL, 4.00 mmol, 4 eq) in dry dichloromethane (40 mL) at 0° C. under argon. After 10 min, the cold bath was removed and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was extracted with water, saturated aqueous sodium bicarbonate and brine. The organic phase was dried over magnesium sulphate, filtered and excess was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; gradient, 10% ethyl acetate in hexane to 20% ethyl acetate in hexane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product 53 (540 mg, 85%). LC/MS, 4.42 min (ES+) m/z (relative intensity) 653.14 ([M+Na].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.20 (s, 1H), 6.71-6.64 (m, J=5.5 Hz, 2H), 5.83 (d, J=9.0 Hz, 1H), 5.80-5.68 (m, J=5.9 Hz, 1H), 5.14-5.06 (m, 2H), 4.58 (dd, J=13.2, 5.2 Hz, 1H), 4.36 (dd, J=13.3, 5.5 Hz, 1H), 3.84 (s, 3H), 3.71 (td, J=10.1, 3.8 Hz, 1H), 2.91 (dd, J=16.9, 10.3 Hz, 1H), 2.36 (d, J=16.8 Hz, 1H), 1.75 (s, 3H), 1.31-1.16 (m, 3H), 1.12-1.01 (m, J=7.4, 2.1 Hz, 18H), 0.89-0.81 (m, 9H), 0.25 (s, 3H), 0.19 (s, 3H).

    (e) (11S,11aS)-allyl 11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (54)

    [1288] Lithium acetate (87 mg, 0.85 mmol) was added to a solution of compound 53 (540 mg, 0.85 mmol) in wet dimethylformamide (6 mL, 50:1 DMF/water). After 4 hours, the reaction was complete and the reaction mixture was diluted with ethyl acetate (25 mL) and washed with aqueous citric acid solution (pH˜3), water and brine. The organic layer was dried over magnesium sulphate filtered and excess ethyl acetate was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; gradient, 25% to 75% ethyl acetate in hexane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product 54 (400 mg, quantitative). LC/MS, (3.33 min (ES+) m/z (relative intensity) 475.26 ([M+H].sup.+, 100).

    (f) (11S,11aS)-allyl 11-((tert-butyldimethylsilyl)oxy)-8-((5-iodopentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (55)

    [1289] Diiodopentane (0.63 mL, 4.21 mmol, 5 eq) and potassium carbonate (116 mg, 0.84 mmol, 1 eq) were added to a solution of phenol 54 (400 mg, 0.84 mmol) in acetone (4 mL, dried over molecular sieves). The reaction mixture was then warmed to 60° C. and stirred for 6 hours. Acetone was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 50/50, v/v, hexane/ethyl acetate,). Pure fractions were collected and combined and excess eluent was removed to provide 55 in 90% yield. LC/MS, 3.90 min (ES+) m/z (relative intensity) 670.91 ([M]+, 100). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.23 (s, 1H), 6.69 (s, 1H), 6.60 (s, 1H), 5.87 (d, J=8.8 Hz, 1H), 5.83-5.68 (m, J=5.6 Hz, 1H), 5.15-5.01 (m, 2H), 4.67-4.58 (m, 1H), 4.45-4.35 (m, 1H), 4.04-3.93 (m, 2H), 3.91 (s, 3H), 3.73 (td, J=10.0, 3.8 Hz, 1H), 3.25-3.14 (m, J=8.5, 7.0 Hz, 2H), 2.92 (dd, J=16.8, 10.3 Hz, 1H), 2.38 (d, J=16.8 Hz, 1H), 1.95-1.81 (m, 4H), 1.77 (s, 3H), 1.64-1.49 (m, 2H), 0.88 (s, 9H), 0.25 (s, 3H), 0.23 (s, 3H).

    (iii) (11S,11aS)-4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl 11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (70)

    [1290] ##STR00138## ##STR00139##

    (a) Allyl 3-(2-(2-(4-((((2-((S)-2-(((tert-butydimethysilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl) carbamoyl)oxy)methyl)phenyl) hydrazinyl) propanamido)-4-methyl-2-oxopentanoate (56)

    [1291] Triethylamine (2.23 mL, 18.04 mmol, 2.2 eq) was added to a stirred solution of the amine 49 (4 g, 8.20 mmol) and triphosgene (778 mg, 2.95 mmol, 0.36 eq) in dry tetrahydrofuran (40 mL) at 5° C. (ice bath). The progress of the isocyanate reaction was monitored by periodically removing aliquots from the reaction mixture and quenching with methanol and performing LC/MS analysis. Once the isocyanate formation was complete a solution of the alloc-Val-Ala-PABOH (4.12 g, 12.30 mmol, 1.5 eq) and triethylamine (1.52 mL, 12.30 mmol, 1.5 eq) in dry tetrahydrofuran (40 mL) was rapidly added by injection to the freshly prepared isocyanate. The reaction mixture was allowed to stir at 40° C. for 4 hours. Excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; gradient, 1% methanol to 5% methanol in dichloromethane). (Alternative chromatography conditions using EtOAc and Hexane have also been successful). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product 56 (3.9 g, 50%). LC/MS, 4.23 min (ES+) m/z (relative intensity) 952.36 ([M+H].sup.+., 100); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.62 (br s, 1H), 8.46 (s, 1H), 7.77 (br s, 1H), 7.53 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.5 Hz, 2H), 6.76 (s, 1H), 6.57 (d, J=7.6 Hz, 1H), 6.17 (s, 1H), 6.03-5.83 (m, 1H), 5.26 (dd, J=33.8, 13.5 Hz, 3H), 5.10 (s, 2H), 4.70-4.60 (m, 2H), 4.58 (dd, J=5.7, 1.3 Hz, 2H), 4.06-3.99 (m, 1H), 3.92 (s, 1H), 3.82-3.71 (m, 1H), 3.75 (s, 3H), 2.79-2.64 (m, 1H), 2.54 (d, J=12.9 Hz, 1H), 2.16 (dq, J=13.5, 6.7 Hz, 1H), 1.67 (s, 3H), 1.46 (d, J=7.0 Hz, 3H), 1.35-1.24 (m, 3H), 1.12 (s, 9H), 1.10 (s, 9H), 0.97 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H), 0.87 (s, 9H), 0.07-−0.02 (m, 6H).

    (b) Allyl 3-(2-(2-(4-((((2-((S)-2-(hydroxymethyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl) carbamoyl)oxy)methyl)phenyl) hydrazinyl) propanamido)-4-methyl-2-oxopentanoate (57)

    [1292] The TBS ether 56 (1.32 g, 1.38 mmol) was dissolved in a 7:1:1:2 mixture of acetic acid/methanol/tetrahydrofuran/water (14:2:2:4 mL) and allowed to stir at room temperature. After 3 hours no more starting material was observed by LC/MS. The reaction mixture was diluted with ethyl acetate (25 mL) and washed sequentially with water, saturated aqueous sodium bicarbonate and brine. The organic phase was dried over magnesium sulphate filtered and excess ethyl acetate removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel, 2% methanol in dichloromethane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to afford the desired product 57 (920 mg, 80%). LC/MS, 3.60 min (ES+) m/z (relative intensity) 838.18 ([M+H].sup.+., 100). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.55 (s, 1H), 8.35 (s, 1H), 7.68 (s, 1H), 7.52 (d, J=8.1 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H), 6.77 (s, 1H), 6.71 (d, J=7.5 Hz, 1H), 6.13 (s, 1H), 5.97-5.82 (m, J=5.7 Hz, 1H), 5.41-5.15 (m, 3H), 5.10 (d, J=3.5 Hz, 2H), 4.76-4.42 (m, 5H), 4.03 (t, J=6.6 Hz, 1H), 3.77 (s, 5H), 2.84 (dd, J=16.7, 10.4 Hz, 1H), 2.26-2.08 (m, 2H), 1.68 (s, 3H), 1.44 (d, J=7.0 Hz, 3H), 1.30 (dt, J=14.7, 7.4 Hz, 3H), 1.12 (s, 9H), 1.10 (s, 9H), 0.96 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8 Hz, 3H).

    (c) (11S,11aS)-4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl 11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (58)

    [1293] Dimethyl sulphoxide (0.2 mL, 2.75 mmol, 2.5 eq) was added dropwise to a solution of oxalyl chloride (0.11 mL, 1.32 mmol, 1.2 eq) in dry dichloromethane (7 mL) at −78° C. (dry ice/acetone bath) under an atmosphere of argon. After 10 minutes a solution of 57 (920 mg, 1.10 mmol) in dry dichloromethane (5 mL) was added slowly with the temperature still at −78° C. After 15 min triethylamine (0.77 mL, dried over 4 Å molecular sieves, 5.50 mmol, 5 eq) was added dropwise and the dry ice/acetone bath was removed. The reaction mixture was allowed to reach room temperature and was extracted with cold hydrochloric acid (0.1 M), saturated aqueous sodium bicarbonate and brine. The organic phase was dried over magnesium sulphate, filtered and excess dichloromethane was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to column flash chromatography (silica gel; gradient 2% methanol to 5% methanol in dichloromethane). Pure fractions were collected and combined and removal of excess eluent by rotary evaporation under reduced pressure afforded the product 58 (550 mg, 60%). LC/MS, 3.43 min (ES+) m/z (relative intensity) 836.01 ([M].sup.+., 100). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.39 (s, 1H), 7.52-7.40 (m, 2H), 7.21-7.08 (m, J=11.5 Hz, 2H), 6.67 (s, 1H), 6.60-6.47 (m, J=7.4 Hz, 1H), 5.97-5.83 (m, 1H), 5.79-5.66 (m, 1H), 5.38-4.90 (m, 6H), 4.68-4.52 (m, J=18.4, 5.5 Hz, 4H), 4.04-3.94 (m, J=6.5 Hz, 1H), 3.87-3.76 (m, 5H), 3.00-2.88 (m, 1H), 2.66-2.49 (m, 2H), 2.21-2.08 (m, 2H), 1.76 (s, 3H), 1.45 (d, J=7.0 Hz, 3H), 1.09-0.98 (m, J=8.9 Hz, 18H), 0.96 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.9 Hz, 3H).

    (d) (11S,11aS)-4-(2-(1-((1-(Allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl) hydrazinyl)benzyl 11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (59)

    [1294] Tert-butyldimethylsilyltriflate (0.38 mL, 1.62 mmol, 3 eq) was added to a solution of compound 58 (450 mg, 0.54 mmol) and 2,6-lutidine (0.25 mL, 2.16 mmol, 4 eq) in dry dichloromethane (5 mL) at 0° C. under argon. After 10 min, the cold bath was removed and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was extracted with water, saturated aqueous sodium bicarbonate and brine. The organic phase was dried over magnesium sulphate, filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to column flash chromatography (silica gel; 50/50 v/v hexane/ethyl acetate). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product 59 (334 mg, 65%). LC/MS, 4.18 min (ES+) m/z (relative intensity) 950.50 ([M].sup.+., 100). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.53 (s, 1H), 8.02 (s, 1H), 7.44 (d, J=7.6 Hz, 2H), 7.21 (s, 1H), 7.08 (d, J=8.2 Hz, 2H), 6.72-6.61 (m, J=8.9 Hz, 2H), 6.16 (s, 1H), 5.97-5.79 (m, J=24.4, 7.5 Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5 Hz, 1H), 4.69-4.60 (m, 1H), 4.57 (s, 1H), 4.03 (t, J=6.7 Hz, 1H), 3.87 (s, 3H), 3.74 (td, J=9.6, 3.6 Hz, 1H), 2.43-2.09 (m, J=34.8, 19.4, 11.7 Hz, 3H), 1.76 (s, 3H), 1.43 (d, J=6.9 Hz, 3H), 1.30-1.21 (m, 3H), 0.97 (d, J=6.7 Hz, 3H), 0.92 (t, J=8.4 Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H), 0.12 (s, 3H).

    (e) (11S,11aS)-4-(2-(1-((1-(Allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl 11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (60)

    [1295] Lithium acetate (50 mg, 0.49 mmol) was added to a solution of compound 59 (470 mg, 0.49 mmol) in wet dimethylformamide (4 mL, 50:1 DMF/water). After 4 hours, the reaction was complete and the reaction mixture was diluted with ethyl acetate and washed with citric acid (pH˜3), water and brine. The organic layer was dried over magnesium sulphate filtered and excess ethyl acetate was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to column flash chromatography (silica gel; gradient, 50/50 to 25/75 v/v hexane/ethyl acetate). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product 60 (400 mg, quantitative). LC/MS, 3.32 min (ES+) m/z (relative intensity) 794.18 ([M+H].sup.+., 100). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.53 (s, 1H), 8.02 (s, 1H), 7.44 (d, J=7.6 Hz, 2H), 7.21 (s, 1H), 7.08 (d, J=8.2 Hz, 2H), 6.72-6.61 (m, J=8.9 Hz, 2H), 6.16 (s, 1H), 5.97-5.79 (m, J=24.4, 7.5 Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5 Hz, 1H), 4.69-4.60 (m, 1H), 4.57 (s, 1H), 4.03 (t, J=6.7 Hz, 1H), 3.87 (s, 3H), 3.74 (td, J=9.6, 3.6 Hz, 1H), 2.43-2.09 (m, J=34.8, 19.4, 11.7 Hz, 3H), 1.76 (s, 3H), 1.43 (d, J=6.9 Hz, 3H), 1.30-1.21 (m, 3H), 0.97 (d, J=6.7 Hz, 3H), 0.92 (t, J=8.4 Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H), 0.12 (s, 3H).

    (iv) (11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (64)

    [1296] ##STR00140##

    (a) (11S)-allyl 8-((5-(((11S)-10-(((4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl) hydrazinyl)benzyl)oxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (61)

    [1297] Potassium carbonate (70 mg, 0.504 mmol, 1 eq) was added to a solution of 55 (370 mg, 0.552 mmol, 1.2 eq) and phenol 60 (400 mg, 0.504 mmol) in dry acetone (25 mL). The reaction was stirred 8 hours at 70° C. The LC/MS showed that all the starting material was not consumed, so the reaction was allowed to stir overnight at room temperature and stirred for an additional 2 hours the next day. Acetone was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 80% ethyl acetate in hexane to 100% ethyl acetate). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product 61 (385 mg, 57%). LC/MS, 4.07 min (ES+) m/z (relative intensity) 1336.55 ([M+H].sup.+., 50).

    (b) (11S)-allyl 8-((5-(((11S)-10-(((4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl) hydrazinyl)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (62)

    [1298] Tetra-n-butylammonium fluoride (1M, 0.34 mL, 0.34 mmol, 2 eq) was added to a solution of 61 (230 mg, 0.172 mmol) in dry tetrahydrofuran (3 mL). The starting material was totally consumed after 10 minutes. The reaction mixture was diluted with ethyl acetate (30 mL) and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess ethyl acetate removed by rotary evaporation under reduced pressure. The resulting residue 62 was used as a crude mixture for the next reaction. LC/MS, 2.87 min (ES+) m/z (relative intensity) 1108.11 ([M+H].sup.+., 100).

    (c) (11S)-4-(2-(1-((1-amino-3-methyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl 11-hydroxy-7-methoxy-8-((5-((7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (63)

    [1299] Tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol, 0.06 eq) was added to a solution of crude 62 (0.172 mmol) and pyrrolidine (36 μL, 0.43 mmol, 2.5 eq) in dry dichloromethane (10 mL). The reaction mixture was stirred 20 minutes and diluted with dichloromethane and washed sequentially with saturated aqueous ammonium chloride and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue 63 was used as a crude mixture for the next reaction. LC/MS, 2.38 min (ES+) m/z (relative intensity) 922.16 ([M+H].sup.+., 40).

    (d) (11S,11aS)-4-((2S,5S)-37-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4, 7,35-trioxo-10, 13,16, 19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (64)

    [1300] 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCl, 33 mg, 0.172 mmol) was added to a solution of crude 63 (0.172 mmol) and Mal-(PEG).sub.8-acid (100 mg, 0.172 mmol) in dry dichloromethane (10 mL). The reaction was stirred for 2 hours and the presence of starting material was no longer observed by LC/MS. The reaction was diluted with dichloromethane and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 100% chloroform to 10% methanol in chloroform). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give 64 (E) (60 mg, 25% over 3 steps).

    Example 8

    [1301] ##STR00141##

    [1302] Compound 65 is compound 79 of WO 2011/130598

    (11S)-4-(1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6, 9, 12, 15-tetraoxa-3, 19,22-triazatetracosanamido)benzyl 11-hydroxy-7-methoxy-8-(3-((7-methoxy-5-oxo-2-((E)-prop-1-en-1-yl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2-((E)-prop-1-en-1-yl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (66)

    [1303] N,N′-diisopropylcarbodiimide (DIC, 4.71 μL, 0.0304 mmol) was added to a solution of amine 65 (0.0276 mmol) and Iodo-(PEG).sub.4-acid (13.1 mg, 0.0304 mmol) in dry dichloromethane (0.8 mL). The reaction was stirred for 3 hours and the presence of starting material was no longer observed by LC/MS. The reaction mixture was directly loaded onto a thin-layer chromatography (TLC) plate and purified by prep-TLC (10% methanol in chloroform). Pure bands were scraped off the TLC plate, taken up in 10% methanol in chloroform, filtered and excess eluent removed by rotary evaporation under reduced pressure to give 66 (D) (20.9 mg, 56%). LC/MS, method 2, 3.08 min (ES+) m/z (relative intensity) 1361.16 ([M+H].sup.+, 100).

