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ANTHRACYCLINS AND CONJUGATES THEREOF

20250312474 · 2025-10-09

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention concerns analogues of nemorubicin as well as PNU-159,682 with a range of substituents other than 2-OMe on the morpholino ring that beneficially affected the toxicity of the toxin over the molecules with the 2-OMe group. In addition, it was found that PNU variants with modified 2-O-alkyl chain show enhanced tolerability in vivo. Thus, by modification of the 2-O-alkyl group, ADCs were generated with carefully tailored potency and tolerability to improve the administered dose in patients. The invention thus concerns compounds according to structure (1) and conjugates therewith, as well as pharmaceutical compositions and methods of targeting tumour cells and treating cancer.

    ##STR00001##

    Claims

    1. A conjugate, comprising a compound according to structure (1) conjugated to a cell-binding agent via a linker, wherein structure (1) is as follows: ##STR00156## wherein: R.sup.1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C.sub.6-15 alkyl, C.sub.2-15 alkenyl, C.sub.2-15 alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, SpX.sup.2R.sup.4, SpN.sub.3, SpX.sup.2SpR.sup.12, or SpN(R.sup.4).sub.2, wherein the optional substituent is selected from a halogen, C.sub.1-12 (hetero)alkyl, (hetero)aryl, C.sub.2-15 alkenyl, C.sub.2-15 alkynyl, X.sup.2R.sup.4, N(R.sup.4).sub.2, or NO.sub.2, and wherein substituents C.sub.1-12 (hetero)alkyl and (hetero)aryl may optionally be further substituted with C.sub.1-6 (hetero)alkyl, X.sup.2R.sup.4 and N(R.sup.4).sub.2; wherein each Sp is individually C.sub.1-12 (hetero)alkylene, (hetero)arylene, C.sub.1-12 (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C.sub.1-12 (hetero)alkylene, wherein the (hetero)alkylene or the (hetero)arylene is optionally substituted with one or more substituents selected from a halogen, X.sup.2R.sup.4, N(R.sup.4).sub.2, C.sub.1-4 alkyl, and NO.sub.2, wherein each R.sup.4 is individually H, C.sub.1-4 alkyl, or adamantly, and X.sup.2 is C(O), C(O)O, C(O)NH, O, S, S(O), S(O).sub.2, S(O)NH, or S(O).sub.2NH, and wherein R.sup.12 is -glucuronide acid, PO.sub.3.sup.(2), OPO.sub.3.sup.(2), CO.sub.2.sup.(), SO.sub.3.sup.(), or N(C.sub.1-4 alkyl).sub.3.sup.(+); R.sup.2 is H, S(O).sub.2OH, or P(O).sub.2OH, and R.sup.3 is OH, or R.sup.2 and R.sup.3 are fused together via an ether moiety to form an oxazolidine ring; R.sup.5 is H or OCH.sub.3; N % is Nor N.fwdarw.O; Y.sup.5 is CH.sub.2Y, C(O)Y, C(N(R.sup.20))Y, or C(R.sup.9)NY, wherein R.sup.9 is selected from C.sub.1-4 alkyl optionally substituted with an OH or O(CO)C.sub.1-6 alkyl group, and R.sup.20 is NR.sup.4C(O)N(R.sup.4).sub.2, NR.sup.4C(O)SpN(R.sup.4).sub.2, NR.sup.4C(O)R.sup.12, or NR.sup.4C(O)SpR.sup.12, wherein Sp, R.sup.4, and R.sup.12 are as defined above; the compound is connected to the linker via Y, or a salt thereof, wherein each ion if present is balanced with one or more pharmaceutically acceptable counter-ions.

    2. The conjugate according to claim 1, having the structure (2) ##STR00157## wherein: CB is the cell-binding agent; D is the compound according to structure (1); L is a linker; Z.sup.1 is a connecting group that connects the cell-binding agent CB to the linker; and Z.sup.2 is a connecting group that connects the compound D to the linker.

    3. The conjugate according to claim 2, wherein the connecting group Z.sup.1 is formed by a conjugation reaction selected from amide bond formation, carbamate bond formation, thiol alkylation, thiol arylation, and cycloaddition reaction.

    4. The conjugate according to claim 2, wherein the connecting group Z.sup.1 is connected to the cell-binding agent CB via a lysine residue of CB, a glutamine residue of CB, a threonine residue of CB, a cysteine residue of CB, a tyrosine residue of CB, or a glycan of CB.

    5. The conjugate according to claim 2, wherein the connecting group Z.sup.2 is an amide moiety, an ester moiety, a thioether moiety, an ether moiety, a carbamate moiety, a [2.2.2]bicyclic structure, a [2.2.1]bicyclic structure, a disulfide, a carbonate moiety, or a (hetero)aryl moiety.

    6. The conjugate according to claim 2, wherein L-Z.sup.2 has the structure: ##STR00158## wherein: the bond labelled with * is connected to the C(O) moiety adjacent to Y of the compound according to structure (1); the bond labelled with ** is connected to connecting group Z.sup.1; Sp.sup.3 is a is C.sub.1-12 (hetero)alkylene, (hetero)arylene, C.sub.1-12 alkylene-(hetero)arylene, or (hetero)arylene-C.sub.1-12 alkylene, wherein the alkylene or the (hetero)arylene may be optionally substituted with one or more substituents selected from a halogen, X.sup.2R.sup.4, N(R.sup.4).sub.2, C.sub.1-4 alkyl, or NO.sub.2, wherein the C.sub.1-4 alkyl substituent may optionally form a cyclic structure by being joined with an NR.sup.4 moiety, and the alkylene may optionally be interrupted with one or more heteroatoms selected from X.sup.2 and NR.sup.4; R.sup.4 and X.sup.2 are as defined in claim 1; L.sup.1, L.sup.2, and L.sup.3 are each individually linkers that together link Z.sup.1 to D; n, o, and p are each individually 0 or 1, provided that n+o+p=1, 2, or 3.

    7. The conjugate according to claim 1, wherein the cell-binding agent is an antibody, a peptide, a small molecule, or an aptamer.

    8. The conjugate according to claim 1, wherein R.sup.1 is selected from Et, i-Pr, t-Bu, Bz, Bn, SpN.sub.3, or SpNH.sub.2, wherein Sp is C.sub.1-4 alkylene or C.sub.1-4 alkylene-arylene.

    9. The conjugate according to claim 1, wherein R.sup.2 and R.sup.3 are joined together via an ether moiety to form an oxazolidine ring.

    10. The conjugate according to claim 1, wherein R.sup.1 is not CH.sub.2CH.sub.2SH, unsubstituted ethyl, or benzyl.

    11. A compound according to structure (1): ##STR00159## wherein: R.sup.1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C.sub.6-15 alkyl, C.sub.2-15 alkenyl, C.sub.2-15 alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, SpX.sup.2R.sup.4, SpN.sub.3, SpX.sup.2SpR.sup.12, or SpN(R.sup.4).sub.2, wherein the optional substituent is selected from a halogen, C.sub.1-12 (hetero)alkyl, (hetero)aryl, C.sub.2-15 alkenyl, C.sub.2-15 alkynyl, X.sup.2R.sup.4, N(R.sup.4).sub.2, or NO.sub.2, and wherein substituents C.sub.1-12 (hetero)alkyl and (hetero)aryl may optionally be further substituted with C.sub.1-6 (hetero)alkyl, X.sup.2R.sup.4, and N(R.sup.4).sub.2; wherein each Sp is individually C.sub.1-12 (hetero)alkylene, (hetero)arylene, C.sub.1-12 (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C.sub.1-12 (hetero)alkylene, wherein the (hetero)alkylene or the (hetero)arylene is optionally substituted with one or more substituents selected from halogen, X.sup.2R.sup.4, N(R.sup.4).sub.2, C.sub.1-4 alkyl, and NO.sub.2, wherein each R.sup.4 is individually H, C.sub.1-4 alkyl, or adamantly, and X.sup.2 is C(O), C(O)O, C(O)NH, O, S, S(O), S(O).sub.2, S(O)NH, or S(O).sub.2NH, and wherein R.sup.12 is -glucuronide acid, PO.sub.3.sup.(2), OPO.sub.3.sup.(2), CO.sub.2.sup.(), SO.sub.3.sup.() or N(C.sub.1-4 alkyl).sub.3.sup.(+); R.sup.2 is H, S(O).sub.2OH, or P(O).sub.2OH, and R.sup.3 is OH, or R.sup.2 and R.sup.3 are fused together via an ether moiety to form an oxazolidine ring; R.sup.5 is H or OCH.sub.3; N % is N or N.fwdarw.O; Y.sup.5 is CH.sub.2Y, C(O)Y, C(N(R.sup.20))Y, C(R.sup.9)NY, or C(R.sup.9)N(R.sup.20), wherein R.sup.9 is selected from C.sub.1-4 alkyl optionally substituted with an OH or O(CO)C.sub.1-6 alkyl group, and R.sup.20 is NR.sup.4C(O)N(R.sup.4).sub.2, NR.sup.4C(O)SpN(R.sup.4).sub.2, NR.sup.4C(O)R.sup.12, or NR.sup.4C(O)SpR.sup.12, wherein Sp, R.sup.4, and R.sup.12 are as defined above; Y is NR.sup.4Sp.sup.3N(R.sup.4).sub.2, NR.sup.4Sp.sup.3X.sup.2(R.sup.4), N(R.sup.4).sub.2, CH.sub.3, R.sup.12, Sp.sup.3R.sup.12, NR.sup.4Sp.sup.3X.sup.2Sp.sup.3R.sup.12, OH, or CH.sub.2OH, wherein each Sp.sup.3 is a spacer; and R.sup.1 is not unsubstituted ethyl, CH.sub.2CH.sub.2SH, or benzyl when Y.sup.5 is C(O)CH.sub.2OH; or a salt thereof, wherein each ion if present is balanced with one or more pharmaceutically acceptable counter-ions.

    12. The compound according to claim 11, wherein: R.sup.1 is selected from i-Pr, t-Bu, Bn, SpN.sub.3, or SpNH.sub.2; and Sp is C.sub.1-4 alkylene or C.sub.1-4 alkylene-arylene.

    13. The compound according to claim 11, wherein R.sup.2 and R.sup.3 are joined together via an ether moiety to form an oxazolidine ring.

    14. The compound according to claim 11, wherein Y is CH.sub.2OH.

    15. The compound according to claim 11, wherein N.sup.% is N.

    16. A conjugate, wherein a compound according to claim 15 is conjugated to a cell-binding agent via a linker.

    17. The conjugate according to claim 16, wherein the compound according to structure (1) is conjugated to the cell-binding agent through: (i) R.sup.1; or (ii) Y.

    18. The conjugate according to claim 16, having structure (2) as defined in claim 2, wherein L-Z.sup.2 has a structure selected from (L1)-(L4): ##STR00160## wherein: the bond labelled with * is connected: (a) for (L1) and (L2) to the C(O) moiety adjacent to Y of the compound according to structure (1), and (b) for (L3) and (L4) to the O atom of the OR.sup.1 moiety of the compound according to structure (1); the bond labelled with ** is connected to the cell-binding agent; R.sup.13 is selected from the group consisting of hydrogen, C.sub.1-C.sub.24 alkyl groups, C.sub.3-C.sub.24 cycloalkyl groups, C.sub.2-C.sub.24 (hetero)aryl groups, C.sub.3-C.sub.24 alkyl(hetero)aryl groups, and C.sub.3-C.sub.24 (hetero)arylalkyl groups, wherein the C.sub.1-C.sub.24 alkyl groups, C.sub.3-C.sub.24 cycloalkyl groups, C.sub.2-C.sub.24 (hetero)aryl groups, C.sub.3-C.sub.24 alkyl(hetero)aryl groups, and C.sub.3-C.sub.24 (hetero)arylalkyl groups are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S, and NR.sup.14, wherein R.sup.14 is independently selected from the group consisting of hydrogen and C.sub.1-C.sub.4 alkyl groups, or R.sup.13 is D connected to N optionally via a spacer moiety, or R.sup.13 is connected to elsewhere in the linker, optionally via a spacer moiety, to form a cyclic structure; L.sup.2 is a dipeptide, a tripeptide, or a tetrapeptide; is 0 or 1; ring A is an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring; z1 is an integer in the range of 1-4; and z2 is 0 or 1.

    19. A pharmaceutical composition comprising: the conjugate according to claim 1; and a pharmaceutically acceptable carrier.

    20. A method for targeting a tumour cell expressing a specific extracellular receptor, the method comprising: contacting the conjugate according to claim 1 with cells that may possibly express the extracellular receptor, wherein the antibody specifically targets the extracellular receptor.

    21. A method for treating cancer, the method comprising: administering to a subject in need thereof the conjugate according to claim 1, wherein the cancer cells specifically express an extracellular receptor.

    22. The method according to claim 20, wherein the extracellular receptor is selected from the group consisting of 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, +ASPHD1, av-integrin, Ax1, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, Brevican, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138/syndecan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD38, CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, fibronectin, FLT3, folate receptor alpha, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanyl cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Ra, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA/B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, nectin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinases (RTK), and tenascin.

    Description

    DESCRIPTION OF THE FIGURES

    [0477] FIG. 1 shows the general scheme for preparation of antibody-drug conjugates by reaction of a monoclonal antibody (in most cases a symmetrical dimer) containing an x number of functionalities F. By incubation of antibody-(F) x with excess of a linker-drug construct (Q-spacer-linker-payload) a conjugate is obtained by reaction of F with Q, forming connecting group Z.

    [0478] FIG. 2 depicts a range of reagents suitable for reaction with cysteine side-chains. Reagents may be monoalkylation type (A) or may be a cross-linker (B) for reaction with two cysteine side-chains.