    General Experimental Methods for Example 9

    [1304] LCMS data were obtained using an Agilent 1200 series LC/MS with an Agilent 6110 quadrupole MS, with Electrospray ionisation. Mobile phase A—0.1% Acetic acid in water. Mobile Phase B—0.1% in acetonitrile. Flow rate of 1.00 ml/min. Gradient from 5% B rising up to 95% B over 3 minutes, remaining at 95% B for 1 minute and then back down to 5% B over 6 seconds. The total run time is 5 minutes. Column: Phenomenex Gemini-NX 3 μm C18, 30×2.00 mm. Chromatograms based on UV detection at 254 nm. Mass Spectra were achieved using the MS in positive mode. Proton NMR chemical shift values were measured on the delta scale at 400 MHz using a Bruker AV400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Coupling constants are reported in Hz. Unless otherwise stated, column chromatography (by the flash procedure) were performed on Merck Kieselgel silica (Art. 9385). Mass spectroscopy (MS) data were collected using a Waters Micromass LCT instrument coupled to a Waters 2795 HPLC separations module. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F.sub.254). All other chemicals and solvents were purchased from Sigma-Aldrich or Fisher Scientific and were used as supplied without further purification.

    [1305] 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. .sup.1H and .sup.13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F.sub.254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the 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 a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C.

    [1306] General LC/MS conditions: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). 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, and then returned to 5% B in 0.3 minutes. Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm

    Example 9

    (i) Key Intermediates

    [1307] ##STR00142##

    (a-i) (S)-2-(allyloxycarbonylamino)-3-methylbutanoic acid (I2)

    [1308] Allyl chloroformate (36.2 ml, 340.59 mmol, 1.2 eq) was added dropwise to a stirred solution of L-valine (I1)(33.25 g, 283.82 mmol, 1.0 eq) and potassium carbonate (59.27 g, 425.74 mmol, 1.5 eq) in water (650 mL) and THF (650 mL). The reaction mixture was stirred at room temperature for 18 hours, then the solvent was concentrated under reduced pressure and the remaining solution extracted with diethyl ether (3×100 mL). The aqueous portion was acidified to pH 2 with conc. HCl and extracted with DCM (3×100 mL). The combined organics were washed with brine, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford the product as a colourless oil (57.1 g, assumed 100% yield). LC/MS (1.966 min (ES.sup.+)), m/z: 202.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.57 (br s, 1H), 7.43 (d, 1H, J=8.6 Hz), 5.96-5.86 (m, 1H), 5.30 (ddd, 1H, J=17.2, 3.4, 1.7 Hz), 5.18 (ddd, 1H, J=10.4, 2.9, 1.6 Hz), 4.48 (dt, 2H, J=5.3, 1.5 Hz), 3.85 (dd, 1H, J=8.6, 6.0 Hz), 2.03 (oct, 1H, J=6.6 Hz), 0.89 (d, 3H, J=6.4 Hz), 0.87 (d, 3H, J=6.5 Hz).

    (a-ii) (S)-2,5-dioxopyrrolidin-1-yl 2-(allyloxycarbonylamino)-3-methylbutanoate (I3)

    [1309] To a stirred solution of the protected acid 12 (60.6 g, 301.16 mmol, 1.0 eq) and N-hydroxysuccinimide (34.66 g, 301.16 mmol, 1.0 eq) in dry THF (800 mL) was added dicyclohexylcarbodiimide (62.14 g, 301.16 mmol, 1 eq). The reaction was stirred for 18 hours at room temperature. The reaction mixture was then filtered, the solid washed with THF and the combined filtrate was concentrated under reduced pressure. The residue was re-dissolved in DCM and left to stand at 0° C. for 30 minutes. The suspension was filtered and washed with cold DCM. Concentration of the filtrate under reduced pressure afforded the product as a viscous colourless oil (84.7 g, assumed 100% yield) which was used in the next step without further purification. LC/MS (2.194 min (ES.sup.+)), m/z: 321.0 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.0 (d, 1H, J=8.3 Hz), 5.97-5.87 (m, 1H), 5.30 (ddd, 1H, J=17.2, 3.0, 1.7 Hz), 5.19 (ddd, 1H, J=10.4, 2.7, 1.4 Hz), 4.52 (dt, 2H, J=5.3, 1.4 Hz), 4.32 (dd, 1H, J=8.3, 6.6 Hz), 2.81 (m, 4H), 2.18 (oct, 1H, J=6.7 Hz), 1.00 (d, 6H, J=6.8 Hz),

    (a-iii) (S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanoic acid (14)

    [1310] A solution of succinimide ester 13(12.99 g, 43.55 mmol, 1.0 eq) in THF (50 mL) was added to a solution of L-alanine (4.07 g, 45.73 mmol, 1.05 eq) and NaHCO.sub.3 (4.02 g, 47.90 mmol, 1.1 eq) in THF (100 mL) and H.sub.2O (100 mL). The mixture was stirred at room temperature for 72 hours when the THF was removed under reduced pressure. The pH was adjusted to 3-4 with citric acid to precipitate a white gum. After extraction with ethyl acetate (6×150 mL), the combined organics were washed with H.sub.2O (200 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. Trituration with diethyl ether afforded the product as a white powder which was collected by filtration and washed with diethyl ether (5.78 g, 49%). LC/MS (1.925 min (ES.sup.+)), m/z: 273.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.47 (br s, 1H), 8.17 (d, 1H, J=6.8 Hz), 7.16 (d, 1H, J=9.0 Hz), 5.95-5.85 (m, 1H), 5.29 (dd, 1H, J=17.2, 1.7 Hz), 5.17 (dd, 1H, J=10.4, 1.5 Hz), 4.46 (m, 2H), 4.18 (quin, 1H, J=7.2 Hz), 3.87 (dd, 1H, J=9.0, 7.1 Hz), 1.95 (oct, 1H, J=6.8 Hz), 1.26 (d, 3H, J=7.3 Hz), 0.88 (d, 3H, J=6.8 Hz), 0.83 (d, 3H, J=6.8 Hz).

    (a-iv) Allyl (S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate (15)

    [1311] EEDQ (5.51 g, 22.29 mmol, 1.05 eq) was added to a solution of p-aminobenzyl alcohol (2.74 g, 22.29 mmol, 1.05 eq) and acid 14 (5.78 g, 21.23 mmol, 1 eq) in dry THF (100 mL). and stirred at room temperature for 72 hours. The reaction mixture was then concentrated under reduced pressure and the resulting brown solid was triturated with diethyl ether and filtered with subsequent washing with an excess of diethyl ether to afford the product as an off-white solid (7.1 g, 88%). LC/MS (1.980 min (ES.sup.+)), m/z: 378.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.89 (br s, 1H), 8.13 (d, 1H, J=7.0 Hz), 7.52 (d, 2H, J=8.5 Hz), 7.26 (m, 1H), 7.23 (d, 2H, J=8.5 Hz), 5.91 (m, 1H), 5.30 (m, 1H), 5.17 (m, 1H), 4.46 (m, 2H), 5.09 (t, 1H, J=5.6 Hz), 4.48 (m, 2H), 4.42 (m, 3H), 3.89 (dd, 1H, J=8.6, 6.8 Hz), 1.97 (m, 1H), 1.30 (d, 3H, J=7.1 Hz), 0.88 (d, 3H, J=6.8 Hz), 0.83 (d, 3H, J=6.7 Hz).

    ##STR00143##

    1-iodo-2-oxo-6, 9, 12, 15-tetraoxa-3-azaoctadecan-18-oic acid (I7)

    [1312] A solution of iodoacetic anhydride (0.250 g, 0.706 mmol, 1.1 eq) in dry DCM (1 mL) was added to amino-PEG.sub.(4)-acid I6 (0.170 g, 0.642 mmol, 1.0 eq) in DCM (1 mL). The mixture was stirred in the dark at room temperature overnight. The reaction mixture was washed with 0.1 M HCl, water, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 3% MeOH and 0.1% formic acid in chloroform to 10% MeOH and 0.1% formic acid in chloroform) to afford the product as an orange oil (0.118 g, 42%). LC/MS (1.623 min (ES.sup.+)), m/z: 433.98 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.069 (s, 1H), 7.22 (br s, 1H), 3.79 (t, 2H, J=5.8 Hz), 3.74 (s, 2H), 3.72-3.58 (m, 14H), 3.50-3.46 (m, 2H), 2.62 (t, 2H, J=5.8 Hz).

    (ii) (11S,11aS)-allyl 11-(tert-butyldimethylsilyloxy)-8-(3-iodopropoxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (74)

    [1313] ##STR00144## ##STR00145##

    (a) (S)-5-((tert-butyldimethylsilyloxy)methyl)-1-(5-methoxy-2-nitro-4-(triisopropylsilyloxy)benzoyl)-4, 5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate (47)

    [1314] Triflic anhydride (28.4 g, 100.0 mmol, 3.0 eq) was added dropwise, over 25 mins, to a vigorously stirred solution of the ketone 46 (19.5 g, 30.0 mmol, 1.0 eq) in DCM (550 mL) containing 2,6-lutidine (14.4 g, 130.0 mmol, 4.0 eq) at −50° C. The reaction mixture was stirred for 1.5 hours when LC/MS indicated complete reaction. The organic phase was washed successively with water (100 mL), saturated sodium bicarbonate (150 mL), brine (50 mL), and the organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 90/10 v/v n-hexane/EtOAc) to afford the product as a pale yellow oil (19.5 g, 82%). LC/MS (4.391 min (ES.sup.+)), m/z: 713.25 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.68 (s, 1H), 6.72 (s, 1H), 6.02 (t, 1H, J=1.9 Hz), 4.75 (m, 1H), 4.05 (m, 2H), 3.87 (s, 3H), 3.15 (ddd, 1H, J=16.2, 10.3, 2.3 Hz), 2.96 (ddd, 1H, J=16.2, 4.0, 1.6 Hz), 1.28-1.21 (m, 3H), 1.07 (d, 18H, J=7.2 Hz), 0.88 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H).

    (b) (S,E)-(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2, 3-dihydro-1H-pyrrol-1-yl) (5-methoxy-2-nitro-4-(triisopropylsilyloxy)phenyl)methanone (67)

    [1315] Tetrakis(triphenylphosphine)palladium(0) (0.41 g, 0.35 mmol, 0.03 eq) was added to a mixture of the triflate 47 (8.4 g, 11.8 mmol, 1.0 eq), E-1-propene-1-ylboronic acid (1.42 g, 16.5 mmol, 1.4 eq) and potassium phosphate (5.0 g, 23.6 mmol, 2.0 eq) in dry dioxane (60 mL) under a nitrogen atmosphere. The mixture was stirred at 25° C. for 120 mins when LC/MS indicated complete reaction. Ethyl acetate (120 mL) and water (120 mL) were added, the organic phase was removed, washed with brine (20 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 95/5 v/v n-hexane/EtOAc to 90/10 v/v n-hexane/EtOAc) to afford the product as a yellow foam (4.96 g, 70%). LC/MS (4.477 min (ES.sup.+)), m/z: 605.0 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.67 (s, 1H), 6.74 (s, 1H), 5.93 (d, 1H, J=15.4 Hz), 5.67 (s, 1H), 4.65 (m, 1H), 4.04 (m, 2H), 3.86 (s, 3H), 2.85 (m, 1H), 2.71 (m, 1H), 1.72 (dd, 3H, J=6.8, 1.0 Hz), 1.30-1.22 (m, 3H), 1.07 (d, 18H, J=7.2 Hz), 0.87 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H). (c) (S,E)-(2-amino-5-methoxy-4-(triisopropylsilyloxy)phenyl) (2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2, 3-dihydro-1H-pyrrol-1-yl) methanone (68) Zinc dust (22.0 g, 0.33 mol, 37 eq) was added, in portions over 20 mins, to a solution of the propenyl intermediate 67 (5.5 g, 9.1 mmol, 1.0 eq) in 5% v/v formic acid/ethanol (55 mL), using an ice bath to maintain the temperature between 25-30° C. After 30 mins, the reaction mixture was filtered through a short bed of Celite®. The Celite® was washed with ethyl acetate (65 mL) and the combined organics were washed successively with water (35 mL), saturated sodium bicarbonate (35 mL) and brine (10 mL). The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 90/10 v/v n-hexane/EtOAc) to afford the product as a pale yellow oil (3.6 g, 69.0%). LC/MS (4.439 min (ES.sup.+)), m/z: 575.2 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) b 6.75 (m, 1H), 6.40 (br s, 1H), 6.28 (m, 1H), 6.11 (d, 1H, J=15.4 Hz), 5.53 (m, 1H), 4.67 (m, 1H), 4.36 (m, 2H), 3.93 (br s, 1H), 3.84 (br s, 1H), 3.73 (s, 3H), 2.86 (dd, 1H, J=15.7, 10.4 Hz), 2.73 (dd, 1H, J=15.9, 4.5 Hz), 1.80 (dd, 3H, J=6.8, 1.3 Hz), 1.35-1.23 (m, 3H), 1.12 (d, 18H, J=7.3 Hz), 0.89 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H).

    (d) (S,E)-allyl 2-(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-(triisopropylsilyloxy)phenylcarbamate (69)

    [1316] Allyl chloroformate (0.83 g, 6.88 mmol, 1.1 eq) was added to a solution of the amine 68 (3.6 g, 6.26 mmol, 1.0 eq) in dry DCM (80 mL) containing dry pyridine (1.09 g, 13.77 mmol, 2.2 eq) at −78° C. The dry ice was removed and the reaction mixture allowed to warm to room temperature. After stirring for a further 15 minutes, LC/MS indicated complete reaction. The organic phase was washed successively with 0.01N HCl (50 mL), saturated sodium bicarbonate (50 mL), brine (10 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure to leave a pale yellow oil which was used in the next step without further purification (4.12 g, assumed 100% yield). LC/MS (4.862 min (ES.sup.+)), m/z: 659.2 [M+H].sup.+.

    (e) (S,E)-allyl 2-(2-(hydroxymethyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-(triisopropylsilyloxy)phenylcarbamate (70)

    [1317] The crude intermediate 69 (assumed 100% yield, 4.12 g, 6.25 mmol, 1.0 eq) was dissolved in a mixture of acetic acid (70 mL), methanol (10 mL), THF (10 mL) and water (20 mL) and allowed to stir at room temperature. After 6 hours the reaction mixture was diluted with ethyl acetate (500 mL) and washed successively with water (2×500 mL), saturated sodium bicarbonate (300 mL) and brine (50 mL). The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 1/99 v/v methanol/DCM to 5/95 v/v methanol/DCM) to afford the product as a yellow oil and a further 1 g of unreacted starting material was recovered. This material was subjected to the same reaction conditions as above, but was left stirring for 16 h. After work up and purification, additional product was isolated (2.7 g, 79%, 2 steps) LC/MS (3.742 min (ES+)), m/z: 545.2 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.38 (m, 1H), 7.72 (m, 1H), 6.81 (s, 1H), 6.37 (m, 1H), 6.10 (d, 1H, J=15.8 Hz), 5.97 (m, 1H), 5.53 (m, 1H), 5.36 (ddd, 1H, J=17.2, 3.1, 1.5 Hz), 5.25 (ddd, 1H, J=10.4, 2.5, 1.3 Hz), 4.78 (m, 1H), 4.65 (dt, 2H, J=5.7, 1.3 Hz), 3.84 (m, 3H), 3.79 (s, 3H), 3.04 (dd, 1H, J=16.7, 10.5 Hz), 2.40 (dd, 1H, J=16.0, 4.5 Hz), 1.82 (dd, 3H, J=6.8, 1.0 Hz), 1.36-1.26 (m, 3H), 1.14 (d, 18H, J=7.3 Hz).

    (f) (11S,11aS)-allyl 11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (71)

    [1318] Dry dimethyl sulfoxide (1.16 g, 14.87 mmol, 3.0 eq) was added dropwise to a solution of oxalyl chloride (0.94 g, 7.43 mmol, 1.5 eq) in DCM (25 mL) at −78° C. under an atmosphere of nitrogen. Maintaining the temperature at −78° C., after 10 mins a solution of the primary alcohol 70 (2.7 g, 4.96 mmol, 1.0 eq) in DCM (20 mL) was added dropwise. After a further 15 mins, dry triethylamine (2.5 g, 24.78 mmol, 5.0 eq) was added, and the reaction mixture allowed to warm to room temperature. The reaction mixture was washed successively with cold 0.1N HCl (50 mL), saturated sodium hydrogen carbonate (50 mL) and brine (10 mL) and the organic layer was dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford the product as a yellow oil which was used in the next step without further purification (2.68 g, assumed 100% yield). LC/MS (3.548 min (ES+)), m/z: 543.2 [M+H].sup.+.

    (g) (11S,11aS)-ally 11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (72)

    [1319] Tert-butyldimethylsilyltrifluoromethane sulfonate (3.93 g, 14.87 mmol, 3.0 eq) was added to a solution of the carbinolamine 71 (assumed 100% yield, 2.68 g, 4.96 mmol, 1.0 eq) and 2,6-lutidine (2.12 g, 19.83 mmol, 4.0 eq) in dry DCM (40 mL) at 0° C. under an atmosphere of nitrogen. After 10 minutes, the reaction mixture was allowed to warm to room temperature and stirred for a further 60 minutes. The organic phase was washed successively with water (10 mL), saturated sodium bicarbonate (10 mL) and brine (5 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, chloroform to 2/98 v/v Methanol/chloroform) to afford the product as a yellow oil (2.0 g, 63%, 2 steps). LC/MS (4.748 min (ES.sup.+)), m/z: 657.2 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.19 (s, 1H), 6.86 (m, 1H), 6.66 (s, 1H), 6.22 (d, 1H, J=15.4 Hz), 5.81 (d, 1H, J=8.8 Hz), 5.78 (m, 1H), 5.48 (m, 1H), 5.11 (d, 1H, J=5.0 Hz), 5.08 (m, 1H), 4.58 (dd, 1H, J=13.4, 5.4 Hz), 4.35 (dd, 1H, J=13.2, 5.7 Hz), 3.83 (s, 3H), 3.76 (s, 1H), 3.00 (dd, 1H, J=15.6, 11.0 Hz), 2.53 (m, 1H), 1.81 (dd, 3H, J=6.8, 0.9 Hz), 1.30-1.18 (m, 3H), 1.08 (d, 9H, J=2.3 Hz), 1.06 (d, 9H, J=2.3 Hz), 0.86 (s, 9H), 0.25 (s, 3H), 0.18 (s, 3H).

    (h) (11S,11aS)-allyl 11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (73)

    [1320] Lithium acetate dihydrate (0.31 g, 3.04 mmol, 1.0 eq) was added to a solution of the diazepine 72 (2.0 g, 3.04 mmol, 1.0 eq) in wet DMF (20 mL) at 25° C. and stirred for 4 hours. The reaction mixture was diluted with ethyl acetate (200 mL) and washed successively with 0.1M citric acid (50 mL, pH 3), water (50 mL) and brine (10 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 50/50 v/v n-hexane/EtOAc to 25/75 v/v n-hexane/EtOAc) to afford the product as a pale yellow solid (0.68 g, 45%). LC/MS (3.352 min (ES.sup.+)), m/z: 501.1 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.02 (s, 1H), 6.66 (m, 1H), 6.53 (s, 1H), 6.03 (d, 1H, J=15.5 Hz), 5.80 (s, 1H), 5.63 (d, 1H, J=8.9 Hz), 5.55 (m, 1H), 5.29 (m, 1H), 4.87 (m, 2H), 4.39 (dd, 1H, J=13.5, 4.2 Hz), 4.20 (dd, 1H, J=13.2, 5.7 Hz), 3.73 (s, 3H), 3.59 (m, 1H), 2.81 (dd, 1H, J=16.1, 10.5 Hz), 2.35 (d, 1H, J=15.7 Hz), 1.61 (d, 3H, J=6.4 Hz), 0.67 (s, 9H), 0.05 (s, 3H), 0.00 (s, 3H).

    (i) (11S,11aS)-allyl 11-(tert-butyldimethylsilyloxy)-8-(3-iodopropoxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (74)

    [1321] Diiodopropane (0.295 g, 1.00 mmol, 5.0 eq) and potassium carbonate (0.028 g, 0.20 mmol, 1.0 eq) were added to a solution of the phenol 33 (0.100 g, 0.020 mmol, 1.0 eq) in dry acetone (5 mL). The reaction mixture was heated at 60° C. for 6 hours when LC/MS showed complete reaction. The reaction mixture was concentrated to dryness under reduced pressure and the residue was purified by flash chromatography (silica gel, 75/25 v/v n-hexane/EtOAc to 50/50 v/v n-hexane/EtOAc) to afford the product as a colourless oil (0.074 g, 56%). LC/MS (3.853 min (ES.sup.+)), m/z: 669.0 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.26 (s, 1H), 6.90 (s, 1H), 6.68 (s, 1H), 6.24 (d, 1H, J=15.3 Hz), 5.87 (d, 1H, J=8.9 Hz), 5.78 (m, 1H), 5.53 (m, 1H), 5.12 (m, 2H), 4.65 (m, 2H), 4.41 (m, 1H), 4.11 (m, 1H), 3.93 (s, 3H), 3.81 (m, 1H), 3.40 (t, 2H, J=6.7 Hz), 3.05 (dd, 1H, J=16.3, 10.1 Hz), 2.57 (m, 1H), 2.34 (m, 2H), 1.84 (d, 3H, J=6.6 Hz), 0.92 (s, 9H), 0.28 (s, 3H), 0.26 (s, 3H).

    (iii) (11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl 11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 79)

    [1322] ##STR00146## ##STR00147##

    (a) Allyl ((S)-1-(((S)-1-((4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-((E)-prop-1-en-1-yl)-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl) carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (75)

    [1323] Triethylamine (0.256 mL, 1.84 mmol, 2.2 eq) was added to a stirred solution of the amine 68 (0.480 g, 0.835 mmol, 1.0 eq) and triphosgene (0.089 g, 0.301 mmol, 0.36 eq) in dry THF (15 mL) at 5° C. (ice bath). The progress of the isocyanate reaction was monitored by periodically removing aliquots from the reaction mixture and quenching with methanol and performing LCMS analysis. Once the isocyanate reaction was complete a solution of Alloc-Val-Ala-PABOH 15 (0.473 g, 1.25 mmol, 1.5 eq) and triethylamine (0.174 mL, 1.25 mmol, 1.5 eq) in dry THF (10 mL) was rapidly added by injection to the freshly prepared isocyanate. The reaction was allowed to stir at 40° C. for 4 hours followed by stirring at room temperature overnight. The mixture was concentrated under reduced pressure, and purified by flash chromatography (silica gel, 20/80 v/v n-hexane/EtOAc to 50/50 v/v n-hexane/EtOAc, then 1/99 v/v DCM/MeOH to 5/95 v/v DCM/MeOH) to afford the product as a yellow solid (0.579 g, 71%). LC/MS (4.468 min (ES.sup.+)), m/z: 978.55 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.63 (br s, 1H), 8.42 (s, 1H), 7.78 (br s, 1H), 7.53 (d, 2H, J=8.1 Hz), 7.31 (d, 2H, J=8.6 Hz), 6.76 (s, 1H), 6.59 (d, 1H, J=7.6 Hz), 6.36 (br s, 1H), 6.04 (d, 1H, J=15.9 Hz), 5.90 (m, 1H), 5.55 (m, 1H), 5.33-5.21 (m, 3H), 5.10 (s, 2H), 4.66 (m, 2H), 4.57 (dd, 2H, J=5.6, 1.0 Hz), 3.98 (dd, 1H, J=7.3, 6.8 Hz), 3.90 (m, 1H), 3.81 (m, 1H), 3.78 (s, 3H), 2.82 (dd, 1H, J=15.4, 9.6 Hz), 2.72 (dd, 1H, J=15.9, 3.5 Hz), 2.17 (m, 1H), 1.78 (dd, 3H, J=6.5, 0.8 Hz), 1.46 (d, 3H, J=7.1 Hz), 1.29 (m, 3H), 1.11 (d, 18H, J=7.1 Hz), 0.97 (d, 3H, J=6.8 Hz), 0.92 (d, 3H, J=6.8 Hz), 0.83 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H).

    (b) Allyl ((S)-1-(((S)-1-((4-((((2-((S)-2-(hydroxymethyl)-4-((E)-prop-1-en-1-yl)-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl) carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (76)

    [1324] The silyl ether 75 (1.49 g, 1.52 mmol, 1.0 eq) was dissolved in a 7:1:1:2 mixture of acetic acid/methanol/tetrahydrofuran/water (14:2:2:4 mL) and allowed to stir at room temperature. After 2 hours the reaction was diluted with EtOAc (100 mL), washed sequentially with water, aq. sodium bicarbonate then brine. The organic phase was then dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 100/0 then 99/1 to 92/8 v/v DCM/MeOH) to afford the product as an orange solid (1.2 g, 92%). LC/MS (3.649 min (ES.sup.+)), m/z: 865.44 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.44 (s, 1H), 8.35 (s, 1H), 7.69 (br s, 1H), 7.53 (d, 2H, J=8.7 Hz), 7.32 (d, 2H, J=8.3 Hz), 6.78 (s, 1H), 6.56 (m, 2H), 6.32 (br s, 1H), 6.05 (d, 1H, J=14.9 Hz), 5.90 (m, 1H), 5.56 (m, 1H), 5.30 (m, 2H), 5.22 (m, 1H), 5.10 (d, 2H, J=3.1 Hz), 4.73 (m, 1H), 4.64 (m, 1H), 4.57 (d, 2H, J=5.8 Hz), 4.01 (m, 1H), 3.79 (m, 2H), 3.76 (s, 3H), 2.98 (dd, 1H, J=16.3, 10.2 Hz), 2.38 (dd, 1H, J=16.6, 4.1 Hz), 2.16 (m, 1H), 1.78 (dd, 3H, J=6.8, 0.9 Hz), 1.46 (d, 3H, J=7.1 Hz), 1.29 (m, 3H), 1.11 (d, 18H, J=7.4 Hz), 0.97 (d, 3H, J=6.7 Hz), 0.92 (d, 3H, J=6.8 Hz).

    (c) (11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido) propanamido)benzyl 11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (77)

    [1325] Dry dimethyl sulfoxide (0.180 g, 2.3 mmol, 3.0 eq) was added dropwise to a solution of oxalyl chloride (0.147 g, 1.1 mmol, 1.5 eq) in DCM (10 mL) at −78° C. under an atmosphere of nitrogen. Maintaining the temperature at −78° C., after 20 minutes, a solution of the primary alcohol 76 (0.666 g, 0.77 mmol, 1.0 eq) in DCM (10 mL) was added dropwise. After a further 15 minutes, dry triethylamine (0.390 g, 3.85 mmol, 5.0 eq) was added, and the reaction mixture allowed to warm to room temperature. The reaction mixture was washed successively with cold 0.1N HCl (10 mL), saturated sodium hydrogen carbonate (10 mL) and brine (5 mL). The organic layer was then dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was then purified by flash chromatography (silica gel, 50/50 v/v n-hexane/EtOAc to 25/75 v/v n-hexane/EtOAc) to afford the product as a white solid (0.356 g, 54%). LC/MS (3.487 min (ES.sup.+)), m/z: 862.2 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.34 (br s, 1H), 7.47 (d, 2H, J=7.6 Hz), 7.17 (s, 1H), 7.14 (d, 2H, J=7.5 Hz), 6.86 (br s, 1H), 6.65 (br s, 1H), 6.42 (d, 1H, J=7.6 Hz), 6.22 (d, 1H, J=14.4 Hz), 5.80 (m, 1H), 5.40 (m, 1H), 5.53 (m, 1H), 5.32 (m, 1H), 5.21 (d, 2H, J=9.6 Hz), 5.06 (d, 1H, J=12.3 Hz), 4.90 (m, 1H), 4.58 (m, 3H), 3.98 (m, 1H), 3.84 (m, 1H), 3.81 (s, 3H), 3.50 (m, 1H), 3.05 (dd, 1H, J=16.0, 10.3 Hz), 2.76 (m, 1H), 2.15 (m, 1H), 1.80 (dd, 3H, J=6.7, 0.8 Hz), 1.44 (d, 3H, J=7.1 Hz), 1.16 (m, 3H), 1.01 (d, 18H, J=6.6 Hz), 0.96 (d, 3H, J=6.8 Hz), 0.92 (d, 3H, J=6.8 Hz).

    (d) (11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl 11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (78)

    [1326] Tert-butyldimethylsilyltrifluoromethane sulfonate (0.46 g, 1.74 mmol, 3.0 eq) was added to a solution of secondary alcohol 77 (0.5 g, 0.58 mmol, 1.0 eq) and 2,6-lutidine (0.25 g, 2.32 mmol, 4.0 eq) in dry DCM (10 mL) at 0° C. under an atmosphere of nitrogen. After 10 minutes, the reaction mixture was allowed to warm to room temperature and stirred for a further 120 mins. The organic phase was then washed successively with water (10 mL), saturated sodium bicarbonate (10 mL) and brine (5 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 50/50 v/v n-hexane/EtOAc) to afford the product as a white solid (0.320 g, 57%). LC/MS (4.415 min (ES.sup.+)), m/z: 976.52 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.31 (br s, 1H), 7.48 (d, 2H, J=8.0 Hz), 7.21 (s, 1H), 7.14 (d, 2H, J=8.3 Hz), 6.89 (s, 1H), 6.65 (s, 1H), 6.38 (d, 1H, J=7.3 Hz), 6.25 (d, 1H, J=14.6 Hz), 5.93 (m, 1H), 5.85 (d, 1H, J=8.8 Hz), 5.50 (m, 1H), 5.34 (m, 1H), 5.24 (m, 2H), 5.15 (d, 1H, J=12.5 Hz), 4.86 (d, 1H, J=12.2 Hz), 4.62 (m, 3H), 4.01 (m, 1H), 3.86 (s, 3H), 3.78 (m, 1H), 3.04 (m, 1H), 2.56 (m, 1H), 2.20 (m, 1H), 1.84 (dd, 3H, J=6.6, 0.7 Hz), 1.48 (d, 3H, J=6.8 Hz), 1.20 (m, 3H), 1.05 (d, 9H, J=2.9 Hz), 1.03 (d, 9H, J=2.9 Hz), 0.99 (d, 3H, J=6.8 Hz), 0.95 (d, 3H, J=6.8 Hz), 0.88 (s, 9H), 0.27 (s, 3H), 0.14 (s, 3H).

    (e) (11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl 11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (79)

    [1327] Lithium acetate dihydrate (0.010 g, 0.10 mmol, 1.0 eq) was added to a solution of the silyl ether 78 (0.100 g, 0.10 mmol, 1.0 eq) in wet DMF (2 mL) at 25° C. for 3 hours. The reaction mixture was then diluted with ethyl acetate (20 mL) and washed successively with 0.1M citric acid (20 mL, pH 3), water (20 mL) and brine (5 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 5/95 v/v methanol/DCM) to afford the product as a pale yellow oil (0.070 g, 83%). LC/MS (3.362 min (ES.sup.+)), m/z: 820.2 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.39 (s, 1H), 7.48 (d, 2H, J=8.2 Hz), 7.25 (s, 1H), 7.12 (d, 2H, J=8.1 Hz), 6.88 (s, 1H), 6.68 (s, 1H), 6.47 (d, 1H, J=7.6 Hz), 6.24 (d, 1H, J=15.2 Hz), 6.03 (s, 1H), 5.92 (m, 1H), 5.84 (d, 1H, J=8.9 Hz), 5.50 (m, 1H), 5.34 (m, 1H), 5.26 (m, 2H), 5.18 (d, 1H, J=12.3 Hz), 4.80 (d, 1H, J=12.4 Hz), 4.66-4.60 (m, 3H), 4.02 (m, 1H), 3.95 (s, 3H), 3.81 (m, 1H), 3.03 (m, 1H), 2.57 (m, 1H), 2.19 (m, 1H), 1.84 (dd, 3H, J=6.8, 0.8 Hz), 1.48 (d, 3H, J=7.1 Hz), 1.00 (d, 3H, J=6.8 Hz), 0.95 (d, 3H, J=6.8 Hz), 0.87 (s, 9H), 0.26 (s, 3H), 0.12 (s, 3H).

    (iv) (11 S,11aS)-4-((20S,23S)-1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl 11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-Prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (66, D)

    [1328] ##STR00148## ##STR00149##

    (a) (11S,11aS)-allyl 8-(3-((11S,11aS)-10-((4-((R)-2-((R)-2-(allyloxycarbonylamino)-3-methylbutanamido) propanamido)benzyloxy)carbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (80)

    [1329] Potassium carbonate (0.030 g, 0.21 mmol, 1.0 eq) was added to a solution of the phenol 79 (0.175 g, 0.21 mmol, 1.0 eq) and the iodo linker 74 (0.214 g, 0.32 mmol, 1.5 eq) in acetone (10 mL). The reaction mixture was heated under a nitrogen atmosphere at 75° C. in a sealed flask for 17 hours. The reaction mixture was concentrated to dryness under reduced pressure and purified by flash chromatography (silica gel, 2/98 v/v methanol/DCM to 5/95 v/v methanol/DCM) to afford the product as a pale yellow solid (0.100 g, 35%). LC/MS (4.293 min (ES.sup.+)), m/z: 1359.13 [M].sup.+.

    (b) (11S,11aS)-allyl 8-(3-((11S,11aS)-10-((4-((R)-2-((R)-2-(allyloxycarbonylamino)-3-methylbutanamido) propanamido)benzyloxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (81)

    [1330] Tetra-n-butylammonium fluoride (1M, 0.22 mL, 0.22 mmol, 2.0 eq) was added to a solution of silyl ether 80 (0.150 g, 0.11 mmol, 1.0 eq) in dry THF (2 mL). The reaction mixture was stirred at room temperature for 20 minutes, after which LC/MS indicated complete reaction. The reaction mixture was diluted with ethyl acetate (10 mL) and washed sequentially with water (5 mL) and brine (5 mL). The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure to leave a yellow solid. Purification by flash chromatography (silica gel, 6/94 v/v methanol/DCM to 10/90 v/v methanol/DCM) afforded the product as a pale yellow solid (0.090 g, 73%). LC/MS (2.947 min (ES.sup.+)), m/z: 1154.0 [M+Na].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.39 (br s, 1H), 7.39 (d, 2H, J=7.6 Hz), 7.18 (d, 2H, J=10.6 Hz), 7.10 (m, 3H), 6.86 (d, 2H, J=10.0 Hz), 6.74 (s, 1H), 6.55 (s, 1H), 6.22 (dd, 2H, J=15.3, 6.6 Hz), 5.85 (m, 2H), 5.74 (m, 3H), 5.52 (m, 2H), 5.22 (m, 1H), 5.00 (m, 2H), 4.57 (m, 6H), 4.41 (m, 2H), 4.09 (m, 4H), 3.85 (m, 11H), 3.06 (m, 2H), 2.76 (m, 2H), 2.20 (m, 2H), 2.08 (m, 1H), 1.79 (d, 6H, J=6.4 Hz), 1.40 (d, 3H, J=6.1 Hz), 0.90 (m, 6H).