    [0479] FIG. 3 shows the general process for non-genetic conversion of a monoclonal antibody (mAb) into an antibody containing probes for click conjugation (F). The click probe may be on various positions in the antibody, depending on the technology employed. For example, the antibody may be converted into an antibody containing two click probes (structure on the left) or four click probes (bottom structure) or eight probes (structure on the right) for click conjugation.

    [0480] FIG. 4 shows a representative (but not comprehensive) set of functional groups (F) that can be introduced into an antibody by engineering, by chemical modification, or by enzymatic means, which upon metal-free click reaction with a complementary reactive group Q lead to connecting group Z. Functional group F may be artificially introduced (engineered) into an antibody at any position of choice. Some functional groups F (e.g. nitrile oxide, quinone), may besides strained alkynes also react with strained alkenes, which as an example is depicted for triazine or tetrazine (bottom line). The pyridine or pyridazine connecting group is the product of the rearrangement of the tetrazabicyclo[2.2.2]octane connecting group, formed upon reaction of triazine or tetrazine with alkyne (but not alkene), respectively, with loss of N.sub.2. Connecting groups Z depicted in FIG. 4 are preferred connecting groups to be used in the present invention.

    [0481] FIG. 5 shows cyclic alkynes suitable for metal-free click chemistry, and preferred embodiments for reactive moiety Q. The list is not comprehensive, for example alkynes can be further activated by fluorination, by substitution of the aromatic rings or by introduction of heteroatoms in the aromatic ring.

    [0482] FIG. 6 depicts a specific example of site-specific conjugation of a payload based on glycan remodeling of a full-length IgG followed by azide-cyclooctyne click chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of azido-sugar onto the core GlcNAc liberated by endoglycosidase. In the next step, the azido-remodeled IgG is subjected to an immune cell-engaging polypeptide, which has been modified with a single cyclooctyne for metal-free click chemistry (SPAAC), leading to a bispecific antibody of 2:2 molecular format. It is also depicted that the cyclooctyne-polypeptide construct will have a specific spacer between cyclooctyne and polypeptide, which enables tailoring of IgG-polypeptide distance or impart other properties onto the resulting bispecific antibody.

    [0483] FIG. 7 depicts a specific example of site-specific conjugation of a payload based on glycan remodeling of a full-length IgG followed by thiol alkylation chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of a thiol-modified (and disulfide-protected) sugar derivative onto the core GlcNAc liberated by endoglycosidase. In the next step, the remodeled IgG is subjected to reduction (to convert the disulfide into thiol), potentially followed by oxidation, then reaction with a payload modified with a suitable thiol-reactive reagent.

    [0484] FIG. 8 depicts the structures of daunorubicin, doxorubicin, nemorubicin (MMDX), PNU-159,696 and PNU-159,682.

    [0485] FIG. 9 depicts two linker-modified PNU-159,682 derivatives, one based on carbamoylation of the hydroxyketone group with a linker containing N,N-dimethylethylenediamine (DMEDA) and maleimide for antibody conjugation to cysteine, the other based on oxidation-amide coupling of the hydroxyketone with a linker containing ethylenediamine (EDA) and glycine-glycine for antibody conjugation under the action of sortase.

    [0486] FIG. 10 shows an ADC obtained by sortase-mediated conjugation of glycine-glycine-EDA-modified oxidized PNU-159,682.

    [0487] FIG. 11 shows the structure of linker-drugs based on PNU-159,682 analogues according to the invention, which can be applied for conjugation to antibodies through reactive moiety Z to generate the corresponding ADCs. Class 1 consists of a PNU-analogue modified at the morpholino ring with a substituent that is different from the methyl group present in PNU-159,682, and has the original hydroxyacetone moiety part oxidized to a carboxylic acid to enable activation/attachment of a linker. Class 2 consists of a PNU-analogue that has the original (hydroxy) acetone moiety of doxorubicin/daunorubicin retained and is modified with a linker at the position of the original methyl group present on the morpholino group of PNU-159,682. In either case, the linker is further modified with the reactive group Z, which can be any functionality that enables attachment to an antibody, e.g. a maleimide, an activated carbonyl, a halogenide, a cycloalkyne, an azide, etc.

    [0488] FIG. 12 shows the synthetic scheme to generate PNU-159,682 analogues 6b-6f with modification at the morpholino ring based on initial TBS-protection of the hydroxyacetone function of doxorubicin,

    [0489] FIG. 13 shows how N-alkylation of the aminosugar of doxorubicin can be achieved for various constructs 8b-8f without prior O-silylation of doxorubicin. This route is also applicable to daunorubicin.

    [0490] FIG. 14A shows the structure of compound 9a based on Val-Cit dipeptide and DMEDA linker.

    [0491] FIG. 14B shows the final step in the preparation of compounds 9c, 9d, 9f and 9g with Val-Ala dipeptide and EDA linker.

    [0492] FIG. 15 shows the structures of compounds 36 and 39 with Val-Ala dipeptide and conjugation through the anthracycline morpholino group.

    [0493] FIG. 16 shows the structures of compounds 47 and 53 based on EDA linkers and Gly-Gly-Phe-Gly or Gly-Gly-Gly peptides, respectively.

    [0494] FIG. 17 shows the in vitro cytotoxicity of trast-9g, trast-9d, trast-9c and trast-36 on four cell lines with variable HER2 expression levels. The To line indicates the number of viable cells at the start of the assay.

    [0495] FIG. 18A shows the time-dependent average body weight of CD-1 mice administered a single bolus of vehicle (PBS), ADC trastuzumab-9d (20 mg/kg), ADC trastuzumab-36 (20 mg/kg) or reference ADC trastuzumab-9g (5 mg/kg).

    [0496] FIG. 18B shows the time-dependent body weight of CD-1 mice administered a single bolus of vehicle (PBS), ADC trastuzumab-47 (15 mg/kg), ADC trastuzumab-9c (40 mg/kg) or reference ADC trastuzumab-9g (5 mg/kg).

    [0497] FIG. 19A shows the tumor volume over time of NOD/SCID mice grafted with JIMT-1 tumor cell line followed by treatment with reference ADC trastuzumab-9g at low (0.3 mg/kg) or high (1 mg/kg) dose.

    [0498] FIG. 19B shows the tumor volume over time of NOD/SCID mice grafted with JIMT-1 tumor cell line followed by treatment with ADC trastuzumab-9d at low (3 mg/kg) or high (5 mg/kg) dose or ADC trastuzumab-36 at low (0.6 mg/kg) or high (2 mg/kg) dose.

    [0499] FIG. 19C shows the tumor volume over time of NOD/SCID mice grafted with JIMT-1 tumor cell line followed by treatment with ADC trastuzumab-47 at low (0.6 mg/kg) or high (2 mg/kg) dose or ADC trastuzumab-9c at high dose (2 mg/kg).

    [0500] FIG. 20 shows the in vitro cytotoxicity of compounds 6a, 6b, 6c, 6d and 6e on four cell lines with variable HER2 expression levels. The To line indicates the number of viable cells at the start of the assay.

    [0501] FIG. 21 shows the in vitro cytotoxicity of trast-9g, trast-63a, trast-63b and trast-75 on three cell lines with variable HER2 expression levels. The To line indicates the number of viable cells at the start of the assay.

    EXAMPLES

    [0502] The invention is illustrated by the following examples.

    General Procedure for Analytical RP-UPLC

    [0503] Prior to RP-UPLC analysis, IgG (10 L, 1 mg/mL in PBS pH 7.4) was added to 12.5 mM DTT, 100 mM TrisHCl pH 8.0 (40 L) and incubated for 15 minutes at 37 C. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 L). RP-UPLC analysis was performed on an H-class Acquity UPLC system (Waters). The sample (5 L) was injected with 0.4 mL/min onto a BioResolve RP mAb Polyphenyl column (450 , 2.7 m, 2.1150 mm, Waters) with a column temperature of 70 C. A linear gradient was applied in 9 minutes from 30 to 55% acetonitrile in 0.1% TFA and water.

    General Procedure for Analytical SEC

    [0504] SE-HPLC analysis was performed on an Agilent 1100 series (Hewlett Packard) using an Xbridge BEH200A column (3.5 M, 7.8300 mm, PN 186007640, Waters). The sample was diluted to 1 mg/mL in PBS and measured with 0.86 mL/min isocratic method (0.1 M sodium phosphate buffer pH 6.9 (NaHPO.sub.4/Na.sub.2PO.sub.4) containing 10% isopropanol) for 16 minutes.

    General Procedure for Mass Spectral Analysis of Monoclonal Antibodies and ADC

    [0505] Prior to mass spectral analysis, IgG was treated with IdeS (Fabricator) for analysis of the Fc/2 fragment. A solution of 20 g (modified) IgG was incubated for 1 hour at 37 C. with 0.5 L IdeS (50 U/L) in phosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 L. Samples were diluted to 40 L followed by analysis on a JEOL AccuTOF LC-plus JMS-T100LP system (ESI-TOF) combined with a HPLC system (Agilent 1100 series, Hewlett Packard). On the HPLC system a MassPREP On-line Desalting Cartridge (Waters P/N 186002785) is installed.

    General Procedure for LC-MS Analysis of Monoclonal Antibodies and ADC

    [0506] For analysis of Fc/2 fragments, IgG was treated with IdeS (Fabricator). A solution of 10 g (modified) IgG was incubated for 1 hour at 37 C. with 0.5 L IdeS (50 U/L) in phosphate-buffered saline (PBS) pH 7.4 in a total volume of 10 L followed by dilution to 100 L using MQ. For analysis of reduced samples, IgG was treated with DTT. A solution of 10 g (modified) IgG was incubated for 15 minutes at 37 C. with DTT (10 mM) in a final volume of 50 L PBS pH 7.4 followed by addition of 50 L quench buffer (49% MQ, 49% acetonitrile, 2% FA). Sample were analyzed on a Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometry system (ESI-QTOF) combined with a UPLC system (Aquity series, Waters). On the UPLC system a bioZen 3.6 m Intact XB-C8, LC column 502.1 mm (cat no: 00B-4766-AN) is installed.

    Synthesis of Bis-Iodo Compound 7b

    ##STR00074##

    Example a1. Synthesis of 2,3,4-tri-O-acetyl--D-arabinopyranosylbromide (10)

    [0507] Tetraacetyl arabinose (24.6 g) was dissolved in a solution of HBr in AcOH (33% HBr, 127 mL). Ac.sub.2O (12 mL) was added, and the mixture was stirred at rt overnight. DCM (200 mL) was added, and the mixture was poured onto ice (300 mL). The two phases were separated, and the aqueous layer was extracted with DCM (2250 mL). The combined organic layers were washed with sat. aq. NaHCO.sub.3 (400 mL) and dried over Na.sub.2SO.sub.4. The mixture was concentrated and recrystallized using Et.sub.2O/heptane to obtain compound 10 (17.66 g, 67.4%). .sup.1H-NMR data was identical to data reported by Grugel et al. Synthesis, 2010, 19, 3248-3258.

    Example a2. Synthesis of 1-(2-azidoethyl)--D-arabinopyranoside (11)

    [0508] Arabinosyl bromide 10 (2.47 g, 6.45 mmol) was dissolved in dry DCM (0.2 M). Molecular sieves, and 2-bromoethanol (5 eq) were added, and the mixture was cooled to 0 C. After addition of Ag.sub.2CO.sub.3 (1 eq), the reaction mixture was warmed to room temperature and stirred for 3 h. The mixture was filtered through celite, the celite pad was washed with Et.sub.2O and the solvent was evaporated. The crude product was purified using flash column chromatography (0.fwdarw.25% EtOAc in heptane). Fractions containing the product were concentrated, dissolved in DMF (0.2 M), after which NaN.sub.3 (4 eq) was added. The mixture was stirred at 80 C. for 1 h and concentrated, after which it was redissolved in MeOH (0.1 M) and NaOMe (5.4 M in MeOH, 0.1 eq) was added. The reaction was stirred overnight, concentrated, and purified using flash column chromatography (0.fwdarw.10% MeOH in EtOAc) to obtain the product 11 (530 mg, 37.5% over three steps). .sup.1H-NMR (500 MHz, CDCl.sub.3) (ppm) 4.28 (d, J=7.1 Hz, 1H), 4.10-3.99 (m, 2H), 3.97 (s, 1H), 3.95-3.90 (m, 1H), 3.80-3.72 (m, 2H), 3.69 (m, 2H), 3.60-3.53 (m, 2H), 3.45 (ddd, J=13.3, 5.8, 3.6 Hz, 2H). .sup.13C-NMR (126 MHZ, CDCl.sub.3) (ppm) 103.28, 72.97, 71.42, 68.22, 68.20, 65.91, 50.84.

    Example a3. Synthesis of 1,5-dihydroxy-2 (S)-(2-azidoethoxy)-3-oxa-pentane (12)

    [0509] Arabinoside 11 (530 mg, 2.42 mmol) was dissolved in H.sub.2O (0.25 M). NaOAc (1.3 eq) was added, followed by NalO.sub.4 (2.5 eq). After stirring for 1 h in the dark, TLC (10% MeOH in EtOAc) showed full consumption of the starting material. The mixture was cooled to 0 C., followed by addition of NaBH.sub.4 in portions. After 1 h, TLC (10% MeOH in EtOAc) showed formation of the diol. EtOAc (10 mL) was added, and the organic layers were separated. The aqueous layer was extracted with EtOAc (10 mL) five times. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated, to obtain product 12 (346 mg, 74.8%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 4.72 (t, J=5.3 Hz, 1H), 3.94-3.83 (m, 2H), 3.82-3.76 (m, 2H), 3.77-3.65 (m, 4H), 3.52-3.38 (m, 2H).

    [0510] .sup.13C-NMR (101 MHZ, CDCl.sub.3) (ppm) 102.88, 68.39, 66.11, 62.34, 61.80, 50.92.