    (c) (11S,11aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl 11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (65)

    [1331] Tetrakis(triphenylphospene)palladium(0) (0.005 g, 0.005 mmol, 0.06 eq) was added to a solution of the bis-carbinolamine 81 (0.090 g, 0.08 mmol, 1.0 eq) and pyrrolidine (16 μL, 0.20 mmol, 2.5 eq) in dry DCM (5 mL). After 20 minutes, the reaction mixture was diluted with DCM (10 mL) and washed sequentially with saturated ammonium chloride (5 mL) and brine (5 mL), dried over MgSO.sub.4, filtered and the solvent was removed under reduced pressure to leave the crude product as a pale yellow solid which was used in the next step without further purification (0.075 g, assumed 100% yield). LC/MS (2.060 min (ES.sup.+)), m/z: 947.2 [M+H].sup.+.

    (d) (11S,11aS)-4-((20S,23S)-1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6,9, 12, 15-tetraoxa-3, 19, 22-triazatetracosanamido)benzyl 11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (66, D)

    [1332] EDCl (0.015 g, 0.08 mmol, 1.0 eq) was added to a solution of amine 65 (assumed 100% yield 0.075 g, 0.08 mmol, 1.0 eq) and iodoacetamide-PEG.sub.4-acid 17 (0.034 g, 0.08 mmol, 1.0 eq) in dry dichloromethane (5 mL) and the reaction was stirred in the dark. After 50 minutes, a further amount of iodoacetamide-PEG.sub.4-acid 17 (0.007 g, 0.016 mmol, 0.2 eq) was added along with a further amount of EDCl (0.003 g, 0.016 mmol, 0.2 eq). After a total of 2.5 hours, the reaction mixture was diluted with dichloromethane (15 mL) and washed sequentially with water (10 mL) and brine (10 mL). The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, Chloroform 100% to 90:10 v/v Chloroform:Methanol). Pure fractions were combined to afford the product (0.0254 g, 23%, 2 steps). The crude fractions were collected and purified by preparative TLC (silica gel, 90:10 v/v Chloroform:Methanol) to afford a second batch of product (0.0036 g, 3%, 2 steps). LC/MS (2.689 min (ES.sup.+)), m/z: 681.0 1/2 [M+2H].sup.+.

    Example 10

    [1333] ##STR00150##

    (a) (S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl trifluoromethanesulfonate (82)

    [1334] Pd(PPh.sub.3).sub.4 (20.6 mg, 0.018 mmol) was added to a stirred mixture of the bis-enol triflate 12 (500 mg, 0.44 mmol), N-methyl piperazine boronic ester (100 mg, 0.4 mmol), Na.sub.2CO.sub.3 (218 mg, 2.05 mmol), MeOH (2.5 mL), toluene (5 mL) and water (2.5 mL). The reaction mixture was allowed to stir at 30° C. under a nitrogen atmosphere for 24 hours after which time all the boronic ester has consumed. The reaction mixture was then evaporated to dryness before the residue was taken up in EtOAc (100 mL) and washed with H.sub.2O (2×50 mL), brine (50 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 80:20 v/v Hexane/EtOAc to 60:40 v/v Hexane/EtOAc) afforded product 82 as a yellowish foam (122.6 mg, 25%). LC/MS 3.15 min (ES+) m/z (relative intensity) 1144 ([M+H].sup.+., 20%).

    (b) (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate (83)

    [1335] PBD-triflate 82 (359 mg, 0.314 mmol), boronic pinacol ester 20 (250 mg, 0.408 mmol) and triethylamine (0.35 mL, 2.51 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (3 mL). The microwave vessel was purged and filled with argon three times before tetrakis(triphenylphosphine)palladium(0) (21.7 mg, 0.018 mmol) was added and the reaction mixture placed in the microwave at 80° C. for 10 minutes. Subsequently, CH.sub.2Cl.sub.2 (100 mL) was added and the organics were washed with water (2×50 mL) and brine (50 mL) before being dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel chromatography column (CHCl.sub.3/MeOH, 100% to 9:1) to afford pure 83 (200 mg, 43% yield). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478 ([M+H].sup.+., 100%).

    (c) (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-4-((S)-7-methoxy-8-(3-(((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)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 (84)

    [1336] A solution of Super-Hydride® (0.34 mL, 1M in THF) was added dropwise to a solution of SEM-dilactam 83 (200 mg, 0.135 mmol) in THF (5 mL) at −78° C. under an argon atmosphere. The addition was completed over 5 minutes in order to maintain the internal temperature of the reaction mixture constant. After 20 minutes, an aliquot was quenched with water for LC/MS analysis, which revealed that the reaction was complete. Water (20 mL) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with EtOAc (3×30 mL) and the combined organics were washed with brine (50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in MeOH (6 mL), CH.sub.2Cl.sub.2 (3 mL), water (1 mL) and enough silica gel to form a thick stirring suspension. After 5 days, the suspension was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (100 mL) until the elution of the product was complete. The organic layer was washed with brine (2×50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCl.sub.3 to 96% CHCl.sub.3/4% MeOH) afforded the product 84 as a yellow solid (100 mg, 63%). LC/MS 2.67 min (ES+) m/z (relative intensity) 1186 ([M+H].sup.+., 5%).

    (d) (S)-2-amino-N—((S)-1-((4-((R)-7-methoxy-8-(3-(((R)-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)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (85)

    [1337] Excess piperidine was added (0.1 mL, 1 mmol) to a solution of PBD 84 (36.4 mg, 0.03 mmol) in DMF (0.9 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL) and the organic phase was washed with H.sub.2O (3×50 mL) until complete piperidine removal. The organic phase was dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude product 85 which was used as such in the next step. LC/MS 2.20 min (ES+) m/z (relative intensity) 964 ([M+H].sup.+., 5%).

    (e) 1-(3-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido)-N-((2S)-1-(((2S)-1-((4-(7-methoxy-8-(3-((7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3, 6, 9,12,15, 18, 21, 24-octaoxaheptacosan-27-amide (86)

    [1338] EDCl hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG.sub.8-acid (25 mg, 0.042 mmol) in dry CH.sub.2Cl.sub.2 (4 mL) under argon atmosphere. PBD 85 (42 mg, crude) was added straight away and stirring was maintained until the reaction was complete (3 hours). The reaction was diluted with CH.sub.2Cl.sub.2 and the organic phase was washed with H.sub.2O and brine before being dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCl.sub.3 up to 9:1 CHCl.sub.3/MeOH) followed by reverse phase HPLC to remove unreacted maleimide-PEG.sub.8-acid. The product 86 was isolated in 10% over two steps (6.6 mg). LC/MS 1.16 min (ES+) m/z (relative intensity) 770.20 ([M+2H].sup.+., 40%).

    Example 11—Alternative Synthesis of Compound 83

    [1339] ##STR00151##

    (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate (83)

    [1340] PBD-triflate 21 (469 mg, 0.323 mmol), boronic pinacol ester (146.5 mg, 0.484 mmol) and Na.sub.2CO.sub.3 (157 mg, 1.48 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (10 mL). The reaction flask was purged with argon three times before tetrakis(triphenylphosphine)palladium(0) (7.41 mg, 0.0064 mmol) was added and the reaction mixture heated to 30° C. overnight. The solvents were removed under reduced pressure and the residue was taken up in H.sub.2O (50 mL) and extracted with EtOAc (3×50 mL). The combined organics were washed with brine (100 mL), dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography (CHCl.sub.3 100% to CHCl.sub.3/MeOH 95%:5%) to afford pure 83 in 33% yield (885 mg). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478 ([M+H].sup.+., 100%).

    Example 12

    [1341] ##STR00152##

    (a) (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methypiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethysilyl)ethoxy)methy)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl trifluoromethanesulfonate (88)

    [1342] Pd(PPh.sub.3).sub.4 (30 mg, 26 mol) was added to a stirred mixture of the bis-enol triflate 87 (1 g, 0.87 mmol), 4-(4-methylpiperazin-1-yl)phenylboronic acid, pinacol ester (264 mg, 0.87 reaction mixture was allowed to stir under a nitrogen atmosphere overnight at room temperature after which time the complete consumption of starting material was observed by TLC (EtOAc) and LC/MS (1.52 min (ES+) m/z (relative intensity) 1171.40 ([M+H].sup.+., 100)). The reaction mixture was diluted with EtOAc (400 mL) and washed with H.sub.2O (2×300 mL), brine (200 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 100:0 v/v EtOAc/MeOH to 85:15 v/v EtOAc/MeOH) afforded the asymmetrical triflate 88 (285 mg, 28%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.39 (s, 1H), 7.37-7.29 (m, 4H), 7.23 (d, J=2.8 Hz, 2H), 7.14 (t, J=2.0 Hz, 1H), 6.89 (d, J=9.0 Hz, 2H), 5.54 (d, J=10.0 Hz, 2H), 4.71 (dd, J=10.0, 2.6 Hz, 2H), 4.62 (td, J=10.7, 3.5 Hz, 2H), 4.13-4.01 (m, 4H), 3.97-3.87 (m, 8H), 3.85-3.75 (m, 2H), 3.74-3.63 (m, 2H), 3.31-3.22 (m, 4H), 3.14 (tdd, J=16.2, 10.8, 2.2 Hz, 2H), 2.73-2.56 (m, 4H), 2.38 (d, J=2.4 Hz, 3H), 2.02-1.92 (m, 4H), 1.73 (dd, J=9.4, 6.0 Hz, 2H), 1.04-0.90 (m, 4H), 0.05-−0.00 (m, 18H). MS (ES+) m/z (relative intensity) 1171.40 ([M+H].sup.+., 100).

    (b) (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (89)

    [1343] Pd(PPh.sub.3).sub.4(8 mg, 7 μmol) was added to a stirred mixture of the asymmetrical triflate 88 (269 mg, 0.23 mmol), Fmoc-Val-Ala-4-aminophenylboronic acid, pinacol ester 20 (210 mg, 0.34 mmol), Na.sub.2CO.sub.3 (36.5 mg, 0.34 mmol), EtOH (5 mL), toluene (10 mL), THF (1 mL), and water (5 mL). The reaction mixture was allowed to stir under a nitrogen atmosphere at 35° C. for 2 hours after which time the complete consumption of starting material was observed by TLC (80:20 v/v EtOAc/MeOH) and LC/MS (1.68 min (ES+) m/z (relative intensity) 1508.10 ([M+H].sup.+., 100)). The reaction mixture was diluted with EtOAc (100 mL) and washed with H.sub.2O (1×100 mL), brine (200 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 100:0 v/v EtOAc/MeOH to 80:20 v/v EtOAc/MeOH) afforded the SEM protected dimer 89 (240 mg, 69%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.42 (s, 1H), 7.76 (d, J=7.5 Hz, 2H), 7.63-7.49 (m, 4H), 7.45-7.28 (m, 9H), 7.25 (d, J=2.9 Hz, 1H), 6.87 (t, J=14.0 Hz, 2H), 6.41 (s, 1H), 5.63-5.49 (m, 2H), 5.25 (s, 1H), 4.71 (d, J=10.1 Hz, 2H), 4.68-4.57 (m, 2H), 4.49 (d, J=6.7 Hz, 2H), 4.20 (s, 1H), 4.16-4.02 (m, 4H), 4.00-3.87 (m, 7H), 3.86-3.61 (m, 7H), 3.30-3.21 (m, 4H), 3.19-3.05 (m, 2H), 2.69-2.54 (m, 4H), 2.37 (s, 3H), 2.04-1.92 (m, 4H), 1.91-1.79 (m, 4H), 1.72 (s, 2H), 1.46 (d, J=6.9 Hz, 3H), 1.04-0.82 (m, 8H), 0.04-−0.02 (m, 18H). MS (ES+) m/z (relative intensity) 1508.10 ([M+H].sup.+., 100).

    (c) (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate (90)

    [1344] Super hydride (0.358 mL, 0.358 mmol, 1.0 M in THF) was added dropwise to a stirred solution of the SEM-tetralactam 89 (216 mg, 0.143 mmol) in anhydrous THF (10 mL) at −78° C. The reaction mixture was allowed to stir for 3 hours after which time the complete conversion of starting material directly was observed by LC/MS (1.37 min (ES+) m/z (relative intensity) 608.15 (([M+2H].sup.2+)/2,100)). The reaction mixture was carefully diluted with H.sub.2O (100 mL) and extracted with DCM (100 mL). The organic layers was washed with brine (100 mL), dried over MgSO.sub.4, filtered and evaporated under reduced pressure to provide the intermediate SEM-carbinolamine. The white solids were immediately dissolved in MeOH (100 mL), DCM (10 mL) and H.sub.2O (20 mL) and treated with flash silica gel (50 g). The thick suspension was allowed to stir at room temperature for 4 days after which time the formation of a significant quantity of desired product was observed by TLC (90:10 v/v CHCl.sub.3/MeOH). The reaction mixture was filtered through a porosity 3 sinter funnel and the pad rinsed slowly and thoroughly with 90:10 v/v CHCl.sub.3/MeOH until no further product eluted (checked by TLC). The filtrate was washed with brine (100 mL), dried (MgSO.sub.4), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 98:2 v/v CHCl.sub.3/MeOH to 88:12 v/v CHCl.sub.3/MeOH) gave 90 as a mixture of carbinolamine ethers and imine (80 mg, 46%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.52 (s, 1H), 7.87 (d, J=3.9 Hz, 2H), 7.75 (d, J=7.5 Hz, 2H), 7.66-7.26 (m, 12H), 6.90 (d, J=8.8 Hz, 2H), 6.81 (s, 1H), 6.64 (d, J=6.0 Hz, 1H), 5.37 (d, J=5.7 Hz, 1H), 4.74-4.58 (m, 2H), 4.54-4.31 (m, 4H), 4.26-3.98 (m, 6H), 3.94 (s, 2H), 3.86 (dd, J=13.6, 6.6 Hz, 1H), 3.63-3.48 (m, 2H), 3.37 (dd, J=16.5, 5.6 Hz, 2H), 3.31-3.17 (m, 4H), 2.66-2.51 (m, 4H), 2.36 (s, 3H), 2.16 (d, J=5.1 Hz, 1H), 2.06-1.88 (m, 4H), 1.78-1.55 (m, 6H), 1.46 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 6H). MS (ES+) m/z (relative intensity) 608.15 (([M+2H].sup.2+)/2,100).

    (d) 1-(3-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3, 6,9,12,15, 18, 21, 24-octaoxaheptacosan-27-amide (91)

    [1345] Piperidine (0.2 mL) was added to a solution of 90 (77 mg, 63.4 μmol) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over MgSO.sub.4, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg).sub.8-acid (56 mg, 95 μmol) and EDCl (18 mg, 95 μmol) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1.19 min (ES+) m/z (relative intensity) 784.25 (([M+2H].sup.2+)/2,100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgSO.sub.4), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCl.sub.3/MeOH to 90:10 v/v CHCl.sub.3/MeOH) gave 91 as a yellow solid (43 mg, 43%). .sup.1H NMR (400 MHz, CDCl.sub.3) b 8.73 (s, 1H), 7.88 (dd, J=7.6, 3.9 Hz, 2H), 7.75 (d, J=8.6 Hz, 2H), 7.52 (d, J=2.0 Hz, 2H), 7.44 (s, 1H), 7.40-7.28 (m, 4H), 6.91 (d, J=8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H), 6.48 (s, 1H), 4.72-4.63 (m, 1H), 4.46-4.34 (m, 2H), 4.25-4.03 (m, 6H), 3.95 (s, 4H), 3.84 (dd, J=17.2, 10.1 Hz, 4H), 3.72-3.46 (m, 30H), 3.44-3.32 (m, 4H), 3.30-3.20 (m, 4H), 2.75-2.63 (m, 1H), 2.59 (s, 4H), 2.55-2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J=12.7, 6.7 Hz, 1H), 2.03-1.89 (m, 4H), 1.72 (d, J=22.7 Hz, 8H), 1.46 (d, J=7.2 Hz, 3H), 1.01 (dd, J=11.5, 6.9 Hz, 6H). MS (ES.sup.+) m/z (relative intensity) 784.25 (([M+2H].sup.2+)/2,100).

    Example 13

    (i) (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4, 1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-23-dihydro-1H-pyrrol-1-yl)methanone) (98)

    [1346] ##STR00153##

    (a) (S, R)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidin-1-yl)methanone) (94)

    [1347] Anhydrous DMF (approx. 0.5 mL) was added dropwise to a stirred suspension of 4,4′-(pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid) (92) (36.64 g, 74.0 mmol) and oxalyl chloride (18.79 mL, 0.222 mol, 3.0 eq.) in anhydrous DCM (450 mL) until vigorous effervescence occurred and the reaction mixture was left to stir overnight. The reaction mixture was evaporated to dryness, and triturated with diethyl ether. The resulting yellow precipitate was filtered from solution, washed with diethyl ether (100 mL) and immediately added to a solution of (3R,5S)-5-((tert-butyldimethylsilyloxy)methyl) pyrrolidin-3-ol (93) (39.40 g, 0.170 mol, 2.3 eq.) and anhydrous triethylamine (82.63 mL, 0.592 mol, 8 eq.) in anhydrous DCM (400 mL) at −40° C. The reaction mixture was allowed to slowly warm to room temperature (over 2.5 hours) after which, LCMS analysis indicated complete reaction. DCM (250 mL) was added and the mixture was transferred into a separating funnel. The organic layer was washed successively with 0.1M HCl (2×800 mL), saturated NaHCO.sub.3 (500 mL) and brine (300 mL). After drying over MgSO.sub.4 and filtration, evaporation of the solvent left the product as a yellow foam (62.8 g, 92%). LC/MS: RT 1.96 min; MS (ES+) m/z (relative intensity) 921.45 ([M+H].sup.+, 100).

    (b) (5S,5'S)-1,1′-(4, 4′-(pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitrobenzoyl))bis(5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-3-one) (95)

    [1348] Trichloroisocyanuric acid (21.86 g, 94.07 mmol, 1.4 eq) was added in one portion to a solution of diol 94 (61.90 g, 67.20 mmol) and TEMPO (2.10 g, 13.44 mmol, 0.2 eq) in anhydrous DCM (500 mL) under an atmosphere of argon at 00° C. The reaction mixture was stirred at 0° C. for 20 minutes after which, LCMS analysis of the reaction mixture showed complete reaction. The reaction mixture was diluted with DCM (400 mL) and washed with saturated sodium bicarbonate (500 mL), 0.2 M sodium thiosulfate solution (600 mL), brine (400 mL) and dried (MgSO.sub.4). Evaporation of the solvent gave the crude product. Flash chromatography [gradient elution 80% n-hexane/20% ethyl acetate to 100% ethyl acetate] gave pure 95 as yellow solid (49.30 g, 80%). LC/MS: RT 2.03 min; MS (ES+) m/z (relative intensity) 917.55 ([M+H].sup.+, 100).

    (c) (5S,5'S)-1,1′-(4, 4′-(pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitrobenzoyl))bis(5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrole-3, 1-diyl)bis(trifluoromethanesulfonate), (96)

    [1349] Triflic anhydride (24.19 mL, 0.144 mol, 6.0 eq) was added dropwise to a vigorously stirred solution of bis-ketone 95 (21.98 g, 23.96 mmol) in anhydrous DCM (400 mL) containing 2,6-lutidine (22.33 mL, 0.192 mol, 8.0 eq) at −40° C. The reaction mixture was stirred at −40° C. for 30 min after which, LCMS analysis indicated complete reaction. Reaction mixture was rapidly diluted with DCM (500 mL) and washed with ice-cold water (600 mL), ice-cold saturated sodium bicarbonate (400 mL) and brine (500 mL), dried over MgSO.sub.4, filtered and evaporated to leave a crude brown oil. Flash chromatography [gradient elution 80% n-hexane/20% ethyl acetate to 66% n-hexane/33% ethyl acetate] gave pure 96 as a brown foam (16.40 g, 58%). LC/MS: RT 2.28 min; MS (ES+) m/z (relative intensity) no data.

    (d) (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4, 1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrol-1-yl) methanone) (97)

    [1350] Triflate 96 (5.06 g, 4.29 mmol), methyl boronic acid (1.80 g, 30.00 mmol, 7 eq) and triphenylarsine (1.05 g, 3.43 mmol, 0.8 eq) were dissolved in anhydrous dioxane and stirred under argon. Pd (II) bisbenzonitrile chloride was then added and the reaction mixture heated rapidly to 80° C. for 20 min. Reaction mixture cooled, filtered through Celite (washed through with ethyl acetate), filtrate washed with water (500 mL), brine (500 mL), dried over MgSO.sub.4, filtered and evaporated. Flash chromatography [gradient elution 50% n-hexane/50% ethyl acetate] gave pure 97 as a brown foam (4.31 g, 59%). LC/MS: RT 2.23 min; MS (ES+) m/z (relative intensity) 913.50 ([M+H].sup.+, 100).

    (e) (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4, 1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrol-1-yl) methanone) (98)

    [1351] Zinc dust (26.48 g, 0.405 mol, 36.0 eq) was added in one portion to a solution of bis-nitro compound 97 (10.26 g, 11.24 mmol) in 5% formic acid/methanol (200 mL) keeping the temperature between 25-30° C. with the aid of a cold water bath. The reaction was stirred at 30° C. for 20 minutes after which, LCMS showed complete reaction. The reaction mixture was filtered through Celite to remove the excess zinc, which was washed with ethyl acetate (600 mL). The organic fractions were washed with water (500 mL), saturated sodium bicarbonate (500 mL) and brine (400 mL), dried over MgSO.sub.4 and evaporated. Flash chromatography [gradient elution 100% chloroform to 99% chloroform/1% methanol] gave pure 98 as an orange foam (6.22 g, 65%). LC/MS: RT 2.20 min; MS (ES+) m/z (relative intensity) 853.50 ([M+H].sup.+, 100).

    (ii) 4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl 4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl ((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5,1-phenylene))dicarbamate (103)

    [1352] ##STR00154##

    (a) Allyl (5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxyphenyl)carbamate (99)

    [1353] Pyridine (1.156 mL, 14.30 mmol, 1.5 eq) was added to a solution of the bis-aniline 98 (8.14 g, 9.54 mmol) in anhydrous DCM (350 mL) at −78° C. under an atmosphere of argon. After 5 minutes, allyl chloroformate (0.911 mL, 8.58 mmol, 0.9 eq) was added and the reaction mixture allowed to warm to room temperature. The reaction mixture was diluted with DCM (250 mL), washed with saturated CuSO.sub.4 solution (400 mL), saturated sodium bicarbonate (400 mL) and brine (400 mL), dried over MgSO.sub.4. Flash chromatography [gradient elution 66% n-hexane/33% ethyl acetate to 33% n-hexane/66% ethyl acetate] gave pure 99 as an orange foam (3.88 g, 43%). LC/MS: RT 2.27 min; MS (ES+) m/z (relative intensity) 937.55 ([M+H].sup.+, 100).

    (b) Allyl 4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl ((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5, 1-phenylene))dicarbamate (100)

    [1354] Triethylamine (0.854 mL, 6.14 mmol, 2.2 eq) was added to a stirred solution of the aniline 99 (2.62 g, 2.79 mmol) and triphosgene (0.30 g, 1.00 mmol, 0.36 eq) in anhydrous THF (50 mL) under argon 0° C. The reaction mixture was stirred at room temperature for 5 minutes. LCMS analysis of an aliquot quenched with methanol, showed formation of the isocyanate. A solution of mPEG.sub.2-Val-Ala-PAB-OH (1.54 g, 3.63 mmol, 1.3 eq) and triethylamine (0.583 mL, 4.19 mmol, 1.5 eq) in dry THF (50 mL) was added in one portion and the resulting mixture was stirred overnight at 40° C. The solvent of the reaction mixture was evaporated leaving a crude product. Flash chromatography [gradient elution 100% chloroform to 98% chloroform/2% methanol] gave pure 100 as a light orange solid (2.38 g, 62%). LC/MS: RT 2.29 min; MS (ES+) m/z (relative intensity) no data.

    (c) 4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl (5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxyphenyl)carbamate (101)

    [1355] Tetrakis(triphenylphosphine)palladium (39 mg, 0.034 mmol, 0.02 eq) was added to a stirred solution of 100 (2.35 g, 1.69 mmol) and pyrrolidine (0.35 mL, 4.24 mmol, 2.5 eq) in anhydrous DCM (25 mL) under argon at room temperature. Reaction mixture allowed to stir for 45 min then diluted with DCM (100 mL), washed with saturated ammonium chloride solution (100 mL), brine (100 mL), dried over MgSO.sub.4, filtered and evaporated. Flash chromatography [gradient elution 100% chloroform to 95% chloroform/5% methanol] gave pure 101 as a yellow solid (1.81 g, 82%). LC/MS: RT 2.21 min; MS (ES+) m/z (relative intensity) 1303.65 ([M+H].sup.+, 100).

    (d) 4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl 4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl ((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5, 1-phenylene))dicarbamate (102)

    [1356] Triethylamine (0.419 mL, 3.01 mmol, 2.2 eq) was added to a stirred solution of the aniline 101 (1.78 g, 1.37 mmol) and triphosgene (0.15 g, 0.49 mmol, 0.36 eq) in anhydrous THF (50 mL) under argon 0° C. The reaction mixture was stirred at room temperature for 5 min. LCMS analysis of an aliquot quenched with methanol, showed formation of the isocyanate. A solution of Alloc-Val-Ala-PAB-OH (0.67 g, 1.78 mmol, 1.3 eq) and triethylamine (0.29 mL, 2.05 mmol, 1.5 eq) in dry THF (45 mL) was added in one portion and the resulting mixture was stirred overnight at 40° C. The solvent of the reaction mixture was evaporated leaving a crude product. Flash chromatography [gradient elution 100% ethyl acetate to 97% ethyl acetate/3% methanol] gave pure 102 as a pale yellow solid (1.33 g, 57%). LC/MS: RT 2.21 min; MS (ES+) m/z (relative intensity) no data.

    (e) 4-((R)-2-(R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl 4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl ((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(hydroxymethyl)-4-methyl-2, 3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5, 1-phenylene))dicarbamate (103)

    [1357] Tetra-n-butylammonium fluoride (1 M, 1.52 mL, 1.52 mmol, 2.0 eq) was added to a solution of the TBS protected compound 102 (1.30 g, 0.76 mmol) in anhydrous THF (15 mL). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with chloroform (100 mL) and washed sequentially with water (40 mL) and brine (40 mL). The organic phase was dried over MgSO.sub.4 and evaporated to leave a yellow solid. Flash chromatography [gradient elution 95% ethyl acetate/5% methanol to 90% ethyl acetate/10% methanol] gave pure 103 as a pale yellow solid (1.00 g, 89%). LC/MS: RT 1.60 min; MS (ES+) m/z (relative intensity) 1478.45 (100).

    (iii) (11S,11aS)-4-((2R,5R)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (106)

    [1358] ##STR00155##

    (a) (11S,11aS)-4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (104)

    [1359] Dess-Martin periodinane (0.59 g, 1.38 mmol, 2.1 eq) was added to a stirred solution of 103 (0.97 g, 0.66 mmol) in anhydrous DCM under argon at room temperature. The reaction mixture was allowed to stir for 4 hours. Reaction mixture diluted with DCM (100 mL), washed with saturated sodium bicarbonate solution (3×100 mL), water (100 mL), brine (100 mL), dried over MgSO.sub.4, filtered and evaporated. Flash chromatography [gradient elution 100% chloroform to 95% chloroform/5% methanol] gave pure 104 as a pale yellow solid (0.88 g, 90%). LC/MS: RT 1.57 min; MS (ES+) m/z (relative intensity) 1473.35 (100).

    (b) (11S,11aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (105)

    [1360] Tetrakis(triphenylphosphine)palladium (5 mg, 0.004 mmol, 0.06 eq) was added to a solution of 104 (105 mg, 0.071 mmol) and pyrrolidine (7 μL, 0.086 mmol, 1.2 eq) in anhydrous DCM (5 mL). The reaction mixture was stirred 15 minutes then diluted with chloroform (50 mL) and washed sequentially with saturated aqueous ammonium chloride (30 mL) and brine (30 mL). The organic phase was dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% chloroform to 90% chloroform/10% methanol] gave pure 105 as a pale yellow solid (54 mg, 55%). LC/MS: RT 1.21 min; MS (ES+) m/z (relative intensity) 1389.50 (100).

    (c) (11S,11aS)-4-((2R,5R)-37-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4, 7,35-trioxo-10, 13, 16, 19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (106)

    [1361] N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 105 (203 mg, 0.146 mmol) and maleimide-PEG.sub.8 acid (87 mg, 0.146 mmol) in chloroform (5 mL). The reaction was stirred for 1.5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/10% methanol] gave 106 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 min; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).

    Example 14: Activity of Released Compounds

    K562 Assay

    [1362] K562 human chronic myeloid leukaemia 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% CO.sub.2 and were incubated with a specified dose of drug for 1 hour or 96 hours at 37° C. in the dark. The incubation was terminated by centrifugation (5 min, 300 g) and the cells were washed once with drug-free medium. Following the appropriate drug treatment, the cells were transferred to 96-well microtiter plates (10.sup.4 cells per well, 8 wells per sample). Plates were then kept in the dark at 37° C. in a humidified atmosphere containing 5% CO.sub.2. 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. Following incubation of the plates for 4 days (to allow 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 the bulk 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 agitated to ensure complete mixing. The optical density was then read at a wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and a dose-response curve was constructed. For each curve, an IC.sub.50 value was read as the dose required to reduce the final optical density to 50% of the control value.

    [1363] Compound RelC has an IC.sub.50 of less than 0.1 pM in this assay.

    [1364] Compound RelE has an IC.sub.50 of 0.425 nM in this assay.

    Example 15: Formation of Conjugates

    General Antibody Conjugation Procedure

    [1365] Antibodies between 1-10 mg/ml in 30 mM Histidine, 200 mM sorbitol, pH 6 is increased in pH from 6 to 7.5 by the addition of a volume of 0.5 M Tris, 25 mM EDTA, pH 8.5, equivalent to 6.7% of the mAb volume. DTT reductant is added to the batch as a 20-fold molar excess with respect to antibody and the reduction reaction is allowed to proceed overnight at room temperature (no agitation). Post reduction the antibody is desalted into 0.1 M sodium phosphate pH 7.5. Reduced antibody is reoxidised by the addition of 25 mM DHAA as a 20 fold molar excess with respect to antibody and the reoxidation reaction is allowed to proceed for a total of 2 hours at 20° C. Conjugation is initiated by the addition of DMA and 10 mM drug-linker is added in that order to achieve a 5% v/v final and 3 fold excess relative to the antibody, respectively. The conjugation reaction is incubated for 60 min. Post conjugation the reaction is quenched with a 3 fold molar excess of N-acetyl cysteine and incubated for an additional 30 mins. The final products can be analysed by SEC, HIC, PLRP and non-reducing gel electrophoresis.

    [1366] Corresponding antibody-drug conjugates can be determined by analysis by High-Performance Liquid Chromatography (HPLC) or Ultra-High-Performance Liquid Chromatography (UHPLC) to assess drug-per-antibody ratio (DAR) using reverse-phase chromatography (RP) or Hydrophobic-Interaction Chromatography (HIC), coupled with UV-Visible, Fluorescence or Mass-Spectrometer detection; aggregate level and monomer purity can be analysed by HPLC or UHPLC using size-exclusion chromatography coupled with UV-Visible, Fluorescence or Mass-Spectrometer detection. Final conjugate concentration is determined by a combination of spectroscopic (absorbance at 280, 214 and 330 nm) and biochemical assay (bicinchonic acid assay BCA; Smith, P. K., et al. (1985) Anal. Biochem. 150 (1): 76-85; using a known-concentration IgG antibody as reference). Antibody-drug conjugates are generally sterile filtered using 0.2 μm filters under aseptic conditions, and stored at +4° C., −20° C. or −80° C.

    DAR Determination

    [1367] Antibody or ADC (ca. 35 μg in 35 μL) was reduced by addition of 10 μL borate buffer (100 mM, pH˜8.4) and 5 μL DTT (0.5 M in water), and heated at 37° C. for 15 minutes. The sample was diluted with 1 volume of acetonitrile: water: formic acid (49%: 49%: 2% v/v), and injected onto a Widepore 3.6μ XB—C18 150×2.1 mm (P/N 00F-4482-AN) column (Phenomenex Aeris) at 80° C., in a UPLC system (Shimadzu Nexera) with a flow rate of 1 ml/min equilibrated in 75% Buffer A (Water, Trifluoroacetic acid (0.1% v/v) (TFA), 25% buffer B (Acetonitrile: water: TFA 90%: 10%: 0.1% v/v). Bound material was eluted using a gradient from 25% to 55% buffer B in 10 min. Peaks of UV absorption at 214 nm were integrated. The following peaks were identified for each ADC or antibody: native antibody light chain (L0), native antibody heavy chain (HO), and each of these chains with added drug-linkers (labelled L1 for light chain with one drug and H1, H2, H3 for heavy chain with 1, 2 or 3 attached drug-linkers). The UV chromatogram at 330 nm was used for identification of fragments containing drug-linkers (i.e., L1, H1, H2, H3).