    Example a4. Synthesis of 1,5-di(p-toluenesulphonyl)oxy-2(S)-(2-azidoethoxy)-3-oxa-pentane (13)

    [0511] Diol 12 (346 mg, 1.81 mmol) was dissolved in dry pyridine (0.1 M), cooled to 0 C. followed by addition of p-TsCl (2.5 eq). The mixture was stirred overnight, concentrated and dissolved in EtOAc (20 mL). The solution was washed with 0.1M HCl (10 mL) and brine (10 mL). The mixture was dried over Na.sub.2SO.sub.4, concentrated, and purified using flash column chromatography (0.fwdarw.50% EtOAc in heptane) to obtain product 13 (368 mg, 40.7%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 7.86-7.77 (m, 4H), 7.47-7.33 (m, 5H), 4.74 (t, J=5.4 Hz, 1H), 4.15 (ddd, J=5.4, 4.0, 1.1 Hz, 2H), 3.97 (dd, J=5.4, 0.8 Hz, 2H), 3.85-3.69 (m, 3H), 3.66-3.58 (m, 1H), 3.36 (dt, J=5.8, 3.9 Hz, 2H), 2.48 (d, J=1.5 Hz, 6H). .sup.13C-NMR (101 MHz, CDCl.sub.3) (ppm) 145.26, 145.08, 132.82, 132.50, 130.00, 129.94, 128.01, 127.96, 99.40, 68.77, 67.86, 65.71, 64.27, 50.67, 21.69, 21.67.

    Example a5. Synthesis of 1,5-diiodo-2 (S)-(2-azidoethoxy)-3-oxa-pentane (7b)

    [0512] Bis-tosylate 13 (491 mg, 0.98 mmol) was dissolved in 2-butanone (0.05 M). Nal (7 eq) was added, and the mixture was stirred at 90 C. for 24 h. The mixture was concentrated and dissolved in EtOAc (20 mL). The organic layer was washed with H.sub.2O (20 mL) and brine (20 mL), dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified using flash column chromatography (0.fwdarw.5% EtOAc in heptane) to obtain compound 7b (260 mg, 64.4%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 4.80 (t, J=5.6 Hz, 1H), 3.98-3.80 (m, 3H), 3.73 (ddd, J=10.4, 5.8, 4.5 Hz, 1H), 3.46 (ddd, J=5.9, 4.2, 1.8 Hz, 2H), 3.37-3.26 (m, 4H). .sup.13C-NMR (101 MHZ, CDCl.sub.3) (ppm) 101.83, 66.95, 65.05, 50.73, 3.77, 2.33.

    ##STR00075##

    Example a6. Synthesis of 1-Isopropyl--D-arabinopyranoside (15)

    [0513] Arabinosyl bromide 10 (3.51 g, 10.3 mmol) was dissolved in dry Et.sub.2O (0.25 M). iPrOH (15 eq) was added, followed by Ag.sub.2O (1 eq). The mixture was stirred in the dark for 3 h. The reaction was filtered over celite, the celite pad was washed with Et.sub.2O and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by addition of NaOMe (5.4 M in MeOH, 0.1 eq.) and stirred overnight at rt. The product was purified using flash column chromatography (0.fwdarw.10% MeOH in EtOAc) to obtain product 15 (1.38 g, 69.4% over two steps). .sup.1H-NMR (400 MHZ, D.sub.2O) (ppm) 5.70-5.66 (m, 1H), 5.38 (hept, J=6.2 Hz, 1H), 5.26 (dd, J=12.4, 2.9 Hz, 1H), 5.22 (dt, J=2.9, 1.5 Hz, 1H), 5.00-4.85 (m, 3H), 2.62 (dd, J=13.0, 6.1 Hz, 6H). .sup.13C-NMR (101 MHZ, D.sub.2O) (ppm) 103.16, 74.35, 72.50, 72.48, 69.72, 66.84, 23.81, 22.07.

    Example a7. Synthesis of 1,5-dihydroxy-2 (S)-isopropyloxy-3-oxa-pentane (16)

    [0514] Arabinoside 15 (585 mg, 3.04 mmol) was dissolved in H.sub.2O (0.25 M). NaOAc (1.3 eq) was added, followed by NalO.sub.4 (2.5 eq). After stirring for 1 h in the dark, TLC (10% MeOH in EtOAc) showed full consumption of the starting material. The mixture was cooled to 0 C., followed by addition of NaBH.sub.4 in portions. After 1 h, TLC (10% MeOH in EtOAc) showed formation of the diol. EtOAc (10 mL) was added, and the organic layers were separated. The aqueous layer was extracted with EtOAc (10 mL) five times. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated, to obtain product 16 (324 mg, 64.8%). .sup.1H-NMR (500 MHZ, CDCl.sub.3) (ppm) 4.72 (dd, J=6.1, 4.7 Hz, 1H), 3.93 (dq, J=12.3, 6.1 Hz, 1H), 3.87-3.75 (m, 3H), 3.71-3.53 (m, 3H), 1.26 (d, J=5.7 Hz, 2H), 1.20 (d, J=6.1 Hz, 3H). .sup.13C-NMR (126 MHZ, CDCl.sub.3) (ppm) 100.99, 70.29, 67.64, 63.21, 62.02, 23.09, 22.32.

    Example a8. Synthesis of 1,5-diiodo-2 (R)-isopropyloxy-3-oxa-pentane (7c)

    [0515] Diol 16 (244 mg, 1.49 mmol) was dissolved in dry THF (0.15 M). Imidazole (7 eq), PPh.sub.3 (3 eq) were added, followed by the addition of I.sub.2 (3 eq). The mixture was stirred overnight at rt in the dark. After dilution with EtOAc (20 mL), the organic layer was washed with 10% aq. sodium thiosulfate (20 mL), brine (20 mL) and dried over Na.sub.2SO.sub.4. The mixture was concentrated and purified using column chromatography (0.fwdarw.5% EtOAc in heptane) to obtain the product 7c (220 mg, 38.5%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 4.76 (t, J=5.5 Hz, 1H), 3.94 (hept, J=6.2 Hz, 1H), 3.87-3.71 (m, 0H), 3.33-3.12 (m, 4H), 1.24 (d, J=6.2 Hz, 3H), 1.20 (d, J=6.1 Hz, 3H). .sup.13C-NMR (101 MHZ, CDCl.sub.3) (ppm) 100.29, 70.13, 65.65, 23.16, 22.06, 5.74, 2.61.

    ##STR00076##

    Example a9. Synthesis of 1-benzyl--D-arabinopyranoside (18)

    [0516] Arabinosyl bromide 10 (2.06 g, 6.09 mmol) was dissolved in dry Et.sub.2O (0.25 M). BnOH (15 eq) was added, followed by Ag.sub.2O (1 eq). The mixture was stirred in the dark for 3 h. The reaction was filtered over celite, the celite pad was washed with Et.sub.2O and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by addition of NaOMe (5.4 M in MeOH, 0.1 eq) and stirred overnight at rt. The product was purified using flash column chromatography (0.fwdarw.10% MeOH in EtOAc) to obtain the product 18 (840 mg, 57.4% over two steps). .sup.1H-NMR (400 MHZ, D2O) (ppm) 7.53-7.37 (m, 5H), 4.92 (dd, J=11.6, 1.4 Hz, 1H), 4.75 (dd, J=11.6, 1.2 Hz, 1H), 4.44 (d, J=7.5 Hz, 1H), 4.02-3.91 (m, 2H), 3.73-3.55 (m, 3H). .sup.13C-NMR (101 MHZ, D2O) (ppm) 136.63, 128.72, 128.64, 128.45, 102.18, 102.13, 72.35, 71.42, 70.72, 70.69, 68.31, 68.24, 66.28.

    Example a10. Synthesis of 1,5-dihydroxy-2 (S)-benzyloxy-3-oxa-pentane (19)

    [0517] Arabinoside 18 (259 mg, 1.08 mmol) was dissolved in H.sub.2O (0.25 M). NaOAc (1.3 eq) was added, followed by NalO.sub.4 (2.5 eq). After stirring for 1 h in the dark, TLC (10% MeOH in EtOAc) showed full consumption of the starting material. The mixture was cooled to 0 C., followed by addition of NaBH.sub.4 in portions. After 1 h, TLC (10% MeOH in EtOAc) showed formation of the diol. EtOAc (10 mL) was added, and the organic layers were separated. The aqueous layer was extracted with EtOAc (10 mL) five times. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated, to obtain product 19 (198 mg, 86.7%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 7.43-7.17 (m, 5H), 4.75-4.66 (m, 2H), 4.57 (d, J=11.7 Hz, 1H), 3.83 (ddd, J=10.6, 5.3, 3.3 Hz, 1H), 3.77-3.71 (m, 2H), 3.68-3.55 (m, 2H). .sup.13C-NMR (101 MHZ, CDCl.sub.3) (ppm) 137.49, 128.55, 127.97, 127.85, 102.29, 69.51, 68.31, 62.44, 61.68.

    Example a11. Synthesis of 1,5-diiodo-2 (S)-benzyloxy-3-oxa-pentane (7d)

    [0518] Diol 19 (579 mg, 2.73 mmol) was dissolved in dry THF (0.15 M). Imidazole (7 eq), PPh3 (3 eq) were added, followed by the addition of 12 (3 eq). The mixture was stirred overnight at rt in the dark. After dilution with EtOAc (20 mL), the organic layer was washed with 10% aq. sodium thiosulfate (20 mL), brine (20 mL) and dried over Na.sub.2SO.sub.4. The mixture was concentrated and purified using column chromatography (0.fwdarw.5% EtOAc in heptane) to obtain the product 7d (723 mg, 61.3%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 7.45-7.31 (m, 5H), 4.83 (t, J=5.6 Hz, 1H), 4.76 (d, J=11.7 Hz, 1H), 4.66 (d, J=11.7 Hz, 1H), 3.94-3.75 (m, 2H), 3.37-3.24 (m, 4H). .sup.13C-NMR (101 MHZ, CDCl.sub.3) (ppm) 137.16, 128.57, 128.05, 127.99, 101.12, 68.66, 66.72, 4.52, 2.50.

    ##STR00077##

    Example a12. Synthesis of Compound 21

    [0519] An ice-cold solution of sodium nitrite (842.3 mg, 12.21 mmol) in water (20 mL) was prepared and transferred to a dropping funnel. This mixture was added over 30 min to a cold solution of 4-aminobenzyl alcohol (1 g, 8.12 mmol) in HCl (5 M, 5 mL). The reaction mixture turned from bright yellow to light yellow and finally to off-white. After 30 min, sodium azide (2.1 g, 32 mmol) was added in 5 portions and the mixture was left stirring. After an hour the ice bath was removed, and solids were observed. After 1.5 h, saturated aqueous NaHCO.sub.3 solution (25 mL) was added followed by EtOAc (25 mL). The reaction was transferred to a separation funnel and the organic layer was separated from the water layer. The organic layer was washed with saturated aqueous NaHCO.sub.3 solution (20 mL), washed with brine (25 mL) and dried over Na.sub.2SO.sub.4. The drying agent was filtered off over a glass filter and the yellow filtrate was concentrated. The crude yellow oil was purified by flash column chromatography over silicagel (5%.fwdarw.80% EtOAc in heptane, column pre-conditioned with 5% EtOAc in heptane) to give product 21 in 89% (1.08 g, 7.24 mmol). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 7.36 (d, J=8.6 Hz, 2H), 7.08-6.97 (m, 2H), 4.67 (d, J=4.3 Hz, 2H), 1.68 (t, J=5.2 Hz, 1H).

    Example a13. Synthesis of Compound 23

    [0520] To a solution of arabinosyl bromide 10 (1.34 mg, 3.95 mmol) and compound 21 (872 mg, 5.85 mmol) in diethyl ether (anhydrous, 20 mL) was added silver(I) oxide (916 mg, 3.95 mmol) and the reaction was stirred in the dark at room temperature. After stirring for 10 days, the reaction mixture was filtered over pre-wetted celite and washed through with diethyl ether and concentrated. The crude oil was dissolved in MeOH (15 mL), and sodium methoxide (134.4 mg, 2.48 mmol) was added. After stirring for 3.5 h at room temperature, the reaction mixture was neutralized with a few drops of 1 M aq. HCl solution and concentrated. The excess 21 was removed by precipitating the desired compound in diethyl ether and filtering it over a glass filter covered with filter paper. Compound 23 was obtained in 65% yield (714.4 mg, 2.54 mmol) as an off-white solid. .sup.1H-NMR (400 MHZ, MeOD) (ppm) 7.47 (d, J=8.6 Hz, 2H), 7.10-7.04 (m, 2H), 4.86 (d, J=11.9 Hz, 1H), 4.63 (d, J=11.9 Hz, 1H), 4.31 (d, J=6.9 Hz, 1H), 3.95-3.79 (m, 3H), 3.67-3.49 (m, 4H).

    Example a14. Synthesis of Compound 24

    [0521] Compound 23 (714.4 mg, 2.54 mmol, 1.0 eq) was dissolved in MeOH (3 mL) and water (5 mL) and cooled down to 0 C. (in the dark). A solution of sodium acetate (270.9 mg, 3.3 mmol, 1.3 eq) in water (3 mL) was added at once followed by sodium periodate (1.35 g, 6.35 mmol, 2.5 eq) in portions. The reaction mixture was stirred on ice for 15 min after which the ice bath was removed and stirring was continued for 4.5 h at room temperature. After stirring for 4.5 h, the reaction mixture was cooled again to 10 C. and sodium borohydride (288.3 mg, 7.62 mmol, 3.0 eq) was added in portions. After stirring for an hour, EtOAc (50 mL) was added, and the reaction mixture was transferred to a separation funnel. The organic layer was separated from the water layer and the water layer was extracted with EtOAc (550 mL). The organic layers were combined and dried over Na.sub.2SO.sub.4, filtered over a glass filter with pre-wetted celite and concentrated. Compound 24 was obtained in 87% yield (637.2 mg, 2.2 mmol). .sup.1H-NMR (400 MHZ, MeOD) (ppm) 7.31 (d, J=8.5 Hz, 2H), 6.98-6.91 (m, 2H), 4.63-4.48 (m, 3H), 3.70-3.65 (m, 1H), 3.60-3.56 (m, 2H), 3.53-3.48 (m, 3H).