    [1368] A PBD/protein molar ratio was calculated for both light chains and heavy chains:

    [00001] Drug Protein .Math. .Math. ratio .Math. .Math. on .Math. .Math. light .Math. .Math. chain = % .Math. .Math. Area .Math. .Math. at .Math. .Math. 214 .Math. .Math. nm .Math. .Math. for .Math. .Math. L .Math. .Math. 1 % .Math. .Math. Area .Math. .Math. at .Math. .Math. .Math. 214 .Math. .Math. nm .Math. .Math. for .Math. .Math. L .Math. .Math. 0 .Math. .Math. and .Math. .Math. L .Math. .Math. 1 Drug Protein .Math. .Math. ratio .Math. .Math. on .Math. .Math. heavy .Math. .Math. chain = .Math. n = 0 3 .Math. n × ( % .Math. .Math. area .Math. .Math. at .Math. .Math. 214 .Math. .Math. for .Math. .Math. Hn ) .Math. n = 0 3 .Math. .Math. % .Math. .Math. area .Math. .Math. at .Math. .Math. 214 .Math. .Math. for .Math. .Math. Hn

    [1369] Final DAR is calculated as:

    [00002] DAR = 2 × ( Drug Protein .Math. .Math. ratio .Math. .Math. on .Math. .Math. light .Math. .Math. chain + Drug Protein .Math. .Math. ratio .Math. .Math. on .Math. .Math. heavy .Math. .Math. chain )

    [1370] DAR measurement is carried out at 214 nm because it minimises interference from drug-linker absorbance.

    Generation of ADCs

    [1371] Mouse 5E5 is an anti-Tn MUC 1 antibody comprising a VH domain having the sequence according to SEQ ID NO. 1 and a VL domain having the sequence according to SEQ ID NO. 30.

    [1372] Ab1 is an anti-Tn MUC 1 antibody comprising a VH domain having the sequence according to SEQ ID NO. 9 and a VL domain having the sequence according to SEQ ID NO. 31.

    [1373] Ab2 is an anti-Tn MUC 1 antibody comprising a VH domain having the sequence according to SEQ ID NO. 19 and a VL domain having the sequence according to SEQ ID NO. 31.

    [1374] Ab3 is an anti-Tn MUC 1 antibody comprising a VH domain having the sequence according to SEQ ID NO. 28 and a VL domain having the sequence according to SEQ ID NO. 31.

    [1375] Ab4 is an anti-Tn MUC 1 antibody comprising a VH domain having the sequence according to SEQ ID NO. 9 and a VL domain having the sequence according to SEQ ID NO. 33.

    [1376] Ab5 is an anti-Tn MUC 1 antibody comprising a VH domain having the sequence according to SEQ ID NO. 19 and a VL domain having the sequence according to SEQ ID NO. 33.

    [1377] Ab6 is an anti-Tn MUC 1 antibody comprising a VH domain having the sequence according to SEQ ID NO. 28 and a VL domain having the sequence according to SEQ ID NO. 33.

    [1378] ADCs targeted to TnMUC1 were generated by conjugating the above antibodies to warhead-linker ConjE as described above. The resulting ADCs are listed in the table below. B12 anti-HIV gp120 antibody was used to generate control non-TnMUC1targeted ADCs.

    TABLE-US-00002 ADC DAR Concentration [mg/ml] Mouse 5E5-ConjE 2.76 1.78 Ab1-ConjE 2.32 2.57 Ab4-ConjE 2.77 2.70 Ab2-ConjE 3.45 1.68 B12-ConjE 2.5  0.53

    Example 12: Development of 5E5 Constructs

    Design

    [1379] Three heavy chain constructs were designed by CDR grafting from mouse 5E5 VH into the FWs of AB066839, AY392978.1 and AF455547.1 respectively. DNA sequences were optimised for expression by GeneArt. Variants were made by back-mutating mismatches at vernier and 5 Å CDR envelope residues or back-mutating mismatched vernier residues only.

    [1380] Two light chain constructs 5E5RKA and 5E5RKB were designed by grafting CDRs from 5E5VK into the acceptor framework (FW) of DQ172590 and DQ841010. Sequence optimised variant light chain constructs 5E5RKA1 and 5E5RKB1 were created by back-mutating mismatches at vernier and 5 Å CDR FW envelope residues.

    [1381] All sequences are set out in the “sequences section”.

    Expression and Testing of Initial VH Constructs

    [1382] The DNA sequences of all constructs were optimised for expression and synthesised by GeneArt. GeneArt plasmids were subcloned into pfuse expression vectors (protocol 1). Pairs of humanised VH and chimeric VK constructs or chimeric VH and humanised VK constructs were used to transiently transfect HEK293T cells using protocol 2. IgG in conditioned medium harvested from these transfected cell cultures was measured using protocol 3 and its Tn-MUC1 binding was measured using protocol 4.

    Expression and Testing of Further Constructs

    [1383] Further V region versions were generated so as to identify residues which are critical to maintaining Tn-MUC1 binding potency. All sequences are set out in the “Sequences section”.

    Final Construct Design and Generation

    [1384] In each V region construct, it was found that some back mutations were critical to binding potency, whereas other back mutations were unnecessary. The final VH and VK designs incorporate these necessary back-mutations and are shown, as protein sequences, in the “Sequences section”.

    Example 13: In Vitro Testing of Final Constructs

    [1385] Expression levels of the constructs were tested as per protocols 2 and 3. Most final constructs expressed well, particularly Ab1, Ab4, and Ab2. In contrast, Ab6 expression is low (see Table 2 below).

    [1386] The expression of the Ab1 and Ab2 antibodes was up to 6× and up to 4× better than the mouse antibody expressed in static transfected HEK293T or shaken HEK293F cells respectively (Table 2).

    [1387] IgG binding potency to Tn-MUC1 was measured as per protocol 4 and binding to the ZR-75-1 cell line, which expresses Tn-MUC1, was measured as per protocol 6. The Tn-MUC1 binding assay data show that the TnMUC1 binding potency of Ab6 is substantially improved over the mouse 5E5 in the (see Table 2 below, along with FIG. 1). The flow cytometry EC.sub.50 data show that the ZR-75-1 binding potency of Ab1, Ab2 and Ab5 is substantially improved over the mouse 5E5 (see Table 2 below, along with FIG. 2).

    [1388] A thermal denaturation assay was performed according to protocol 5 in order to investigate molecular stability. The most heat-stable humanised 5E5 versions are Ab1 and Ab2.

    TABLE-US-00003 TABLE 2 Static Shaken HEK293T HEK293F Tn-MUC1 ZR-75-1 cell transient transient binding EC.sub.50 binding EC.sub.50 IgG IgG ng/ml ng/ml μg/ml μg/ml T.sub.m ° C. Mouse 31.10 28.34 4.0 18.9 69.97 5E5 Ab1 24.57 9.86 23.1 63.2 69.37 Ab2 34.46 16.83 24.9 27.2 68.32 Ab3 61.48 26.78 3.0 7.0 65.32 Ab4 75.49 42.29 10.9 18.9 65.23 Ab5 26.87 18.42 3.3 17.2 62.58 Ab6 19.80 41.30 0.18 1.0 n.d.

    Example 14: In Vivo Testing of Final Constructs

    [1389] The Tn-MUC1(+ve) cell line ZR75-1 was used in a mouse xenograft model to test the in vivo efficacy of ADCs comprising the humanised 5E5 constructs. The anti-HIV gp120 antibody, B12, linked to ConjE was used as a null-binder isotype control in the same model.

    Study Design

    Drugs and Treatment:

    [1390]

    TABLE-US-00004 Group Animals Dose level Dose volume No per group ADC (mg/kg) (mg/kg) 1 15 [vehicle only] — — 2 15 Mouse 5E5-ConjE 0.3 Qwkx3 10 3 15 Mouse 5E5-ConjE 1.0 Qwkx3 10 4 15 Ab1-ConjE 0.3 Qwkx3 10 5 15 Ab1-ConjE 1.0 Qwkx3 10 6 15 Ab4-ConjE 0.3 Qwkx3 10 7 15 Ab4-ConjE 1.0 Qwkx3 10 8 15 Ab2-ConjE 0.3 Qwkx3 10 9 15 Ab2-ConjE 1.0 Qwkx3 10 10 15 isotype control-ConjE 1.0 Qwkx3 10

    Procedures:

    [1391] Set up CR female CB.17 SCID mice with 1×10.sup.7 ZR-75-1 tumor cells in 50% Matrigel sc in flank. Cell Injection Volume is 0.1 mL/mouse. Age at Start Date: 8 to 12 weeks.

    [1392] Perform a pair match when tumors reach an average size of 100-150 mm.sup.3, and begin treatment. Body Weight: qd×5 then biwk to end. Caliper Measurement: biweekly to end. Implant estradiol pellets, s.c. between the scapulae, 3-7 days prior to cell implantation.

    [1393] Report any adverse reactions or death immediately. Any individual animal with a single observation of >than 30% body weight loss or three consecutive measurements of >25% body weight loss will be euthanized. Any group with two measurements of mean body weight loss of >20% or >10% mortality will stop dosing. The group is not euthanized and recovery is allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint will be euthanized. If the group treatment related body weight loss is recovered to within 10% of the original weights, dosing may resume at a lower dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery may be allowed on a case-by-case basis.

    [1394] Endpoint TGD. Animals are to be monitored individually. The endpoint of the experiment is a tumor volume of 1000 mm.sup.3 or 45 days, whichever comes first. Responders can be followed longer. When the endpoint is reached, the animals are to be euthanized.

    General Methodological Approach

    [1395] For the calculation of group mean tumor volumes the following rule was applied: when an animal exited the study due to tumor size, the final tumor volume recorded for the animal was included with the data used to calculate the mean volume at subsequent time points. Error bars indicate standard error of the mean (SEM). Tumor volumes values were not used to calculate group mean tumor volumes when fewer than 50% of the animals in a group remained in the study. Prism (GraphPad, San Diego, Calif.) was used for graphical presentations and statistical analyses.

    Results

    [1396] FIGS. 4 and 5 shows ADCs comprising the humanised 5E5 in a ZR-75-1 xenograft model.

    [1397] Mice were dosed when the mean tumor volume per experimental group reached 0.1 cm.sup.3 and they were treated with 3× weekly doses of the ADC at 0.3 or 1 mg/kg (for 5E5 ADCs) and 1 mg/kg (for B12 ADC) via the tail vein. Data represent the mean tumour volume (+/−SEM) in each group of fifteen mice.

    [1398] The ADCs dosed at 1.0 mg/kg uniformly exhibited more potent anti-tumor activity than those dosed at 0.3 mg/kg. In addition, all of the 5E5 ADCs exhibited significantly higher anti-tumour activity than the isotype control ADC, even when the 5E5 ADC was dosed at 0.3 mg/kg compared to the B12 ADC's 1.0 mg/kg. The anti-tumor activity of the 5E5 ADCs was broadly comparable to that of mouse 5E5.

    Example 15: Further In Vivo Testing of Final Constructs

    [1399] Female SWISS nude mice were subcutaneously implanted with ODS-BRE-407 (human primary breast tumor) fragments, into the right flank, on day 0.

    [1400] On day 33, when the tumors reached a mean volume of 124±52 mm3, treatments were started. ADC were administered i.v., weekly for 3 consecutive weeks (q7d×3).

    [1401] The group receiving Ab1-ConjE; 0.3 mg/kg, q7d x 3, was re-challenged with a single dose of Ab1-ConjE, 1 mg/kg on day 76.

    [1402] Tumor volumes were measured and recorded twice a week and the study was terminated on day 112.

    [1403] FIG. 6 shows that, as for the data in Example 14, the ADCs dosed at 1.0 mg/kg uniformly exhibited more potent anti-tumor activity than the ADC dosed at 0.3 mg/kg. In addition, all of the 5E5 ADCs exhibited significantly higher anti-tumour activity than the isotype control ADC.

    Abbreviations

    [1404] 5E5 VH VH of mouse 5E5 antibody [1405] 5E5 VK VK of mouse 5E5 antibody [1406] 5E5RHA1 Humanised versionA1, of 5E5 VH [1407] 5E5RKA1 Humanised version, A1, of 5E5 VK [1408] A Adenine [1409] Å Angstrom [1410] Ac acetyl [1411] Acm acetamidomethyl [1412] Alloc allyloxycarbonyl [1413] B7 The anti-LPA antibody product of mouse hybridoma clone B7 [1414] Boc di-tert-butyl dicarbonate [1415] bp base pairs [1416] Bzl benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is methylbenzene [1417] C Cytosine [1418] Cbz or Z benzyloxy-carbonyl, where Z—Cl and Z—Br are chloro- and bromobenzyloxy carbonyl respectively [1419] CDR Complementarity determining region in the immunoglobulin variable regions, defined using the Kabat numbering system [1420] D-gene Diversity gene [1421] DMF N,N-dimethylformamide [1422] DNA Deoxyribonucleic acid [1423] Dnp dinitrophenyl [1424] DTT dithiothreitol [1425] Fmoc 9H-fluoren-9-ylmethoxycarbonyl [1426] FW Framework region: the immunoglobulin variable regions excluding the CDR regions [1427] G Guanine [1428] IgG Immunoglobulin G [1429] imp N-10 imine protecting group: 3-(2-methoxyethoxy)propanoate-Val-Ala-PAB [1430] MC-OSu maleimidocaproyl-O—N-succinimide [1431] J-gene Joining gene [1432] Kabat an immunoglobulin alignment and numbering system pioneered by Elvin A Kabat [1433] mAb monoclonal antibody [1434] Moc methoxycarbonyl [1435] MP maleimidopropanamide [1436] Mtr 4-methoxy-2,3,6-trimethtylbenzenesulfonyl [1437] PAB para-aminobenzyloxycarbonyl [1438] PEG ethyleneoxy [1439] PNZ p-nitrobenzyl carbamate [1440] Psec 2-(phenylsulfonyl)ethoxycarbonyl [1441] T Thymine [1442] TBDMS tert-butyldimethylsilyl [1443] TBDPS tert-butyldiphenylsilyl [1444] t-Bu tert-butyl [1445] Teoc 2-(trimethylsilyl)ethoxycarbonyl [1446] Tos tosyl [1447] Troc 2,2,2-trichlorethoxycarbonyl chloride [1448] Trt trityl [1449] V region The segment of IgG chains which is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain. [1450] VCl Framework residue classified as vernier or canonical or VH-VL interface [1451] V-gene The gene segment that is rearranged, together with a J (and D for VH) gene, to generate a complete VK (or VH) [1452] VH Immunoglobulin heavy chain variable region [1453] VK Immunoglobulin kappa light chain variable region [1454] Xan xanthyl

    REFERENCES

    [1455] [1] C. Chothia, et al., “Domain association in immunoglobulin molecules. The packing of variable domains,” J Mol. Biol. 186(3), 651 (1985). [1456] [2] J. Foote and G. Winter, “Antibody framework residues affecting the conformation of the hypervariable loops,” J Mol. Biol. 224(2), 487 (1992). [1457] [3] E. A Kabat, et al., sequences of proteins of immunological interest, 5 ed. (NIH National Technical Information Service, 1991). [1458] [4] V. Morea, A. M. Lesk, and A. Tramontano, “Antibody modeling: implications for engineering and design,” Methods 20(3), 267 (2000).

    STATEMENTS OF DISCLOSURE

    [1459] 1. An isolated humanized antibody that binds to Tn-MUC1, wherein the isolated humanized antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, a light chain variable region having the amino acid sequence of SEQ ID NO: 31, 32, 33, or 34, and optionally comprises a constant region derived from one or more human antibodies.

    [1460] 2. The isolated humanized antibody according to statement 1, wherein the isolated humanized antibody comprises:

    [1461] (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1462] (ii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1463] (iii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1464] (iv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1465] (v) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1466] (vi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1467] (vii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1468] (viii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1469] (ix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1470] (x) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1471] (xi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1472] (xii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1473] (xiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1474] (xiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1475] (xv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1476] (xvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1477] (xvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1478] (xviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1479] (xix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1480] (xx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1481] (xxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1482] (xxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1483] (xxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1484] (xxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1485] (xxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1486] (xxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1487] (xxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1488] (xxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30;

    [1489] (xxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1490] (xxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1491] (xxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1492] (xxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1493] (xxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1494] (xxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1495] (xxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1496] (xxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1497] (xxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1498] (xxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1499] (xxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1500] (xl) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1501] (xli) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1502] (xlii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1503] (xliii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1504] (xliv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1505] (xlv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1506] (xlvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1507] (xlvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1508] (xlviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1509] (xlix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1510] (l) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1511] (li) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1512] (lii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1513] (liii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1514] (liv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1515] (lv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1516] (lvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1517] (lvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31;

    [1518] (lviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1519] (lix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1520] (lx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1521] (lxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1522] (lxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1523] (lxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1524] (lxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1525] (lxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1526] (lxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1527] (lxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1528] (lxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1529] (lxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1530] (lxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1531] (lxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1532] (lxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1533] (lxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1534] (lxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1535] (lxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1536] (lxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1537] (lxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1538] (lxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1539] (lxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1540] (lxxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1541] (lxxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1542] (lxxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1543] (lxxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1544] (lxxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1545] (lxxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1546] (lxxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32;

    [1547] (lxxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1548] (lxxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1549] (lxxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1550] (xc) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1551] (xci) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1552] (xcii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1553] (xciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1554] (xciv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1555] (xcv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1556] (xcvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1557] (xcvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1558] (xciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1559] (xciv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1560] (xcv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1561] (xcvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1562] (xcvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1563] (xcviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1564] (xciv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1565] (xcv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1566] (xcvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1567] (xcvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1568] (xcviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1569] (xcix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1570] (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1571] (ci) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1572] (cii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1573] (ciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1574] (civ) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1575] (cv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33;

    [1576] (cvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1577] (cvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1578] (cviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1579] (cix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1580] (cx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1581] (cxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1582] (cxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1583] (cxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1584] (cxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1585] (cxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1586] (cxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1587] (cxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1588] (cxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1589] (cxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1590] (cxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1591] (cxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1592] (cxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1593] (cxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1594] (cxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1595] (cxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1596] (cxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1597] (cxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1598] (cxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1599] (cxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1600] (cxxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1601] (cxxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1602] (cxxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34;

    [1603] (cxxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; or

    [1604] (cxxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34.

    [1605] 3. The humanized antibody according to any one of statements 1 to 2, wherein said antibody binds Tn-MUC1 with an affinity (Kd) of at least 10.sup.−6 M.

    [1606] 4. The humanized antibody according to statement 3, wherein said antibody binds Tn-MUC1 with an affinity (Kd) of at least 10.sup.−9 M.

    [1607] 5. The humanized antibody according to any one of statements 1 to 4, wherein said antibody competitively inhibits the binding to Tn-MUC1 of an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30.

    [1608] 6. The humanized antibody according to any one of statements 1 to 5, wherein said antibody competitively inhibits the binding to Tn-MUC1 of the mouse 5E5 antibody.

    [1609] 7. The humanized antibody according to any one of statements 1 to 6, wherein said antibody or antibody fragment substantially neutralizes at least one activity of at least one Tn-MUC1.

    [1610] 8. The humanized antibody according to any one of statements 1 to 7, wherein said antibody or antibody fragment expresses at a level of at least 10 micrograms/ml in a static HEK293T transient expression system.

    [1611] 9. The humanized antibody according to any one of statements 1 to 7, wherein said antibody or antibody fragment expresses at a level of at least 20 micrograms/ml in a shaken HEK293T transient expression system.

    [1612] 10. The humanized antibody according to any one of statements 1 to 9, wherein said antibody has a melting temperature (T.sub.m) of at least 62° C.

    [1613] 11. The humanized antibody according to any one of statements 1 to 10, wherein said antibody or antibody fragment has a constant region of either isotype IgG1, IgG2, IgG3 or IgG4, or a mutated IgG constant region, and optionally a light chain constant region of isotype kappa or lambda.

    [1614] 12. The humanized antibody according to any one of statements 1 to 11, wherein the humanized antibody fragment is a scFv, Fab or F(ab′).sub.2.

    [1615] 13. A conjugate of formula L—(D.sup.L).sub.p, where DL is of formula I or II:

    ##STR00156##

    [1616] wherein:

    [1617] L is an isolated humanized antibody that binds to Tn-MUC1 (Ab) according to any one of statements 1 to 12; [1618] when there is a double bond present between C2′ and C3′, R.sup.12 is selected from the group consisting of:

    [1619] (ia) C-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;

    [1620] (ib) C.sub.1-5 saturated aliphatic alkyl;

    [1621] (ic) C.sub.3-6 saturated cycloalkyl;

    ##STR00157##

    wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5;

    ##STR00158##

    wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

    ##STR00159##

    where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
    when there is a single bond present between C2′ and C3′,

    [1622] R.sup.12 is

    ##STR00160##

    where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;

    [1623] R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo;

    [1624] where R and R′ are independently selected from optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups;

    [1625] R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo;

    [1626] R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;

    [1627] Y and Y′ are selected from O, S, or NH;

    [1628] R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively;

    [Formula I]

    [1629] R.sup.L1′ is a linker for connection to the antibody (Ab);

    [1630] R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;

    [1631] R.sup.20 and R.sup.21 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;

    [1632] R.sup.20 is selected from H and R.sup.C, where R.sup.C is a capping group;

    [1633] R.sup.21 is selected from OH, OR.sup.A and SO.sub.zM;

    [1634] when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of:

    [1635] (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.13 alkylene;

    [1636] (ib) C.sub.1-5 saturated aliphatic alkyl;

    [1637] (ic) C.sub.3-6 saturated cycloalkyl;

    ##STR00161##

    wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;

    ##STR00162##

    wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

    ##STR00163##

    where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

    [1638] when there is a single bond present between C2 and C3,

    [1639] R.sup.2 is

    ##STR00164##

    where R.sup.116 and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;

    [Formula II]

    [1640] R.sup.22 is of formula IIIa, formula IIIb or formula IIIc:

    ##STR00165##

    [1641] where A is a C.sub.5-7 aryl group, and either

    [1642] (i) Q.sup.1 is a single bond, and Q.sup.2 is selected from a single bond and —Z—(CH.sub.2).sub.n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or

    [1643] (ii) Q.sup.1 is —CH═CH—, and Q.sup.2 is a single bond;

    ##STR00166##

    [1644] where;

    [1645] R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and unsubstituted C.sub.1-2 alkyl;

    ##STR00167##

    [1646] where Q is selected from O—R.sup.L2′, S—R.sup.L2′ and NR.sup.N—R.sup.L2′, and R.sup.N is selected from H, methyl and ethyl

    [1647] X is selected from the group comprising: O—R.sup.L2′, S—R.sup.L2′, CO.sub.2—R.sup.L2′, CO—R.sup.L2′, NH—C(═O)—R.sup.L2′, NHNH—R.sup.L2′, CONHNH—R.sup.L2′,

    ##STR00168##

    NR.sup.NR.sup.L2′, wherein R.sup.N is selected from the group comprising H and C.sub.1-4 alkyl;

    [1648] R.sup.L2′ is a linker for connection to the antibody (Ab);

    [1649] R.sup.10 and R.sup.11 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;

    [1650] R.sup.10 is H and R.sup.11 is selected from OH, OR.sup.A and SO.sub.zM;

    [1651] R.sup.30 and R.sup.31 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;

    [1652] R.sup.30 is H and R.sup.31 is selected from OH, OR.sup.A and SO.sub.zM.

    [1653] 14. The conjugate according to statement 13, wherein R.sup.7 is selected from H, OH and OR.

    [1654] 15. The conjugate according to statement 14, wherein R.sup.7 is a C.sub.1-4 alkyloxy group.

    [1655] 16. The conjugate according to any one of statements 13 to 15, wherein Y is O.

    [1656] 17. The conjugate according to any one of statements 13 to 16, wherein R″ is C.sub.3-7 alkylene.

    [1657] 18. The conjugate according to any one of statements 13 to 17, wherein R.sup.9 is H.

    [1658] 19. The conjugate according to any one of statements 13 to 18, wherein R.sup.6 is selected from H and halo.

    [1659] 20. The conjugate according to any one of statements 13 to 29, wherein there is a double bond between C2′ and C3′, and R.sup.12 is a C.sub.5-7 aryl group.

    [1660] 21. The conjugate according to statement 20, wherein R.sup.12 is phenyl.

    [1661] 22. The conjugate according to any one of statements 13 to 19, wherein there is a double bond between C2′ and C3′, and R.sup.12 is a C.sub.8-10 aryl group.

    [1662] 23. The conjugate according to any one of statements 20 to 22, wherein R.sup.12 bears one to three substituent groups.

    [1663] 24. The conjugate according to any one of statements 20 to 23, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

    [1664] 25. The conjugate according to any one of statements 13 to 19, wherein there is a double bond between C2′ and C3′, and R.sup.12 is a C.sub.1-5 saturated aliphatic alkyl group.

    [1665] 26. A compound according to statement 25, wherein R.sup.12 is methyl, ethyl or propyl.

    [1666] 27. The conjugate according to any one of statements 13 to 19, wherein there is a double bond between C2′ and C3′, and R.sup.12 is a C.sub.3-6 saturated cycloalkyl group.

    [1667] 28. The conjugate according to statement 27, wherein R.sup.12 is cyclopropyl.

    [1668] 29. The conjugate according to any one of statements 13 to 19, wherein there is a double bond between C2′ and C3′, and R.sup.12 is a group of formula:

    ##STR00169##

    [1669] 30. The conjugate according to statement 29, wherein the total number of carbon atoms in the R.sup.12 group is no more than 4.

    [1670] 31. The conjugate according to statement 30, wherein the total number of carbon atoms in the R.sup.12 group is no more than 3.

    [1671] 32. The conjugate according to any one of statements 29 to 31, wherein one of R.sup.21, R.sup.22 and R.sup.23 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.

    [1672] 33. The conjugate according to any one of statements 29 to 31, wherein two of R.sup.21, R.sup.22 and R.sup.23 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.

    [1673] 34. The conjugate according to any one of statements 13 to 19, wherein there is a double bond between C2′ and C3′, and R.sup.12 is a group of formula:

    ##STR00170##

    [1674] 35. The conjugate according to statement 34, wherein R.sup.12 is the group:

    ##STR00171##

    [1675] 36. The conjugate according to any one of statements 13 to 19, wherein there is a double bond between C2′ and C3′, and R.sup.12 is a group of formula:

    ##STR00172##

    [1676] 37. The conjugate according to statement 36, wherein R.sup.24 is selected from H, methyl, ethyl, ethenyl and ethynyl.

    [1677] 38. The conjugate according to statement 37, wherein R.sup.24 is selected from H and methyl.

    [1678] 39. The conjugate according to any one of statements 13 to 19, wherein there is a single bond between C2′ and C3′, R.sup.12 is

    ##STR00173##

    and R.sup.26a and R.sup.26b are both H.

    [1679] 40. The conjugate according to any one of statements 13 to 19, wherein there is a single bond between C2′ and C3′, R.sup.12 is

    ##STR00174##

    and R.sup.26a and R.sup.26b are both methyl.

    [1680] 41. The conjugate according to any one of statements 13 to 19, wherein there is a single bond between C2′ and C3′, R.sup.12 is

    ##STR00175##

    one of R.sup.26a and R.sup.26b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.

    [Formula I]

    [1681] 42. The conjugate according to any one of statements 13 to 41, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.5-7 aryl group.

    [1682] 43. The conjugate according to statement 42, wherein R.sup.2 is phenyl.

    [1683] 44. The conjugate according to any one of statements 13 to 41, wherein there is a double bond between C2 and C3, and R.sup.1 is a C.sub.8-10 aryl group.

    [1684] 45. A compound according to any one of statements 42 to 44, wherein R.sup.2 bears one to three substituent groups.

    [1685] 46. The conjugate according to any one of statements 42 to 45, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

    [1686] 47. The conjugate according to any one of statements 13 to 41, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.1-5 saturated aliphatic alkyl group.

    [1687] 48. The conjugate according to statement 47, wherein R.sup.2 is methyl, ethyl or propyl.

    [1688] 49. The conjugate according to any one of statements 13 to 41, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.3-6 saturated cycloalkyl group.

    [1689] 50. The conjugate according to statement 49, wherein R.sup.2 is cyclopropyl.

    [1690] 51. The conjugate according to any one of statements 13 to 41, wherein there is a double bond between C2 and C3, and R.sup.2 is a group of formula:

    ##STR00176##

    [1691] 52. The conjugate according to statement 51, wherein the total number of carbon atoms in the R.sup.2 group is no more than 4.

    [1692] 53. The conjugate according to statement 52, wherein the total number of carbon atoms in the R.sup.2 group is no more than 3.

    [1693] 54. The conjugate according to any one of statements 51 to 53, wherein one of R.sup.11, R.sup.12 and R.sup.13 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.

    [1694] 55. The conjugate according to any one of statements 51 to 53, wherein two of R.sup.11, R.sup.12 and R.sup.13 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.

    [1695] 56. The conjugate according to any one of statements 13 to 41, wherein there is a double bond between C2 and C3, and R.sup.2 is a group of formula:

    ##STR00177##

    [1696] 57. The conjugate according to statement 56, wherein R.sup.2 is the group:

    ##STR00178##

    [1697] 58. The conjugate according to any one of statements 13 to 41, wherein there is a double bond between C2 and C3, and R.sup.2 is a group of formula:

    ##STR00179##

    [1698] 59. The conjugate according to statement 59, wherein R.sup.14 is selected from H, methyl, ethyl, ethenyl and ethynyl.

    [1699] 60. The conjugate according to statement 59, wherein R.sup.14 is selected from H and methyl.

    [1700] 61. The conjugate according to any one of statements 13 to 41, wherein there is a single bond between C2 and C3, R.sup.2 is

    ##STR00180##

    and R.sup.16a and R.sup.16b are both H.

    [1701] 62. The conjugate according to any one of statements 13 to 41, wherein there is a single bond between C2 and C3, R.sup.2 is

    ##STR00181##

    and R.sup.16a and R.sup.16b are both methyl.

    [1702] 63. The conjugate according to any one of statements 13 to 41, wherein there is a single bond between C2 and C3, R.sup.2 is

    ##STR00182##

    one of R.sup.16a and R.sup.16b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.

    [1703] 64. The conjugate according to any one of statements 13 to 63, wherein R.sup.11a is OH.

    [1704] 65. The conjugate according to any one of statements 13 to 64, wherein R.sup.21 is OH.

    [1705] 66. The conjugate according to any one of statements 13 to 64, wherein R.sup.21 is OMe.

    [1706] 67. The conjugate according to any one of statements 13 to 66, wherein R.sup.20 is H.

    [1707] 68. The conjugate according to any one of statements 13 to 66, wherein R.sup.20 is R.sup.C.

    [1708] 69. The conjugate according to statement 68, wherein R.sup.C is selected from the group consisting of: Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.

    [1709] 70. The conjugate according to statement 68, wherein R.sup.C is a group:

    ##STR00183## [1710] where the asterisk indicates the point of attachment to the N10 position, G.sup.2 is a terminating group, L.sup.3 is a covalent bond or a cleavable linker L.sup.1, L.sup.2 is a covalent bond or together with OC(═O) forms a self-immolative linker.

    [1711] 71. The conjugate according to statement 70, wherein G.sup.2 is Ac or Moc or is selected from the group consisting of: Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.

    [1712] 72. The conjugate according to any one of statements 1 to 64, wherein R.sup.20 and R.sup.21 together form a double bond between the nitrogen and carbon atoms to which they are bound.

    [Formula II]

    [1713] 73. The conjugate according to any one of statements 13 to 41, wherein R.sup.22 is of formula IIIa, and A is phenyl.

    [1714] 74. The conjugate according to any one of statements 13 to 41 and statement 73, wherein R.sup.22 is of formula IIa, and Q.sup.1 is a single bond.

    [1715] 75. The conjugate according to statement 73, wherein Q.sup.2 is a single bond.

    [1716] 76. The conjugate according to statement 73, wherein Q.sup.2 is —Z—(CH.sub.2).sub.n—, Z is O or S and n is 1 or 2.

    [1717] 77. The conjugate according any one of statements 13 to 41 and statement 73, wherein R.sup.22 is of formula IIIa, and Q.sup.1 is —CH═CH—.

    [1718] 78. The conjugate according to any one of statements 13 to 41, wherein R.sup.22 is of formula IIIb,

    [1719] and R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and methyl.

    [1720] 79. The conjugate according to statement 78, wherein R.sup.C1, R.sup.C2 and R.sup.C3 are all H.

    [1721] 80. The conjugate according to statement 78, wherein R.sup.C1, R.sup.C2 and R.sup.C3 are all methyl.

    [1722] 81. The conjugate according to any one of statements 13 to 41 and statements 73 to 80, wherein R.sup.22 is of formula IIIa or formula IIIb and X is selected from O—R.sup.L2′, S—R.sup.L2′, CO.sub.2—R.sup.L2′, —N—C(═O)—R.sup.L2′ and NH—R.sup.L2′.

    [1723] 82. The conjugate according to statement 81, wherein X is NH—R.sup.L2′.

    [1724] 83. The conjugate according to any one of statements 13 to 41, wherein R.sup.22 is of formula IIIc, and Q is NR.sup.N—R.sup.L2′.

    [1725] 84. The conjugate according to statement 83, wherein R.sup.N is H or methyl.

    [1726] 85. The conjugate according to any one of statements 13 to 41, wherein R.sup.22 is of formula IIIc, and Q is O—R.sup.L2′ or S—R.sup.L2′.

    [1727] 86. The conjugate according to any one of statements 13 to 41 and statements 73 to 85, wherein R.sup.11 is OH.

    [1728] 87. The conjugate according to any one of statements 13 to 41 and statements 73 to 85, wherein R.sup.11 is OMe.

    [1729] 88. The conjugate according to any one of statements 13 to 41 and statements 73 to 85, wherein R.sup.10 is H.

    [1730] 89. The conjugate according to any one of statements 13 to 41 and statements 73 to 85, wherein R.sup.10 and R.sup.11 together form a double bond between the nitrogen and carbon atoms to which they are bound.

    [1731] 90. The conjugate according to any one of statements 13 to 41 and statements 73 to 89, wherein R.sup.31 is OH.

    [1732] 91. The conjugate according to any one of statements 13 to 41 and statements 73 to 89, wherein R.sup.31 is OMe.

    [1733] 92. The conjugate according to any one of statements 13 to 41 and statements 73 to 91, wherein R.sup.30 is H.

    [1734] 93. The conjugate according to any one of statements 13 to 41 and statements 73 to 89, wherein R.sup.30 and R.sup.31 together form a double bond between the nitrogen and carbon atoms to which they are bound.

    [1735] 94. The conjugate according to any one of statements 13 to 93, wherein R.sup.6′, R.sup.7′, R.sup.9′, and Y′ are the same as R.sup.6, R.sup.7, R.sup.9, and Y.