    Example a15. Synthesis of Compound 25

    [0522] A solution of 24 (637.2 mg, 2.21 mmol) in dry DCM (8 mL) was cooled to 0 C. (under flow of nitrogen). Pyridine (537 L, 6.64 mmol, 3.0 eq), methanesulfonic anhydride (964.2 mg, 5.53 mmol, 2.5 eq) and DMAP (27.0 mg, 221.4 mmol, 0.1 eq) were added. After stirring for 3.5 h, the reaction mixture was washed with aqueous saturated NaHCO.sub.3 solution (11 mL). The water layer was extracted twice with DCM (10 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude orange oil was purified by flash column chromatography over silicagel (10%.fwdarw.80% EtOAc in heptane, column pre-conditioned with 10% EtOAc in heptane) to give compound 25 as a clear light-yellow oil in 61% (571.6 mg, 1.4 mmol). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 7.34 (d, J=8.5 Hz, 2H), 7.06-7.00 (m, 2H), 4.91 (t, J=5.2 Hz, 1H), 4.72 (d, J=11.7 Hz, 1H), 4.61 (d, J=11.7 Hz, 1H), 4.37 (t, J=4.5 Hz, 2H), 4.24 (dd, J=5.2, 2.1 Hz, 2H), 3.96-3.88 (m, 1H), 3.86-3.80 (m, 1H), 3.06 (s, 3H), 3.05 (s, 3H).

    Example a16. Synthesis of Compound 7e

    [0523] A solution of compound 25 (571.2 mg, 1.4 mmol, 1.0 eq) and sodium iodide (1.42 g, 9.48 mmol, 7 eq) in 2-butanone (15 mL) was refluxed in the dark. After refluxing for 72 h, the reaction mixture was diluted with EtOAc (30 mL) and washed with water (20 mL). The layers were separated, and the organic layer was washed with brine (20 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude orange oil was purified by flash column chromatography over silicagel (0%.fwdarw.7% EtOAc in heptane) to give compound 7e as an opaque light-yellow oil in 47.5% (313.2 mg, 0.64 mmol). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 7.37 (d, J=8.6 Hz, 2H), 7.11-6.93 (m, 2H), 4.79 (t, J=5.6 Hz, 1H), 4.70 (d, J=11.6 Hz, 1H), 4.59 (d, J=11.7 Hz, 1H), 3.90-3.72 (m, 2H), 3.35-3.18 (m, 4H).

    ##STR00078##

    Example a17. Synthesis of 1-ethyl--D-arabinopyranoside (27)

    [0524] Arabinosyl bromide 10 (5.31 g, 15.66 mmol) was dissolved in dry Et.sub.2O (0.25 M). EtOH (15 eq) was added, followed by Ag.sub.2O (1 eq). The mixture was stirred in the dark for 3 h. The reaction was filtered over celite, the celite pad was washed with Et.sub.2O and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by addition of NaOMe (5.4 M in MeOH, 0.1 eq) and stirred overnight at rt. The product was purified using flash column chromatography (0.fwdarw.10% MeOH in EtOAc) to obtain the product 27 (1.80 g, 64.5% over two steps). .sup.1H-NMR (400 MHZ, MeOD) (ppm) 4.22 (d, J=7.1 Hz, 1H), 3.97-3.79 (m, 3H), 3.67-3.50 (m, 4H), 1.26 (t, J=7.1 Hz, 3H). .sup.13C-NMR (101 MHZ, MeOD) (ppm) 104.65, 74.33, 72.43, 69.70, 66.91, 65.94, 15.49.

    Example a18. Synthesis of 1,5-dihydroxy-2(S)-ethoxy-3-oxa-pentane (28)

    [0525] Arabinoside 27 (721 mg, 4.05 mmol) was dissolved in H.sub.2O (0.25 M). NaOAc (1.3 eq) was added, followed by NalO.sub.4 (2.5 eq). After stirring for 1 h in the dark, TLC (10% MeOH in EtOAc) showed full consumption of the starting material. The mixture was cooled to 0 C., followed by addition of NaBH.sub.4 in portions. After 1 h, TLC (10% MeOH in EtOAc) showed formation of the diol. EtOAc (15 mL) was added, and the organic layers were separated. The aqueous layer was extracted with EtOAc (15 mL) five times. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated, to obtain product 28 (381 mg, 62.7%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 4.62 (dd, J=6.1, 4.3 Hz, 1H), 3.89-3.81 (m, 1H), 3.79-3.69 (m, 3H), 3.67-3.53 (m, 4H), 1.24-1.19 (m, 3H). .sup.13C-NMR (101 MHZ, CDCl.sub.3) (ppm) 102.65, 68.44, 63.66, 62.61, 61.83, 15.29.

    Example a19. Synthesis of 1,5-diiodo-2 (S)-ethoxy-3-oxa-pentane (7f)

    [0526] Diol 28 (381 mg, 2.54 mmol) was dissolved in dry THF (0.15 M). Imidazole (7 eq), PPh3 (3 eq) were added, followed by the addition of 12 (3 eq). The mixture was stirred overnight at rt in the dark. After dilution with EtOAc (20 mL), the organic layer was washed with 10% sodium thiosulfate (20 mL), brine (20 mL) and dried over Na.sub.2SO.sub.4. The mixture was concentrated and purified using column chromatography (0.fwdarw.5% EtOAc in heptane) to obtain the product 7f (170 mg, 18.1%). .sup.1H-NMR (400 MHZ, CDCl.sub.3) (ppm) 4.72 (t, J=5.5 Hz, 1H), 3.93-3.70 (m, 3H), 3.66-3.56 (m, 1H), 3.32-3.23 (m, 4H), 1.26 (t, J=7.1 Hz, 3H). .sup.13C-NMR (101 MHZ, CDCl.sub.3) (ppm) 101.76, 66.90, 62.52, 15.10, 4.78, 2.48.

    General Scheme from Doxorubicin to PNU-159,682 Analogues

    [0527] General schemes are depicted in FIGS. 12 and 13. Similar analogues can be prepared from daunorubicin by omitting silylation and desilylation steps.

    Example a20. Synthesis of Compound 2

    [0528] A solution of doxorubicin HCl (3.09 g, 5.33 mmol) in anhydrous DMF (35 mL) was cooled to 0 C. and imidazole (1.47 g, 21.6 mmol) was added. After stirring for a few minutes, TBDMS-Cl (1.90 g, 12.65 mmol) was added. The reaction mixture was stirred at 0 C. for 5 min after which it was allowed to warm up to room temperature. After stirring for 3.5 h at room temperature, the reaction mixture was purified by flash column chromatography over silicagel (column preconditioned 1% MeOH/DCM, 1%.fwdarw.30% MeOH in DCM) to give compound 2 as a dark red thick oil (3.59 g, 5.4 mmol, 100%). LCMS (ESI+) calculated for C.sub.33H.sub.44NO.sub.11Si.sup.+ (M+H.sup.+) 658.27 found 658.44.

    Example a21. Synthesis of Compound 3b

    ##STR00079##

    [0529] To a stock solution of compound 2 (550 mg, 836 mol) in anhydrous DMF (1 mL) was added (S)-1-(2-azidoethoxy-2-iodo-1-(2-iodoethoxy)ethane 7b (828.5 mg, 2.06 mmol) and DIPEA (437 L, 2.51 mmol). The reaction mixture was heated at 42 C. and stirred for 25 minutes after which the heating device was removed, and the reaction mixture was left to react at room temperature. After 72 hours at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by flash column chromatography over silicagel (0%.fwdarw.3% MeOH in DCM) to give compound 3b as a dark red oil (303 mg, 367 mol, 43.9%). LCMS (ESI+) calculated for C.sub.39H.sub.53N.sub.4O.sub.13Si.sup.+ (M+H.sup.+) 813.34 found 813.51.

    Example a22. Synthesis of Compound 4b

    ##STR00080##

    [0530] A solution of compound 3b (303 mg, 253 mol, 68 wt %) in anhydrous DCM (31 mL) was cooled by dry ice/acetone cooling-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in anhydrous DCM (70 mg, 580 mM, 699 L, 406 mol) was added dropwise. After stirring for 7 minutes, full conversion was reached. The RM was quenched with an ice-cold solution of acetone (reagent grade, 3.33 mL) and the RM was stirred. After 20 min the cold bath was removed, and the RM was allowed to warm up to room temperature. The RM was transferred to a separation funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (6 mL). The water layers were combined and extracted once with DCM (8 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered through a filter paper, and concentrated until a volume of 30 mL was obtained. No further purification was performed and compound 4b was used as such in the next step. LCMS (ESI+) calculated for C.sub.39H.sub.53N.sub.4O.sub.14Si.sup.+ (M+H.sup.+) 829.33 found 829.57.

    Example a23. Synthesis of Compound 5b

    ##STR00081##

    [0531] To a solution of compound 4b (188.7 mg, 227.7 mol) in DCM (27 mL) was added anhydrous acetonitrile (25 mL). The RM was partially concentrated to remove the DCM and to obtain the compound in anhydrous acetonitrile (25 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (5 mL). Then, potassium carbonate (198 mg, 1.43 mmol) was added, and the RM was cooled to 0 C. after which cyanuric chloride (247.7 mg, 12.1 mL, 111 mM, 1.35 mmol) as a stock solution in anhydrous acetonitrile was added. After stirring for 4 h at 0 C., the RM was quenched with a solution of 3-aminopropane-1,2 diol (489.6 mg, 2.9 mL, 1.85 M, 5.37 mmol) in water. The ice bath was removed after 30 min after which it was allowed to warm up to room temperature. To the RM was added DMF (1.5 mL) and the RM was concentrated until only a solution of DMF/water (8 mL) was left, which was purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). The collected fractions were combined and concentrated until a volume of 6 mL acetonitrile was left. Then it was dried over Na.sub.2SO.sub.4, filtered and the residue was washed through with anhydrous THF (3750 L). The combined organic layers were partially concentrated to a volume of 5 mL (acetonitrile/THF) and to give compound 5b as a red solution, which was used without further purification. LCMS (ESI+) calculated for C.sub.39H.sub.51N.sub.4O.sub.13Si.sup.+ (M+H.sup.+) 811.32 found 811.51.

    Example a24. Synthesis of Compound 6b

    ##STR00082##

    [0532] To a solution of compound 5b (185 mg, 228 mol) in a mixture of acetonitrile and THF (5 mL) was added triethylamine acetate (731 L, 4.56 mmol) and the rm was cooled to 15 C. Then, while the RM was vigorously stirred TBAF (1 M in THF, 1.03 g, 4 mL, 4 mmol) was added in portions (color change from red to green observed and back to dark red). After stirring for 2.5 h, the RM was quenched with water (15 mL), solution changed from dark red to light red. The RM was stirred on ice for 1 min and then left at room temperature for an additional 90 min. The RM was transferred to a separation funnel and extracted with DCM (18 mL). The water layer was extracted with additional DCM (26 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and further diluted with DCM to 55 mL, and purified by flash column chromatography over silicagel (0%.fwdarw.10% MeOH in DCM). Additional prep-HPLC purification (30%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm) was required. The collected fractions were combined and partially concentrated to give compound 6b (0.22 mM based on a doxorubicin-based calibration line for HPLC, 45 mL, 9.82 mol, 4.3% yield over 3 steps) as a solution in 45 mL acetonitrile/water. LCMS (ESI+) calculated for C.sub.33H.sub.37N.sub.4O.sub.13Si.sup.+ (M+H.sup.+) 697.24 found 697.45.

    Example a25. Synthesis of Compound 3c

    ##STR00083##

    [0533] To a stock solution of compound 2 (650 mg, 612 mol) in anhydrous DMF (1.6 mL) was added (R)-2-(2-iodo-1-(2-iodoethoxy)ethoxy) propane 7c (1.13 g, 2.93 mmol) and DIPEA (516 UL, 2.96 mmol). The reaction mixture was heated to 40 C. and stirred for 10 minutes after which the heating device was removed, and the reaction mixture was left to react at room temperature. After 72 at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by flash column chromatography over silicagel (0%.fwdarw.3% MeOH in DCM) to give compound 3c as a dark red oil (241.8 mg, 307.6 mol, 31.1%). LCMS (ESI+) calculated for C.sub.40H.sub.56NO.sub.13Si.sup.+ (M+H.sup.+) 786.96 found 786.64.

    Example a26. Synthesis of Compound 4c

    ##STR00084##

    [0534] A solution of 3c (125 mg, 159 mol) in anhydrous DCM (15 mL) was cooled with a dry ice/acetone-cooling bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in anhydrous DCM (52.1 mg, 580 mM, 521 L, 302 mol) was dropwise added. After stirring 78 C. for 7 minutes, full conversion was reached. The RM was quenched with an ice-cold solution of acetone (reagent grade, 1.46 mL) and the RM was stirred. After an hour the cold bath was removed, and the RM was allowed to warm up to room temperature. The RM was transferred to a separation funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (12 mL). The water layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered through a filter paper, and concentrated until a volume of 12 mL was obtained, providing 4c as a red solution. No further purification was performed and compound 4c was used as such. LCMS (ESI+) calculated for C.sub.40H.sub.56NO.sub.14Si.sup.+ (M+H.sup.+) 802.96 found 802.63.

    Example a27. Synthesis of Compound 5c

    ##STR00085##

    [0535] To a solution of 4c (128 mg, 159 mol) in DCM (12 mL) was added anhydrous acetonitrile (6 mL). The RM was partially concentrated to remove the DCM and to obtain the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (20 mL). Then, potassium carbonate (86 mg, 622 mol) was added, and the RM was cooled to 7 C. after which cyanuric chloride (73.6 mg, 3.59 mL, 111 mM, 399 mol) as a stock solution in anhydrous acetonitrile was added. After stirring for 2 h at 7 C., the RM was quenched with a solution of 3-aminopropane-1,2 diol (179 mg, 854 L, 2.3 molar, 1.96 mmol) in water. The ice bath was removed after 15 min after which it was allowed to warm up to room temperature. To the RM was added DMF (1 mL) and the RM was concentrated until only a solution of DMF/water was left, which was purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm). The collected fractions were combined and concentrated until a volume of 4 mL was left. The resulting solution consisting of mostly acetonitrile was then dried over Na.sub.2SO.sub.4, filtered and the drying agent was washed through with anhydrous THF (3750 L). It was concentrated again to a volume of 3 mL and compound 5c was used as such. LCMS (ESI+) calculated for C.sub.40H.sub.54NO.sub.13Si.sup.+ (M+H.sup.+) 784.94 found 786.57.

    Example a28. Synthesis of Compound 6c

    ##STR00086##

    [0536] To compound 5c (121 mg, 154 mol) in a mixture of acetonitrile and THF (2.88 mL) was added additional dry THF (2.0 mL), and the resulting red solution was cooled to 40 C. Then, while the RM was vigorously stirred TBAF (1 M in THF, 484.5 mg, 1.85 mL, 1.85 mmol) was added (color change from red to green observed). After stirring for 4 h, the RM was quenched with water (3.0 mL), solution changed from green to red. The RM was transferred to a separation funnel and extracted with DCM (310 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and purified by flash column chromatography over silicagel (0%.fwdarw.5% MeOH in DCM) followed by prep-HPLC (40%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). Compound 6c was obtained as a red solution in DCM (70 mL, 0.086 mM based on a doxorubicin-based calibration line for HPLC, 6,0 mol, 3,9% yield over 3 steps). LCMS (ESI+) calculated for C.sub.34H.sub.40NO.sub.13.sup.+ (M+H.sup.+) 670.25 found 670.51.

    Example a29. Synthesis of Compound 3d

    ##STR00087##

    [0537] To a stock solution of compound 2 (650 mg, 612 mol) in anhydrous DMF (1.6 mL) was added (S)-((2-iodo-1-(2-iodoethoxy)ethoxy)methyl)benzene 7d (1.27 g, 2.94 mmol) and DIPEA (516 L, 2.96 mmol). The reaction mixture was heated to 42 C. and stirred for 10 minutes after which the heating device was removed, and the reaction mixture was left to react at room temperature. After 72 at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by flash column chromatography over silicagel (0%.fwdarw.3% MeOH in DCM) to give compound 3d as a dark red oil (413.5 mg, 479 mol, 48.5%). LCMS (ESI+) calculated for C.sub.44H.sub.56NO.sub.13Si.sup.+ (M+H.sup.+) 834.35 found 834.50.

    Example a30. Synthesis of Compound 4d

    ##STR00088##

    [0538] A solution of compound 3d (125 mg, 159 mol) in anhydrous DCM (15 mL) was cooled by dry ice/acetone cooling-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in anhydrous DCM (52.0 mg, 580 mM, 519 L, 301.5 mol) was dropwise added. After stirring for 36 minutes, full conversion was reached. The RM was quenched with an ice-cold solution of acetone (reagent grade, 1.38 mL) and the RM was stirred. After 12 min the cooling-bath was removed, and the RM was allowed to warm up to room temperature. The RM was transferred to a separation funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (12 mL). The water layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered through a filter paper, and concentrated until a volume of 12 mL was obtained. No further purification was performed and compound 4d was used as such. LCMS (ESI+) calculated for C.sub.44H.sub.56NO.sub.14Si.sup.+ (M+H.sup.+) 850.35 found 850.60.

    Example a31. Synthesis of Compound 5d

    ##STR00089##

    [0539] To a solution of compound 4d (127 mg, 134 mol) in DCM (12 mL) was added anhydrous acetonitrile (6 mL). The RM was partially concentrated to remove the DCM and to obtain the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (20 mL). Then, potassium carbonate (73 mg, 530 mol) was added, and the RM was cooled to 7 C. after which a solution of cyanuric chloride in anhydrous acetonitrile (62.0 mg, 3.01 mL, 111.8 mM, 336 mol) was added. After stirring for 2 h at 7 C., the RM was quenched with a solution of 3-aminopropane-1,2 diol (151 mg, 1.85 mL, 894 mM, 1.65 mmol) in water. The ice bath was removed after 15 min after which it was allowed to warm up to room temperature. To the RM was added DMF (2 mL) and the RM was concentrated until only a solution of DMF/water was left and purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm). The collected fractions were combined and concentrated until a volume of 4 mL was left. The resulting solution consisting of mostly acetonitrile was dried over Na.sub.2SO.sub.4, filtered and the drying agent was washed through with anhydrous THF (3750 L). The combined organic layers were again partially concentrated to a volume of 4 mL (acetonitrile/THF) and compound 5d was used as such. LCMS (ESI+) calculated for C.sub.44H.sub.54NO.sub.13Si.sup.+ (M+H.sup.+) 832.34 found 832.53.

    Example a32. Synthesis of Compound 6d

    ##STR00090##

    [0540] To compound 5d (25 mg, 30 mol) in a mixture of acetonitrile and THF (4 mL) was added triethylammonium acetate (97 mg, 96 L, 600 mol) and the resulting red solution was cooled to-20 C. Then, while the RM was vigorously stirred TBAF (1 M in THF, 35 mg, 340 L, 340 mmol) was added (color change from red to green and back to red observed). After stirring for 3 h, the RM was quenched with water (2.4 mL). The RM was transferred to a separation funnel and extracted with DCM (35 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated until approximately 10 mL was left (water bath rotary evaporator set 32 C.) and purified by flash column chromatography over silicagel (0%.fwdarw.10% MeOH in DCM) to give compound 6d as a red solution in DCM (4 mL, 0.82 mM based on a doxorubicin-based calibration line for HPLC, 2.36 mg, 3.29 mol, 11%). LCMS (ESI+) calculated for C.sub.38H.sub.40NO.sub.13.sup.+ (M+H.sup.+) 718.25 found 718.50.

    Example a33. Synthesis of Compound 3e

    ##STR00091##

    [0541] To a stock solution of compound 2 (165 mg, 612 mol) in anhydrous DMF (410 L) was added(S)-1-azido-4-((2-iodo-1-(2-iodoethoxy)ethoxy)methyl)benzene 7e (294 mg, 603 mol) and DIPEA (131 L, 753 mol). The reaction mixture was vortexed and was left to react at room temperature. After 120 h at room temperature, the reaction mixture was diluted with DCM (8 mL) and purified by flash column chromatography over silicagel (0%.fwdarw.3% MeOH in DCM) to give compound 3e as a dark red oil (53.9 mg, 61.6 mol, 24.6%). LCMS (ESI+) calculated for C.sub.44H.sub.55N.sub.4O.sub.13Si.sup.+ (M+H.sup.+) 875.35 found 875.61.

    Example a34. Synthesis of Compound 4e

    ##STR00092##

    [0542] A solution of compound 3e (53.9 mg, 61.6 mol) in anhydrous DCM (10 mL) was cooled with a dry ice/acetone cooling-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in anhydrous DCM (20.2 mg, 580 mM, 202 L, 117 mol) was added dropwise. After stirring for 8 minutes 78 C., full conversion was reached. The RM was quenched with an ice-cold solution of acetone (reagent grade, 565 L) and the RM was stirred at 78 C. After 45 min the cooling-bath was removed, and the RM was allowed to warm up to room temperature. The RM was transferred to a separation funnel and washed twice with a saturated aqueous NaHCO.sub.3 solution (10 mL). The water layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered through a filter paper, and concentrated until a volume of 10 mL was obtained. No further purification was performed and compound 4e was used as such. LCMS (ESI+) calculated for C.sub.44H.sub.55N.sub.4O.sub.14Si.sup.+ (M+H.sup.+) 891.35 found 891.52.

    Example a35. Synthesis of Compound 5e

    ##STR00093##

    [0543] To a solution of compound 4e (54.9 mg, 61.6 mol) in DCM (10 mL) was added anhydrous acetonitrile (6 mL). The RM was partially concentrated to remove the DCM and to obtain the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (10 mL). Then, potassium carbonate (33.2 mg, 240 mol) was added, and the RM was cooled to 7 C. after which a solution of cyanuric chloride in anhydrous acetonitrile (28.4 mg, 1.39 mL, 111 mM, 154 mol) was added. After stirring for 6 h at 7 C., the RM was quenched with a solution of 3-aminopropane-1,2 diol (69 mg, 330 L, 2.3 M, 758 mol) in water. The ice bath was removed after 15 min after which it was allowed to warm up to room temperature. To the RM was added DMF (1 mL) and the RM was concentrated until only a solution of DMF/water was left and purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm). The collected fractions were combined and concentrated until a volume of 4 mL acetonitrile was left. Then it was dried over Na.sub.2SO.sub.4, filtered and the residue was washed through with anhydrous THF (3750 L). The combined organic layers were again partially concentrated to a volume of 4 mL (acetonitrile/THF) and compound 5e was used as such. LCMS (ESI+) calculated for C.sub.44H.sub.53N.sub.4O.sub.13Si.sup.+ (M+H.sup.+) 873.34 found 873.46.

    Example a36. Synthesis of Compound 6e

    ##STR00094##

    [0544] Compound 5e (27 mg, 31 mol) in a mixture of acetonitrile and THF (4 mL) was cooled to 40 C. Then, while the RM was vigorously stirred TBAF (1 M in THF, 80 mg, 300 L, 300 mmol) was added (color change from red to green observed). After stirring for 2.5 h, the RM was quenched with water (2.5 mL), solution changed from green to red. The RM was transferred to a separation funnel and extracted with DCM (37 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and purified by flash column chromatography over silicagel (0%.fwdarw.10% MeOH in DCM) to give compound 6e as a red solution in DCM (10 mL, 0.31 mM based on a doxorubicin-based calibration line for HPLC, 2.3 mg, 3.0 mol, 9.8% over 3 steps). LCMS (ESI+) calculated for C.sub.38H.sub.39N.sub.4O.sub.13.sup.+ (M+H.sup.+) 759.25 found 759.40.

    Example a37. Synthesis of Compound 3f

    ##STR00095##

    [0545] A stock solution of compound 2 (150 mg, 228 mol) in anhydrous DMF (373 L) was added to a vial containing 1,5-diiodo-2 (S)-ethoxy-3-oxa-pentane 7f (1.27 g, 2.94 mmol), followed by DIPEA (119 L, 684 mol). The reaction mixture was heated to 42 C. and swirled for 1 minute and then left at room temperature in the dark. After 96 h at room temperature, the reaction mixture was diluted with DCM (4 mL) and purified by flash column chromatography over silicagel (0%.fwdarw.3% MeOH in DCM) to give 3f as a dark red oil (43.3 mg, 56.1 mol, 24.6%). LCMS (ESI+) calculated for C.sub.39H.sub.54NO.sub.13Si.sup.+ (M+H.sup.+) 772.34 found 772.65.

    Example a38. Synthesis of Compound 4f

    ##STR00096##

    [0546] A solution of compound 3f (43.3 mg, 56.1 mol) in anhydrous DCM (700 L) was cooled with a dry ice/acetone-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in anhydrous DCM (6.19 mg, 462 mM, 77.6 L, 35.9 mol) was added dropwise. After stirring for 20 minutes, a 2.sup.nd solution of mCPBA in anhydrous DCM (26.5 L, 462 mM, 12.2 mol) was added dropwise and the resulting red solution was stirred for another 15 minutes. Finally, a 3rd batch of mCPBA in anhydrous DCM (0.24 mg, 462 mM, 3.0 L, 1.4 mol) was added. The RM was stirred for another 2 minutes and was then quenched with a pre-cooled (78 C.) solution of acetone (reagent grade, 300 L) and the RM was stirred at 78 C. After 12 min the cooling-bath was removed, and the RM was allowed to warm up to room temperature and diluted with additional DCM (3.5 mL). The RM was transferred to a separation funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (2 mL). The water layers were combined and extracted twice with DCM (2 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered through a membrane filter. The filtrate, a red solution containing compound 4f, was used without further purification. LCMS (ESI+) calculated for C.sub.39H.sub.54NO.sub.14Si.sup.+ (M+H.sup.+) 788.33 found 788.64.

    Example a39. Synthesis of Compound 5f

    ##STR00097##

    [0547] To a solution of compound 4f (44.3 mol) in a mixture (circa 10 mL) of mainly DCM and minimal acetone was added anhydrous acetonitrile (2 mL). The RM was partially concentratedto remove the DCMto a volume of roughly 6 mL. Next, additional anhydrous acetonitrile (4.0 mL) was added, and the mixture was again partially concentrated to a volume of 4.4 mL. A stirring bar was added, and the RM was analyzed by HPLC-MS to assess the concentration of the starting material 4f (Indicating 44.3 mol of 4f based on a doxorubicin-based calibration line). Next, potassium carbonate (31.3 mg, 226 mol) was added, and the RM was cooled to 0 C. after which a solution of cyanuric chloride in anhydrous acetonitrile (20.5 mg, 1.00 mL, 111 mM, 111 mol) was added. After stirring for 2.5 h at 0 C., the RM was again treated with a solution of cyanuric chloride in anhydrous acetonitrile (80 L, 111 mmolar, 8.9 mol). The RM was stirred at 0 C. for another 23 minutes and was then quenched with a solution of 3-aminopropane-1,2 diol (62.9 mg, 345 L, 2.0 M, 690 mol) in water. The resulting dark red solution was allowed to slowly warm to room temperature. To the RM was added DMF (3 mL) to give a red solution with a mostly white precipitate. The solution was decanted, and the residue was washed a few times with additional DMF, which was filtered over a membrane-filter before combining with the decanted solution. The solution was partially concentrated in vacuo to mostly remove acetonitrile and was then purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm). The collected fractions were combined and concentrated until a volume of 4 mL was left. The resulting solution consisting of mostly acetonitrile was dried over Na.sub.2SO.sub.4, filtered and the drying agent was washed through with anhydrous THF (3). The combined organic layers were again partially concentrated to a volume of 1.7 mL (acetonitrile/THF) and compound 5f (9.32 mM based on a doxorubicin-based calibration line for HPLC, 15.8 mM, 35.7%) was used as such. LCMS (ESI+) calculated for C.sub.39H.sub.52NO.sub.13Si.sup.+ (M+H.sup.+) 770.32 found 770.65.

    Example a40. Synthesis of Compound 6f

    ##STR00098##

    [0548] To compound 5f (12.9 mg, 15.8 mol) in a mixture of acetonitrile and THF (1.7 mL) was added triethylammonium acetate (13.8 mg, 13.7 L, 85.5 mol) and the resulting red solution was cooled to 15 C. Then, while the RM was stirred TBAF (1 M in THF, 22.4 mg, 85.5 L, 85.5 mol) was added (color change from red to green and back to red observed). After stirring for 46 minutes additional triethylammonium acetate (13.8 mg, 13.7 L, 85.5 mol) and TBAF (1 M in THF, 22.4 mg, 85.5 L, 85.5 mol) were added. The RM was stirred for another 20 minutes before a third batch of TBAF (1 M in THF, 22.4 mg, 85.5 L, 85.5 mol) was added, which was stirred for another 30 minutes. Finally, a fourth batch of TBAF (1 M in THF, 10.0 mg, 40.0 L, 40.0 mol) was added and the RM was stirred at 10 C. for another 35 minutes before the reaction was quenched with water (1.0 mL). The RM was allowed to warm to rt, diluted with DCM (7.5 mL) and transferred to a separation funnel. The biphasic system was separated, and the water layer was extracted with DCM (21 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered then purified by flash column chromatography over silicagel (0%.fwdarw.6% MeOH in DCM). The pure fractions were combined and partially concentrated to a volume of 4.5 mL and then diluted with MeOH (7 mL). Next, the solution was again partially concentrated to a volume of 6 mL and diluted until a volume of 6.7 mL to give compound 6f as a red solution in mostly MeOH (6.7 mL, 1.55 mM based on a doxorubicin-based calibration line for HPLC, 10.36 mol, 65.6%). LCMS (ESI+) calculated for C.sub.33H.sub.38NO.sub.13.sup.+ (M+H.sup.+) 656.23 found 656.59.

    Example a41. Synthesis of Compound 3a

    ##STR00099##

    [0549] To a solution of nemorubicin (75 mg, 0.12 mmol) and imidazole (40 mg, 0.58 mmol) in anhydrous DMF (2.0 mL) was added TBDMS-CI (53 mg, 0.35 mmol, 3 equiv.). The resulting red solution was mixed and left at rt for 15 minutes. Next, the mixture was diluted with DCM (22 mL) and purified by flash column chromatography over silicagel (1%.fwdarw.5% MeOH in DCM) to give compound 3a as a dark red oil (134 mg, quant.). LCMS (ESI+) calculated for C.sub.38H.sub.52NO.sub.13Si.sup.+ (M+H.sup.+) 758.32 found 758.59.

    Example a42. Synthesis of Compound 4a

    ##STR00100##

    [0550] A solution of compound 3a (120 mol) in anhydrous DCM (15.0 mL) was cooled with a dry ice/acetone cooling-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in anhydrous DCM (33.1 mg, 580 mM, 331 L, 192 mol) was added dropwise, while stirring vigorously. After stirring for 18 minutes the mixture was quenched with a pre-cooled (78 C.) solution of acetone (reagent grade, 1.1 mL. After 23 min the cooling-bath was removed, and the RM was allowed to warm up to room temperature. The RM was transferred to a separation funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (3 mL). The water layers were combined and extracted once with DCM (4 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and partially concentrated in vacuo to a volume of 12 mL, affording compound 4a as a red solution in mostly DCM, which was used without further purification. LCMS (ESI+) calculated for C.sub.38H.sub.52NO.sub.14Si.sup.+ (M+H.sup.+) 774.32 found 774.50.

    Example a43. Synthesis of Compound 5a

    ##STR00101##

    [0551] To a solution of compound 4a (100.6 mol) in a mixture (10.1 ml) of mainly DCM and minimal acetone was added anhydrous acetonitrile (8 mL). The RM was partially concentrated to remove the DCM to a volume of circa 4 mL. Next, potassium carbonate (54.5 mg, 394 mol) was added, and the RM was cooled to 0 C. after which a solution of cyanuric chloride in anhydrous acetonitrile (46.4 mg, 2.266 mL, 111 mM, 251.5 mol) was added. After stirring for 150 minutes at 0 C., the RM was quenched with a solution of 3-aminopropane-1,2 diol (1.33 mL, 929 mM, 1.24 mmol) in water. The resulting dark red solution was allowed to slowly warm to room temperature over 20 minutes. To the RM was added DMF (1.33 mL) and the resulting mixture was partially concentrated and then purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm). The collected fractions were combined and concentrated until a volume of 4 mL was left. The resulting solution consisting of mostly acetonitrile was dried over Na.sub.2SO.sub.4, filtered and the drying agent was washed through with anhydrous THF (3). The combined organic layers were again partially concentrated to a volume of 4.5 mL (acetonitrile/THF) to give compound 5a as a red solution, which was used as such in the next step. LCMS (ESI+) calculated for C.sub.38H.sub.48NO.sub.13Si.sup. (MH.sup.+) 754.29 found 754.52.

    Example a44. Synthesis of Compound 6a

    ##STR00102##

    [0552] To compound 5a (40 mg, 53 mol) in a mixture of acetonitrile and THF (4.5 mL) was added triethylammonium acetate (170 L, 1.06 mmol) and the resulting red solution was cooled to 15 C. Then, while the RM was stirred TBAF (1 M in THF, 65 mg, 0.25 mL, 0.25 mmol) was added (color change from red to green and back to red observed). After stirring for 1 minute additional TBAF (1 M in THF, 63 mg, 0.24 mL, 0.24 mmol) were added. The RM was stirred for another 14 minutes before a third batch of TBAF (1 M in THF, 46 mg, 0.17 mL, 0.17 mmol) was added, which was stirred for another 41 minutes. The reaction was quenched with water (2.4 mL). The RM was allowed to warm to rt over 25 minutes and then combined with a 2.sup.nd batch of crude compound 6a, which was obtained in the same manner as described above starting with compound 5a (10 mg, 13.0 mol). After combining both quenched reaction mixtures DCM (6 mL) was added, and the resulting biphasic system was separated. The water layer was extracted two times (4 mL then 2 mL) and the combined organic layers were dried (Na.sub.2SO.sub.4) and then purified by flash column chromatography over silicagel (0%.fwdarw.6% MeOH in DCM). The pure fractions were combined and partially concentrated to a volume of 8-9 mL and then diluted with additional DCM to a volume of 10.0 mL to give compound 6a as a red solution in mostly DCM (10.0 mL, 1.66 mM based on a doxorubicin-based calibration line for HPLC, 16.6 mol, 16.5% over 2 steps (assuming quantitative conversion during N-oxide formation)). LCMS (ESI+) calculated for C.sub.32H.sub.36NO.sub.13.sup.+ (M+H.sup.+) 642.22 found 642.46.

    Example a45. Synthesis of Compound 8b

    ##STR00103##

    [0553] A mixture of doxorubicin.Math.HCl salt (48.6 mg, 83.8 mol, 1.00 eq) and(S)-1-(2-azidoethoxy)-2-iodo-1-(2-iodoethyoxy)ethane 7b (101.0 mg, 245.8 mol, 2.93 eq) were dissolved in anhydrous DMF (175 L) and DIPEA (58.7 L, 335 mol, 4 eq) was added. The red suspension was stirred in the dark at room temperature for 3 days, diluted with DCM (1.8 mL) and purified by flash column chromatography over silicagel (column preconditioned 2% MeOH/DCM, 2%.fwdarw.8% MeOH in DCM) to give compound 8b as a red oil (10.8 mg, 15.5 mol, 18.4%). LCMS (ESI+) calculated for C.sub.33H.sub.39N.sub.4O.sub.13.sup.+ (M+H.sup.+) 669.25 found 699.62.

    Example a46. Synthesis of Compound 9

    ##STR00104##

    [0554] To a solution of 8b (8.1 mg, 12 mol, 1.0 equiv.) in a 1:1 mixture of MeOH/DCM (150 L) was added a 200 mmolar solution of PPh3 in DCM (177 L, 31.9 mol, 2.7 equiv.) and H.sub.2O (80 L). The resulting biphasic system was stirred at rt in the dark for 8 hours. Next, the RM was stored in the freezer for 8 days and was then concentrated in vacuo. The residue was taken up in DMF and purified by prep-HPLC purification (5%.fwdarw.90% acetonitrile in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm), affording compound 9 as a red residue (0.5 mg, 0.7 mol, 6% yield). The impure fractions from the prep-HPLC purification were combined and purified by a 2.sup.nd prep-HPLC purification (5%.fwdarw.90% acetonitrile in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm), affording additional compound 9 as a red residue (0.6 mg, 0.9 mol, 8% yield). LCMS (ESI+) calculated for C.sub.33H.sub.41N.sub.2O.sub.13.sup.+ (M+H.sup.+) 673.26 found 673.65.

    Example a47. Synthesis of Compound 8c

    ##STR00105##

    [0555] A mixture of doxorubicin.Math.HCl salt (51.3 mg, 88.5 mol, 1.00 eq) and (R)-2-(2-iodo-1-(2-iodoethoxy)ethoxy) propane 7c (104 mg, 271 mol, 3.06 eq) were dissolved in anhydrous DMF (175 L) and DIPEA (61.6 L, 354 mol, 4 eq) was added. The red suspension was stirred in the dark at room temperature for 3 days, diluted with DCM (1.8 mL) and purified by flash column chromatography over silicagel (column preconditioned 2% MeOH/DCM, 2%.fwdarw.8% MeOH in DCM) to give compound 8c as a red oil (12.2 mg, 18.2 mol, 20.5%). LCMS (ESI+) calculated for C.sub.34H.sub.42NO.sub.13.sup.+ (M+H.sup.+) 672.27 found 672.60.

    Example a48. Synthesis of Compound 8d

    ##STR00106##

    [0556] A mixture of doxorubicin.Math.HCl salt (49.7 mg, 85.7 mol, 1.00 eq) and (S)-((2-iodo-1-(2-iodoethoxy)ethoxy)methyl)benzene 7d (106.5 mg, 246.5 mol, 2.88 eq) were dissolved in anhydrous DMF (175 L) and DIPEA (59.7 L, 343 mol, 4 eq) was added. The red suspension was stirred in the dark at room temperature for 3 days, diluted with DCM (1.8 mL) and purified twice by flash column chromatography over silicagel (column preconditioned 2% MeOH/DCM, 0%->13% MeOH in DCM) to give compound 8d as a red oil (6.4 mg, 8.9 mol, 10%). LCMS (ESI+) calculated for C.sub.38H.sub.42NO.sub.13.sup.+ (M+H.sup.+) 720.27 found 720.64.

    Example a49. Synthesis of Compound 8f

    ##STR00107##

    [0557] A mixture of doxorubicin.Math.HCl salt (52.3 mg, 90.2 mol, 1.00 eq) and(S)-1-ethoxy-2-iodo-1-(2-iodoethyoxy)ethane 7f (102.1 mg, 276.0 mol, 3.06 eq) were dissolved in anhydrous DMF (175 L) and DIPEA (62.8 L, 361 mol, 4 eq) was added. The red suspension was stirred in the dark at room temperature for 4 days, diluted with DCM (1.8 mL) and purified by flash column chromatography over silicagel (column preconditioned 2% MeOH/DCM, 2%.fwdarw.8% MeOH in DCM) to give compound 8f as a red oil (11.1 mg, 16.9 mol, 18.7%). LCMS (ESI+) calculated for C.sub.3H.sub.40NO.sub.13.sup.+ (M+H.sup.+) 658.25 found 658.70.

    Example a50. Synthesis of Compound 31

    ##STR00108##

    [0558] To a solution of Fmoc-N-ethylene-1,2-diamine.Math.HCl (26 mg, 83 mol) in anhydrous DMF (200 L) was added a solution of compound 29 (67 mg, 75 mol) in anhydrous DCM (800 L) and triethylamine (32 L, 23 mg, 230 mol). After stirring for 1 hour at room temperature, the reaction mixture was purified by flash column chromatography over silicagel (0%.fwdarw.30% EtOAc in DCM (to remove p-nitrophenol) followed by 0%.fwdarw.25% MeOH in DCM) to give intermediate 30 as a colorless oil (32.7 mg, 32 mol, 43%). LCMS (ESI+) calculated for C.sub.49H.sub.62N.sub.7O.sub.13S.sup.+ (M+H.sup.+) 988.41 found 988.78.

    [0559] To a solution of intermediate 30 (16.3 mg, 16.5 mol) in DMF (150 L) was added triethylamine (13.8 L, 10 mg, 99.0 mol). After stirring for 18 hours at room temperature complete conversion was obtained and the reaction mixture was concentrated to obtain compound 31 as an oil (12.6 mg, 16.5 mol, 100%). LCMS (ESI+) calculated for C.sub.34H.sub.52N.sub.7O.sub.11S.sup.+ (M+H.sup.+) 766.34 found 766.65.

    ##STR00109##

    Example a51. Synthesis of Compound 9c

    [0560] To a solution of compound 6c (3.26 mg, 4.87 mol) in DCM was added MeOH (3 mL) and the mixture was concentrated until only MeOH (1.5 mL) was left. Water (200 L) was added and a solution of sodium periodate in water (60 mM, 282 L, 16.9 mol) was added and the reaction mixture was stirred for 41 hours in the dark. Once complete conversion was achieved, DMF (800 L) was added and the reaction mixture was concentrated till there was 400 L in DMF was left (2.19 mg, 3.34 mol, 68.6%). This intermediate was then added to compound 31 (10.2 mg, 13.2 mol) followed by DiPEA (2 L, 10 mol) and HATU (2.1 mg, 28 L, 200 mM, 5.4 mol). After stirring for 20.5 hours at room temperature, the reaction mixture was purified by prep-HPLC (40%->95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). Compound 9c was obtained as a red solution in DMF (250 L, 3.83 mM based on a doxorubicin-based calibration line for HPLC, 1.34 mg, 0.95 mol, 28.6%). LCMS (ESI+) calculated for C.sub.67H.sub.87N.sub.8O.sub.23S.sup.+ (M+H.sup.+) 1404.51 found 1404.06.

    Example a52. Synthesis of Compound 9d

    [0561] To a solution of compound 6d (3.4 mg, 4.7 mol) in DCM was added MeOH (5 mL) and the mixture was concentrated until only MeOH (2.1 mL) was left. Then a solution of sodium periodate in water (60 mM, 288 L, 17.5 mol) was added and the reaction mixture was stirred for 68 hours in the dark. Once complete conversion was achieved, DMF (600 L) was added and the reaction mixture was concentrated till there was 490 L in DMF was left (1.78 mg, 2.53 mol, 53%). This intermediate was then added to compound 31 (6.2 mg, 8.1 mol) followed by DiPEA (1.32 L, 7.59 mol) and HATU (1.15 mg, 15.2 L, 200 mM, 3.04 mol). After stirring for 21.5 hours at room temperature, the reaction mixture was purified by prep-HPLC (40%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). Compound 9d was obtained as a red solution in DMF (165 L, 10.14 mM based on a doxorubicin-based calibration line for HPLC, 2.42 mg, 1.67 mol, 65.9%). LCMS (ESI+) calculated for C.sub.71H.sub.87N.sub.8O.sub.23S.sup.+ (M+H.sup.+) 1452.56 found 1452.03.

    Example a52-2. Synthesis of Compound 9f

    [0562] To a solution of compound 6f (6.8 mg, 10.4 mol) in a mixture of MeOH (6.7 mL) and water (1.4 mL) was added a solution of sodium periodate in water (62.9 mM, 206 L, 13.0 mol) was added and the reaction mixture was stirred at rt for 3 hours in the dark. Additional sodium periodate in water (62.9 mM, 210 L, 13.2 mol) was added and the reaction mixture was stirred at rt for another 17 hours. Finally, a 3rd batch of sodium periodate in water (62.9 mM, 50 L, 3.1 mol) was added and the mixture was stirred at rt for 80 minutes and was then partially concentrated in vacuo to a volume of 5.4 mL and then left at rt for another 5 hours. DMF (670 L) was then added, and the resulting red solution was partially concentrated to a volume of circa 350 L, affording a white residue and a red solution containing crude intermediate. The mixture was diluted with additional DMF to 666 L and 222 L (3.45 mol) of this solution was then treated with a stock solution of compound 31 in DMF (110 mmolar, 62.7 L, 6.9 mol) followed by DiPEA (1.79 L, 10.4 mol) and a solution of HATU in dry DMF (204 mM, 16.9 L, 3.45 mol). The resulting mixture was vortexed and left at room temperature for 31 minutes. Next, additional compound 31 in DMF (110 mmolar, 13.9 L, 1.53 mol) and HATU in dry DMF (204 mM, 33.8 L, 6.90 mol) were added. The mixture was again vortexed and left at room temperature for 13 minutes and then stored in the freezer for 17 hours. Finally, the mixture was removed from the freezer and purified by prep-HPLC (40%->95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). Compound 9f was obtained as a red solution in DMF (300 L, 8.2 mM based on a doxorubicin-based calibration line for HPLC, 3.9 mg, 2.47 mol, 71.6%). LCMS (ESI+) calculated for C.sub.66H.sub.85N.sub.8O.sub.23S.sup.+ (M+H.sup.+) 1389.54 found 1390.07.

    Example a53. Synthesis of Compound 9g (OMe-PNU)

    [0563] To a solution of compound 6a (2.17 mg, 3.38 mol) in DCM was added MeOH (3 mL) and the mixture was concentrated until only MeOH (1.35 mL) was left. Water (300 L) was added and a solution of sodium periodate in water (60 mM, 112.4 L, 6.8 mol) was added and the reaction mixture was stirred for 19 hours in the dark. The stirring bar was removed, and the RM was concentrated in vacuo, DMF (200 L) was added to the residue, affording a white residue and a red solution containing crude intermediate. To this intermediate was then added compound 31 (6 mg, 7.8 mol) followed by DiPEA (1.8 L, 10.5 mol) and HATU (2.05 mg, 5.4 L, 196 mM, 5.4 mol). After 25 minutes at room temperature, the reaction mixture was purified by prep-HPLC (40%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). Compound 9g was obtained as a red solution in DMF (122 L, 14.24 mM based on a doxorubicin-based calibration line for HPLC, 2.4 mg, 1.74 mol, 80.9%). LCMS (ESI+) calculated for C.sub.65H.sub.83N.sub.8O.sub.23S.sup.+ (M+H.sup.+) 1375.53 found 1376.01.

    Example a54. Synthesis of Compound 32

    ##STR00110##

    [0564] To a solution of compound 3b (80.4 mg, 98.9 mol) in MeOH (600 L) was added a solution of triphenylphosphine in DCM (88.2 mg, 967 L, 348 mM, 336 mol) and water (450 L). The biphasic mixture was stirred for 3 hours at room temperature after which Fmoc-Val-Ala-PAB-OPNP (79.4 mg, 117 mol) was added. After stirring the biphasic mixture for an additional 16 hours at room temperature, an extraction with DCM (21 mL) was done to remove the water. The combined organic layers were dried over Na.sub.2SO.sub.4 and immediately purified by flash column chromatography over silicagel (0%.fwdarw.40% EtOAc in DCM (to remove excess Fmoc-Val-Ala-PAB-OPNP) followed by 0%.fwdarw.15% MeOH in DCM) to give compound 32 as a clear red solution in DCM (7.7 mL, 123.6 mg, 93 mol, 94.1%). LCMS (ESI+) calculated for C.sub.70H.sub.86N.sub.5O.sub.19Si.sup.+ (M+H.sup.+) 1329.54 found 1329.00.

    Example a55. Synthesis of Compound 33

    ##STR00111##

    [0565] A solution of compound 32 (123.6 mg, 93 mol) in anhydrous DCM (7.7 mL) was concentrated until a 2 mL solution was left. Next, the reaction mixture was cooled with dry ice/acetone cooling-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of m-CPBA in anhydrous DCM (17.66 mg, 580 mM, 176 L, 102.3 mol) was dropwise added. After stirring for 17 minutes, full conversion was obtained. The RM was quenched with an ice-cold solution of acetone (reagent grade, 2.5 mL) and the RM was stirred. After an hour the cold bath was removed, and the RM was allowed to warm up to room temperature. The RM was transferred to a separation funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (10 mL). The water layers were combined and extracted twice with DCM (20 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered through a filter paper, and concentrated till a volume of 19 mL was obtained. No further purification was performed and compound 33 (108.3 mg, 80.5 mol, 86.6%) was used as such. LCMS (ESI+) calculated for C.sub.70H.sub.86N.sub.5O.sub.20Si.sup.+ (M+H.sup.+) 1345.54 found 1345.14.

    Example a56. Synthesis of Compound 34 and 35

    ##STR00112##

    [0566] To a solution of compound 33 (108.3 mg, 80.5 mol) in DCM (19 mL) was added anhydrous acetonitrile (10 mL). The RM was concentrated to remove the DCM and to obtain the compound in anhydrous acetonitrile (5 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (45 mL). Then, potassium carbonate (65 mg, 463 mol) was added, and the RM was cooled to 10 C. after which cyanuric chloride (55.7 mg, 2.44 mL, 120 mM, 302.2 mol) as a stock solution in anhydrous acetonitrile was added. After stirring for 5.5 h at 0 C., the RM was quenched with a solution of 3-aminopropane-1,2 diol (90.3 mg, 1.1 mL, 900 mM, 990 mol) in water. The ice bath was removed after 15 min, and it was allowed to warm up to room temperature. To the RM was added DMF (3 mL) and the RM was concentrated till only a solution of DMF/water was left and purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). The collected fractions were combined and concentrated till a volume of 11 mL acetonitrile was left. Then it was dried over Na.sub.2SO.sub.4, filtered and the drying agent was washed through with anhydrous THF (31 mL). Intermediate 34 was obtained as a solution in acetonitrile/THF (14 mL, 8.7 mg, 6.6 mol, 8.1%). LCMS (ESI+) calculated for C.sub.70H.sub.84N.sub.5O.sub.19Si.sup.+ (M+H.sup.+) 1327.52 found 1327.07.

    [0567] A solution of intermediate 34 (8.7 mg, 6.6 mol) in anhydrous acetonitrile/THF (14 mL) was concentrated till 7 mL solution was left and anhydrous THF (1 mL) was added. To this was added triethylammonium acetate (5.3 mg, 5.3 mol, 33 mol) and the resulting red solution was cooled to 15 C. Then, while the RM was vigorously stirred TBAF (1 M in THF, 17.2 mg, 66 L, 66 mol) was added (color change from red to green observed). After stirring for 3 h, the RM was quenched with water (1.5 mL) and RM turned red again. The RM was transferred to a separation funnel and extracted with DCM (20 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and directly purified by flash column chromatography over silicagel (0%.fwdarw.10% MeOH in DCM) to give compound 35 as a red solution in DCM (8 mL, 2.3 mg, 1.9 mol, 29%). LCMS (ESI+) calculated for C.sub.64H.sub.70N.sub.5O.sub.19.sup.+ (M+H.sup.+) 1212.47 found 1212.93.

    Example a57. Synthesis of Compound 36

    ##STR00113##

    [0568] Synthesis of BCN-HS-PEG2-OPNP has been described in WO2021144314A1, which is incorporated herein by reference. To a solution of compound 35 (2.3 mg, 1.9 mol) in DCM (8 mL) was added DMF (100 L) and the RM was concentrated to remove the DCM. Then, BCN-HS-PEG2-OPNP (1.2 mg, 25 l, 93 mM, 2.3 mol) a solution in DMF was added followed by triethylamine (2.6 L, 19 mol). After 30 hours at room temperature, the RM was further diluted with DCM (300 L) and purified by flash column chromatography over silicagel (0%.fwdarw.15% MeOH in DCM) to give compound 36 as a red solution in DMF (150 L, 0.64 mM based on a doxorubicin-based calibration line for HPLC, 0.13 mg, 0.09 mol, 5%). LCMS (ESI+) calculated for C.sub.65H.sub.82N.sub.7O.sub.24S.sup.+ (M+H.sup.+) 1377.44 found 1377.04.

    Example a58. Synthesis of Compound 38

    ##STR00114##

    [0569] This compound was synthesized according to a literature procedure described in WO2017137457A1.

    Example a59. Synthesis of Compound 39

    ##STR00115##

    [0570] To a solution of compound 6e (1.0 mg, 1.3 mol, 1.00 equiv.) in non-dry THF (90 L) was added a solution of PPh3 (200 mmolar, 13 L, 2.6 mol, 2.0 equiv.) in THF and H.sub.2O (13 L). The resulting red solution was vortexed and then left at room temperature for 19 hours. The mixture was then transferred to an Eppendorf vial and put in an Eppendorf shaker at 37 C. and 1400 RPM for 6.5 hours and then for another 19 hours at room temperature at 1400 RPM. The reaction mixture was then stored in the freezer for 11 days and then treated with a solution of compound 38 (790 mM, 2.0 L, 1.6 mol, 1.2 equiv.) in DMF, followed by the addition of HATU in DMF (517 mM, 1.4 mol, 1.1 equiv.) and finally a solution of DMAP in DMF (500 mM, 1.1 L, 0.53 mol, 0.4 equiv.). The resulting mixture was vortexed and left at room temperature for 3 hours and was then stored in the freezer for 21 hours. The mixture was removed from the freezer and purified by prep-HPLC (40%.fwdarw.100% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). The pure fractions were combined and diluted with DMF and partially concentrated to give compound 39 as a red solution in DMF (85 L, 1.5 mM based on a doxorubicin-based calibration line for HPLC, 0.17 mg, 0.13 mol, 10%). LCMS (ESI+) calculated for C.sub.62H.sub.77N.sub.6O.sub.22S.sup.+ (M+H.sup.+) 1289.48 found 1289.99.

    Example a60. Synthesis of Compound 42 and 43

    ##STR00116##

    [0571] To vial containing Boc-Gly-Gly-Phe-Gly-OH (500 mg, 1.15 mmol, 1.00 equiv.) and Fmoc-EDA-H (476 mg, 1.69 mmol, 1.47 equiv.) was added DMF (1.25 mL), followed by Et.sub.3N (479 UL, 3.44 mmol, 3.00 equiv.) and finally HATU (479 mg, 1.26 mmol, 1.10 equiv.). The resulting mixture was stirred at room temperature for 2 minutes before adding DCM (2 mL), generating a clear yellow solution, which was stirred at room temperature for 110 minutes. The reaction mixture was then stored in the freezer for 16 hours. The mixture was then removed from the freezer and diluted with DCM (15 mL) and purified directly via flash column chromatography (50.fwdarw.100% EtOAc in heptane). Fractions containing the product were concentrated, affording compound 42 as a white solid (357 mg, 509 mol, 45%). LCMS (ESI+) calculated for C.sub.37H.sub.45N.sub.6O.sub.8.sup.+ (M+H.sup.+) 701.33 found 701.67. Compound 42 (357 mg, 509 mol, 1.00 equiv.) was suspended in DCM (3.0 mL) and cooled to 0 C. with an ice-bath. Next, TFA (1.37 mL, 17.8 mmol, 35 equiv.) was added dropwise, generating a light-yellow solution that was stirred at 0 C. for 47 minutes. The ice-bath was then removed, and the RM was warmed to room temperature and stirred for another 75 minutes. The mixture was concentrated in vacuo and the residue was taken up in a mixture of DCM (1.5 mL) and toluene (2 mL) and concentrated a 2.sup.nd time, affording compound 43 (TFA-salt) as a brittle solid, which was used without further purification in the next step. Alternatively, compound 43 was subjected to a prep-HPLC purification (5%.fwdarw.95% acetonitrile with 1% AcOH in water with 1% AcOH, column Xbridge prep C18, 5 M OBD, 30100 mm) to obtain compound 43 as an acetate-salt. LCMS (ESI+) calculated for C.sub.32H.sub.37N.sub.6O.sub.6.sup.+ (M+H.sup.+) 601.28 found 601.60.

    Example a61. Synthesis of Compound 45 and 46

    ##STR00117##

    [0572] To a solution of BCN-OH 44 (40 mg, 90 wt % according to .sup.1H-qNMR, 240 mol, 1.00 equiv.) in dry DCM (2.5 mL) at 40 C. in a dry ice/MeCN bath was added chlorosulfonyl isocyanate (22 L, 250 mol, 1.05 equiv.) in one portion, resulting in a pale-yellow solution. The mixture was stirred at 40 C. for 17 minutes, followed by the addition of Et.sub.3N (67 L, 480 mol, 2.00 equiv.). The resulting solution was stirred for circa 5 minutes at 40 C. before adding a solution of the acetate-salt of compound 43 (80 mg, 120 mol, 0.51 equiv.) and DIPEA (23 L) in DMF (250 L), followed after 10 minutes by another addition with the TFA-salt of compound 43 (115.4 mg, 161.5 mol, 0.67 equiv.) as a suspension in a mixture of DIPEA (46 L) and Et.sub.3N (23 L) in DMF (5 mL). Finally, dimethylacetamide (1 mL) was added and the reaction mixture was allowed to warm to room temperature over 5 hours. The reaction mixture was then stored in the freezer for 5 days and was then removed from the freezer and concentrated in vacuo. The residue was purified via by prep-HPLC (40%.fwdarw.90% acetonitrile with 1% AcOH in water with 1% AcOH, column Xbridge prep C18, 5 M OBD, 30100 mm) to give compound 45 (8.9 mg, 9.5 mol, 4% yield). LCMS (ESI+) calculated for C.sub.43H.sub.50N.sub.7O.sub.10S.sup.+ (M+H.sup.+) 856.33 found 856.66. To a solution of compound 45 (8.9 mg, 9.5 mol in non-dry DMF (400 L) was added Et.sub.3N (58 L, 0.42 mmol, 40 equiv.). The resulting mixture was mixed and left at room temperature for circa 18 hours and was then concentrated in vacuo to a volume of circa 25 L. This solution was transferred to an Eppendorf vial with additional DMF to give compound 46 as a solution in DMF with final volume of 150 UL, which was used without further purification in the next step. LCMS (ESI+) calculated for C.sub.28H.sub.40NO.sub.8S.sup.+ (M+H.sup.+) 634.27 found 634.54.

    Example a62. Synthesis of Compound 47

    ##STR00118##

    [0573] To a solution of compound 6f (6.8 mg, 10.4 mol) in a mixture of MeOH (6.7 mL) and water (1.4 mL) was added a solution of sodium periodate in water (62.9 mM, 206 L, 13.0 mol) and the reaction mixture was stirred at rt for 3 hours in the dark. Additional sodium periodate in water (62.9 mM, 210 L, 13.2 mol) was added and the reaction mixture was stirred at rt for another 17 hours. Finally, a 3rd batch of sodium periodate in water (62.9 mM, 50 L, 3.1 mol) was added and the mixture was stirred at rt for 80 minutes and was then partially concentrated in vacuo to a volume of 5.4 mL and then left at rt for another 5 hours. DMF (670 L) was then added, and the resulting red solution was partially concentrated to a volume of circa 350 L, affording a white residue and a red solution containing crude intermediate. The mixture was diluted with additional DMF to 666 L and 222 L (3.45 mol) of this solution was then treated with a stock solution of compound 46 in DMF (66.6 mmolar, 150 L, 9.99 mol) followed by DiPEA (1.80 L, 10.4 mol) and a solution of HATU in dry DMF (490 mM, 7.1 L, 3.5 mol). The resulting mixture was vortexed and left at room temperature for 17 minutes. Next, additional HATU in dry DMF (490 mM, 7.1 L, 3.5 mol) was added. The mixture was again vortexed and left at room temperature for 23 minutes. A 3rd batch of HATU in dry DMF (490 mM, 7.1 L, 3.5 mol) was added. The resulting mixture was vortexed and left at room temperature for 14 minutes. Next, additional compound 46 (2.4 mg, 3.8 mol) was added to the RM, followed after 14 minutes by additional compound 46 (2.4 mg, 3.8 mol) and a 4th batch of HATU in dry DMF (490 mM, 7.1 L, 3.5 mol). The mixture was stirred at rt for another 29 minutes before adding additional DiPEA (1.80 L, 10.4 mol). The mixture was then pushed over a membrane-filter and then treated with a 5th and final batch of HATU in dry DMF (490 mM, 7.1 L, 3.5 mol). The mixture was vortexed and left at room temperature for 80 minutes and was then purified by prep-HPLC (40%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C18, 5 M OBD, 30100 mm). Compound 47 was obtained as a red solution in DMF (196 L, 3.5 mM based on a doxorubicin-based calibration line for HPLC, 1.0 mg, 0.684 mol, 20%). LCMS (ESI+) calculated for C.sub.60H.sub.73N.sub.8O.sub.20S.sup.+ (M+H.sup.+) 1257.47 found 1357.86.

    Example a63. Synthesis of Compound 49 and 50

    ##STR00119##

    [0574] To a suspension of (tert-butoxycarbonyl) glycylglycylglycine (337.4 mg, 1.17 mmol, 1.00 equiv.) and Fmoc-EDA-H (368.8 mg, 1.306 mmol, 1.12 equiv.) in DMF (1 mL) was added triethylamine (488 L, 3.50 mmol, 3.00 equiv.). To the resulting suspension was added HATU (494.8 mg, 1.301 mmol, 1.12 equiv.). The resulting yellow mixture was stirred at room temperature for 2 minutes, followed by the addition of DCM (2 mL), generating a yellow solution which was stirred at room temperature for another 90 minutes. The mixture was then stored in the freezer for 1 day. The mixture was removed from the freezer and then purified via flash column chromatography (0.fwdarw.10% MeOH in DCM), affording the product 50 (443.5 mg, 801.1 mol, 68.7%) as a white solid. LCMS (ESI+) calculated for C.sub.28H.sub.36N.sub.5O.sub.7.sup.+ (M+H.sup.+) 554.26 found 554.60. Compound 50 was then dissolved in DCM (2.00 mL) and the resulting mixture was cooled in an ice-bath to 0 C. Next, TFA (400 L, 5.19 mmol, 7.05 equiv.) was added dropwise to the reaction mixture while stirring. Upon complete addition the ice-bath was removed, and the resulting solution was stirred at room temperature for 85 minutes. Additional TFA (1.6 mL, 21 mmol, 28 equiv.) was added portion-wise and the resulting mixture was stirred at room temperature for another 200 minutes. The mixture was then concentrated in vacuo, and the residue was then purified by prep-HPLC (5%.fwdarw.90% acetonitrile in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). Compound 50 was obtained as white powder (84.9 mg, 186 mol, 25%). LCMS (ESI+) calculated for C.sub.23H.sub.28N.sub.5O.sub.5.sup.+ (M+H.sup.+) 454.21 found 454.54.

    Example a64. Synthesis of Compound 51 and 52

    ##STR00120##

    [0575] To a solution BCN-OH 44 (30.5 mg, 90 wt % according to .sup.1H-qNMR, 183 mol, 1.00 equiv.) in dry DCM (2.1 mL) at 40 C. in a dry ice/MeCN bath was added chlorosulfonyl isocyanate (16.7 L, 192 mol, 1.05 equiv.) in one portion resulting in a pale-yellow solution. The mixture was stirred at 40 C. for 5 minutes, followed by the addition of Et.sub.3N (76.4 L, 548 mol, 3.00 equiv.). The resulting solution was stirred for circa 14 minutes at 40 C. before adding a solution of compound 50 (84.9 mg, 187 mol, 1.02 equiv.) in DMF (450 L). This reaction mixture was stirred at 40 C. for 10 minutes and was then allowed to warm to room temperature over 2 hours. The reaction mixture was then stored in the freezer for 1 day and then removed from the freezer and diluted with DCM and purified via flash column chromatography (0.fwdarw.13% MeOH in DCM), affording impure intermediate 51 (containing Et.sub.3N) as two batches (in total 45 mol, 25% yield). The two batches were combined and co-evaporated with DMF. The resulting oil was diluted with DCM (10 mL) and washed twice with sat. aq. NH.sub.4Cl (2 mL, 2). During the extraction a gummy oil was formed which was combined with the organic layer and concentrated in vacuo, affording intermediate 51 as a brown gummy solid. The residue was taken up in 600 L DMF, followed by the addition of Et.sub.3N (240 L, 1.70 mmol, 40 equiv.). The resulting mixture was stirred at room temperature for 1 day and was then stored in the freezer for 3 days. The reaction mixture was removed from the freezer and filtered over a membrane-filter, which was washed with additional DMF (3). The resulting yellow solution was partially concentrated to a volume of circa 240 L to give compound 52 as a yellow solution in DMF, which was used without further purification in the next step. LCMS (ESI+) calculated for C.sub.19H.sub.31N.sub.6O.sub.7S.sup.+ (M+H.sup.+) 487.20 found 487.55.

    Example a65. Synthesis of Compound 53

    ##STR00121##

    [0576] To a solution of compound 6f (6.8 mg, 10.4 mol) in a mixture of MeOH (6.7 mL) and water (1.4 mL) was added a solution of sodium periodate in water (62.9 mM, 206 L, 13.0 mol) and the reaction mixture was stirred at rt for 3 hours in the dark. Additional sodium periodate in water (62.9 mM, 210 L, 13.2 mol) was added and the reaction mixture was stirred at rt for another 17 hours. Finally, a 3rd batch of sodium periodate in water (62.9 mM, 50 L, 3.1 mol) was added and the mixture was stirred at rt for 80 minutes and was then partially concentrated in vacuo to a volume of 5.4 mL and then left at rt for another 5 hours. DMF (670 L) was then added, and the resulting red solution was partially concentrated to a volume of circa 350 L, affording a white residue and a red solution containing crude intermediate. The mixture was diluted with additional DMF to 666 L and 222 L (3.45 mol) of this solution was then treated with a stock solution of 52 in DMF (175 mmolar, 39.4 L, 6.9 mol) followed by DiPEA (1.79 L, 10.4 mol) and a solution of HATU in dry DMF (500 mM, 6.90 L, 3.45 mol). The resulting mixture was vortexed and left at room temperature for 17 minutes. Next, additional 52 in DMF (175 mmolar, 157.6 L, 27.6 mol) was added. The mixture was again vortexed and left at room temperature for 14 minutes, followed by the addition of additional HATU in dry DMF (500 mM, 13.80 L, 6.90 mol). The mixture was again vortexed and left at room temperature for 13 minutes and then stored in the freezer for 2 days. Finally, the mixture was removed from the freezer and purified by prep-HPLC (40%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). Compound 53 was obtained as a red solution in DMF (165 L, 2.5 mM based on a doxorubicin-based calibration line for HPLC, 0.5 mg, 0.41 mol, 12%). LCMS (ESI+) calculated for C.sub.51H.sub.64N.sub.7O.sub.19S.sup.+ (M+H.sup.+) 1110.40 found 1110.85.

    Synthesis of Compound 57

    ##STR00122## ##STR00123##

    Example a66. Synthesis of Compound 54

    [0577] This compound was synthesized according to a literature procedure described by Pawar et al. in the International Journal of Pharmaceutics, Volume 436, Issues 1-2, Pages 183-193.

    Example a67. Synthesis of Compound 55

    [0578] To a round-bottom flask containing compound 54 (5.241 g, 1 Eq, 6.844 mmol) was added a solution of THF (160 mL) and water (160 mL). The resulting red suspension was cooled to 0 C. and then a cold solution of sodium periodate in H2O (1.464 g, 34.22 mL, 200 mmolar, 1.00 Eq, 6.844 mmol) was added dropwise over 15 minutes. The resulting red solution with some solids at the bottom was stirred on ice for a total of 10 minutes. The ice-bath was then removed, and the RM was allowed to warm to room temperature and stirred for 23 hours. The RM was partially concentrated in vacuo (removing all THF and circa 50% of the water, until 80 mbar), affording a red suspension in mostly water. The mixture was treated with DCM (200 mL) and the resulting suspension was rotated at 43 C. degrees for a few minutes. The bi-phasic system was transferred to a separation funnel. To the remaining residue in the round bottom flask was added additional 300 ml DCM and the resulting mixture was rotated at 43 C. again until the remaining solids had dissolved. The solution was also added to the separation funnel and the resulting bi-phasic system was shaken and separated. The aqueous layer was extracted twice with additional DCM (2200 ml). The separatory funnel-containing small amounts of dark red residualswas washed with 10% MeOH in DCM (150 mL), which completely solubilized the residue. This organic layer was washed with the water layer once. The resulting organic layer was then combined with the other organic layers. To the combined organic layers was added MeOH (40 mL) generating a clear solution. The combined organic layers were dried (Na.sub.2SO.sub.4) and then filtered over a glass-