    [1736] 95. The conjugate according to any one of statements 13 to 94 wherein, wherein L-R.sup.L1′ or L-R.sup.L2′ is a group:

    ##STR00184## [1737] where the asterisk indicates the point of attachment to the PBD, Ab is the antibody, L.sup.1 is a cleavable linker, A is a connecting group connecting L.sup.1 to the antibody, L.sup.2 is a covalent bond or together with —OC(═O)— forms a self-immolative linker.

    [1738] 96. The conjugate of statement 95, wherein L.sup.1 is enzyme cleavable.

    [1739] 97. The conjugate of statement 95 or statement 96, wherein L.sup.1 comprises a contiguous sequence of amino acids.

    [1740] 98. The conjugate of statement 97, wherein L.sup.1 comprises a dipeptide and the group —X.sub.1—X.sub.2— in dipeptide, —NH—X.sub.1—X.sub.2—CO—, is selected from: [1741] -Phe-Lys-, [1742] -Val-Ala-, [1743] -Val-Lys-, [1744] -Ala-Lys-, [1745] -Val-Cit-, [1746] -Phe-Cit-, [1747] -Leu-Cit-, [1748] -Ile-Cit-, [1749] -Phe-Arg-, [1750] -Trp-Cit-.

    [1751] 99. The conjugate according to statement 98, wherein the group —X.sub.1—X.sub.2— in dipeptide, —NH—X.sub.1—X.sub.2—CO—, is selected from: [1752] -Phe-Lys-, [1753] -Val-Ala-, [1754] -Val-Lys-, [1755] -Ala-Lys-, [1756] -Val-Cit-.

    [1757] 100. The conjugate according to statement 99, wherein the group —X.sub.1—X.sub.2— in dipeptide, —NH—X.sub.1—X.sub.2—CO—, is -Phe-Lys-, -Val-Ala- or -Val-Cit-.

    [1758] 101. The conjugate according to any one of statements 98 to 100, wherein the group X.sub.2—CO— is connected to L.sup.2.

    [1759] 102. The conjugate according to any one of statements 98 to 101, wherein the group NH—X.sub.1— is connected to A.

    [1760] 103. The conjugate according to any one of statements 98 to 102, wherein L.sup.2 together with OC(═O) forms a self-immolative linker.

    [1761] 104. The conjugate according to statement 102, wherein C(═O)O and L.sup.2 together form the group:

    ##STR00185## [1762] where the asterisk indicates the point of attachment to the PBD, the wavy line indicates the point of attachment to the linker L.sup.1, Y is NH, O, C(═O)NH or C(═O)O, and n is 0 to 3.

    [1763] 105. The conjugate according to statement 104, wherein Y is NH.

    [1764] 106. The conjugate according to statement 104 or statement 105, wherein n is 0.

    [1765] 107. The conjugate according to statement 105, wherein L.sup.1 and L.sup.2 together with —OC(═O)— comprise a group selected from:

    ##STR00186## [1766] where the asterisk indicates the point of attachment to the PBD, and the wavy line indicates the point of attachment to the remaining portion of the linker L.sup.1 or the point of attachment to A.

    [1767] 108. The conjugate according to statement 107, wherein the wavy line indicates the point of attachment to A.

    [1768] 109. The conjugate according to any one of statements 95 to 108, wherein A is:

    ##STR00187## [1769] where the asterisk indicates the point of attachment to L.sup.1, the wavy line indicates the point of attachment to the antibody, and n is 0 to 6; or

    ##STR00188## [1770] where the asterisk indicates the point of attachment to L.sup.1, the wavy line indicates the point of attachment to the antibody, n is 0 or 1, and m is 0 to 30.

    [1771] 110. A conjugate according to statement 13 of formula

    ##STR00189## ##STR00190## ##STR00191##

    [1772] 111. The drug-conjugate according to any one of statements 13 to 110 wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1 to about 8.

    [1773] 112. The drug-conjugate according to statement 111 wherein p is 1, 2, 3, or 4.

    [1774] 113. The drug-conjugate according to statement 111 comprising a mixture of the antibody-drug conjugate compounds, wherein the average drug loading per antibody in the mixture of antibody-drug conjugate compounds is about 2 to about 5.

    [1775] 114. The drug-conjugate according to any one of statements 13 to 113, for use in therapy.

    [1776] 115. The drug-conjugate according to any one of statements 13 to 113, for use in the treatment of a proliferative disease in a subject.

    [1777] 116. The drug-conjugate according to any one of statements 13 to 113, for use in the treatment of a proliferative disease in a subject, wherein the subject has raised levels of Tn-MUC1, CA 27.29, or CA 15-3 and wherein the method comprises identifying that the subject has raised levels of Tn-MUC1, CA 27.29, or CA 15-3 and administering the conjugate to the patient.

    [1778] 117. The drug-conjugate according to any one of statements 13 to 113, for use in the treatment of a proliferative disease in a subject, wherein the proliferative disease is associated with raised levels of Tn-MUC1, CA 27.29, or CA 15-3, the method comprising administering the conjugate to the patient.

    [1779] 118. The drug-conjugate according to any one of statements 115 to 117, wherein the disease is cancer.

    [1780] 119. A pharmaceutical composition comprising the drug-conjugate of any one of statements 13 to 113 and a pharmaceutically acceptable diluent, carrier or excipient.

    [1781] 120. The pharmaceutical composition of statement 119 further comprising a therapeutically effective amount of a chemotherapeutic agent.

    [1782] 121. Use of a drug-conjugate according to any one of statements 13 to 113 in the preparation of a medicament for use in the treatment of a proliferative disease in a subject.

    [1783] 122 A method of treating cancer comprising administering to a patient the pharmaceutical composition according to either one of statements 119 or 120.

    [1784] 123. The method of statement 122 wherein the patient is administered a chemotherapeutic agent, in combination with the drug-conjugate.

    [1785] 124. A polynucleotide encoding a humanized antibody according to any one of statements 1 to 12.

    [1786] 125. A vector comprising the polynucleotide of statement 124.

    [1787] 126. The vector of statement 125 wherein the vector is an expression vector.

    [1788] 127. A host cell comprising a vector according to either one of statements 125 or 126.

    [1789] 128. The host cell according to statement 127 wherein the host cell is prokaryotic, eukaryotic, or mammalian.

    [1790] 129. A conjugate comprising the humanized antibody according to any one of statements 1 to 12 coupled to a functional moiety.

    [1791] 130. The conjugate according to statement 129, wherein the functional moiety is selected from a drug, a reporter, a toxin, an organic moiety, and a binding member.

    [1792] 131. The conjugate according to statement 130 wherein the reporter is a fluorescent compound, a radionuclide, or an enzyme.

    [1793] 132. The conjugate according to statement 130 wherein the binding member is an antibody or antibody fragment.

    [1794] 133. The conjugate according to any one of statements 129 to 133, wherein the humanized antibody is covalently bonded to the functional moiety.

    [1795] 134. A method of selecting an individual for treatment with the drug-conjugate according to any one of statements 13 to 113, or with the pharmaceutical composition of either one of statements 119 or 120, which method comprises assessing the level of TnMUC1; [1796] wherein individuals having TnMUC1 are selected for treatment.

    [1797] 135. A method of timing the application of treatment of an individual with the drug-conjugate according to any one of statements 13 to 113, or with the pharmaceutical composition of either one of statements 119 or 120, which method comprises assessing the level of TnMUC1; [1798] wherein the treatment is applied if the individual has TnMUC1.

    [1799] 136. The method according to either one of statements 134 or 135, wherein the individual has cancer and treatment reduces tumour volume.

    TABLE-US-00005 SEQUENCES  SEQ ID NO: 1 [5E5VH] QVQLQQSDAELVKPGSSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGH FSPGNTDIKYNDKFKGKATLTVDRSSSTAYMQLNSLTSEDSAVYFCKTST FFFDYWGQGTTLTVSS  SEQ ID NO: 2 [5E5RHA] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEVVMG  HFSPGNTDIKYNDKFKGRVTITRDRSMSTAYMELSSLRSEDTAMYYCATS TFFFDYWGQGTMVTVSS  SEQ ID NO: 3 [5E5RHA2] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEWIGH  FSPGNTDIKYNDKFKGRATLTVDRSMSTAYMELSSLRSEDTAMYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 4 [5E5RHA3] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEVVMG  HFSPGNTDIKYNDKFKGRATLTVDRSMSTAYMELSSLRSEDTAMYYCKTS TFFFDYWGQGTMVTVSS  SEQ ID NO: 5 [5E5RHA4] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEWIGH FSPGNTDIKYNDKFKGRVTLTVDRSMSTAYMELSSLRSEDTAMYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 6 [5E5RHA5] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEWIGH  FSPGNTDIKYNDKFKGRATITVDRSMSTAYMELSSLRSEDTAMYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 7 [5E5RHA6] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEWIGH FSPGNTDIKYNDKFKGRATLTRDRSMSTAYMELSSLRSEDTAMYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 8 [5E5RHA7] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEWIGH FSPGNTDIKYNDKFKGRATLTVDRSMSTAYMELSSLRSEDTAMYYCATST FFFDYWGQGTMVTVSS  SEQ ID NO: 9 [5E5RHA8] QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHWVRQAPGQALEVVMG HFSPGNTDIKYNDKFKGRVTLTVDRSMSTAYMELSSLRSEDTAMYYCKTS TFFFDYWGQGTMVTVSS  SEQ ID NO: 10 [5E5RHB] EVQLVQSGAEVKKPGESLKISCKISGYIFTDHAIHWVRQMPGKGLEVVMG HFSPGNTDIKYNDKFKGQVTFSVDRSINTAYLQWSSLKASDTAIYFCARS TFFFDYWGQGTRVTVSS  SEQ ID NO: 11 [5E5RHB2] EVQLVQSGAEVKKPGESLKISCKISGYTFTDHAIHWVRQMPGKGLEWIGH  FSPGNTDIKYNDKFKGQATLSVDRSINTAYLQWSSLKASDTAIYFCKTST FFFDYWGQGTRVTVSS  SEQ ID NO: 12 [5E5RHB3] EVQLVQSGAEVKKPGESLKISCKISGYIFTDHAIHWVRQMPGKGLEWIGH  FSPGNTDIKYNDKFKGQATLSVDRSINTAYLQWSSLKASDTAIYFCKTST FFFDYWGQGTRVTVSS  SEQ ID NO: 13 [5E5RHB4] EVQLVQSGAEVKKPGESLKISCKISGYTFTDHAIHWVRQMPGKGLEWMGH  FSPGNTDIKYNDKFKGQATLSVDRSINTAYLQWSSLKASDTAIYFCKTST FFFDYWGQGTRVTVSS  SEQ ID NO: 14 [5E5RHB5] EVQLVQSGAEVKKPGESLKISCKISGYTFTDHAIHWVRQMPGKGLEWIGH  FSPGNTDIKYNDKFKGQVTLSVDRSINTAYLQWSSLKASDTAIYFCKTST FFFDYWGQGTRVTVSS  SEQ ID NO: 15 [5E5RHB6] EVQLVQSGAEVKKPGESLKISCKISGYTFTDHAIHWVRQMPGKGLEWIGH  FSPGNTDIKYNDKFKGQATFSVDRSINTAYLQWSSLKASDTAIYFCKTST FFFDYWGQGTRVTVSS  SEQ ID NO: 16 [5E5RHB7] EVQLVQSGAEVKKPGESLKISCKISGYTFTDHAIHWVRQMPGKGLEWIGH  FSPGNTDIKYNDKFKGQATLSVDRSINTAYLQWSSLKASDTAIYFCATST FFFDYWGQGTRVTVSS  SEQ ID NO: 17 [5E5RHB8] EVQLVQSGAEVKKPGESLKISCKISGYTFTDHAIHWVRQMPGKGLEWIGH  FSPGNTDIKYNDKFKGQATLSVDRSINTAYLQWSSLKASDTAIYFCKRST FFFDYWGQGTRVTVSS  SEQ ID NO: 18 [5E5RHB9] EVQLVQSGAEVKKPGESLKISCKISGYIFTDHAIHWVRQMPGKGLEVVMG  HFSPGNTDIKYNDKFKGQATLSVDRSINTAYLQWSSLKASDTAIYFCKTS TFFFDYWGQGTRVTVSS  SEQ ID NO: 19 [5E5RHB10] EVQLVQSGAEVKKPGESLKISCKISGYIFTDHAIHWVRQMPGKGLEVVMG  HFSPGNTDIKYNDKFKGQVTLSVDRSINTAYLQWSSLKASDTAIYFCKTS TFFFDYWGQGTRVTVSS  SEQ ID NO: 20 [5E5RHC] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWMGH  FSPGNTDIKYNDKFKGRVTMTRDTSINTAYMELRRLRSDDTAVYYCATST FFFDYWGQGTMVTVSS  SEQ ID NO: 21 [5E5RHC2] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWIGH  FSPGNTDIKYNDKFKGRATLTVDRSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 22 [5E5RHC3] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWMGH  FSPGNTDIKYNDKFKGRATLTVDRSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 23 [5E5RHC4] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWIGH  FSPGNTDIKYNDKFKGRVTLTVDRSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 24 [5E5RHC5] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWIGH  FSPGNTDIKYNDKFKGRATMTVDRSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 25 [5E5RHC6] FSPGNTDIKYEVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQA PGQGLEWIGHNDKFKGRATLTRDRSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 26 [5E5RHC7] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWIGH  FSPGNTDIKYNDKFKGRATLTVDTSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 27 [5E5RHC8] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWIGH  FSPGNTDIKYNDKFKGRATLTVDRSINTAYMELRRLRSDDTAVYYCATST FFFDYWGQGTMVTVSS  SEQ ID NO: 28 [5E5RHC9] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWMGH  FSPGNTDIKYNDKFKGRVTMTVDRSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 29 [5E5RHC9 + V67A] EVQLVESGAEVKKPGASVEVSCQASGYTFTDHAIHWVRQAPGQGLEWMGH  FSPGNTDIKYNDKFKGRATMTVDRSINTAYMELRRLRSDDTAVYYCKTST FFFDYWGQGTMVTVSS  SEQ ID NO: 30 [5E5VK] DIVMTQSPSSLTVTAGEKVTMICKSSQSLLNSGDQKNYLTWYQQKPGQPP  KLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSY  PLTFGAGTKLELK SEQ ID NO: 31 [5E5RKA] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGDQKNYLTWYQQKPGQPP  KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY  PLTFGQGTKVEIK SEQ ID NO: 32 [5E5RKA1] DIVMTQSPDSLAVSLGERATIICKSSQSLLNSGDQKNYLTWYQQKPGQPP  KLLIFWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY  PLTFGQGTKVEIK SEQ ID NO: 33 [5E5RKB] DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGDQKNYLTWYLQKPGQSP  QLLIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQNDYSY PLTFGGGTKVEIK  SEQ ID NO: 34 [5E5RKB1] DIVMTQSPLSLPVTPGEPASIICKSSQSLLNSGDQKNYLTWYLQKPGQSP  QLLIFWASTRESGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCQNDYSY  PLTFGGGTKVEIK SEQ ID NO. 160 (IgG1 CH3):  GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN  YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK  SEQ ID NO. 161 (IgG2 CH3):  GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENN  YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS  LSLSPGK SEQ ID NO. 162 (IgG3 CH3):  GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENN  YNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKS  LSLSPGK SEQ ID NO. 163 (IgG4 CH3):  GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN  YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS  LSLSLGK SEQ ID NO. 164 (IgG1m CH3):  GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN  YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK  SEQ ID NO. 165 (IgG1m[non-1] CH3):  GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN  YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK  SEQ ID NO. 166 (IgG1 CH3 S.fwdarw.4C):  GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LCLSPGK  SEQ ID NO. 167 (IgG2 CH3 S.fwdarw.C):  GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENN  YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS  LCLSPGK SEQ ID NO. 168 (IgG3 CH3 S.fwdarw.C):  GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENN  YNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKS  LCLSPGK SEQ ID NO. 169 (IgG4 CH3 S.fwdarw.C):  GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LCLSLGK  SEQ ID NO. 170 (IgG1 m CH3 S.fwdarw.C):  GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LCLSPGK  SEQ ID NO. 171 (IgG1 m[non-1] CH3 S.fwdarw.C):  GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LCLSPGK  SEQ ID NO. 180 (IgG1 Fc):  DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK  SEQ ID NO. 181 (IgG2 Fc):  KCCVECPPCPAPPVAG-PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED  PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK  SEQ ID NO. 182 (IgG3 Fc):  DTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED  PEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRFTQKSLSLSPGK  SEQ ID NO. 183 (IgG4 Fc):  KYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED  PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK  SEQ ID NO. 184 (IgG1m Fc):  DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED  VPEVKFNWYVDGEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK  SEQ ID NO. 185 (IgG1m[non-1] Fc):  DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK  SEQ ID NO. 186 (IgG1 Fc S.fwdarw.C):  DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED  PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLCLSPGK  SEQ ID NO. 187 (IgG2 Fc S.fwdarw.C):  KCCVECPPCPAPPVAG-PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLCLSPGK  SEQ ID NO. 188 (IgG3 Fc S.fwdarw.C):  DTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED  PEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRFTQKSLCLSPGK  SEQ ID NO. 189 (IgG4 Fc S.fwdarw.C):  KYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED  PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLCLSLGK  SEQ ID NO. 190 (IgGlm Fc S.fwdarw.C):  DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEDP  EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLCLSPGK  SEQ ID NO. 191 (IgG1m[non-1] Fc S.fwdarw.C):  DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLCLSPGK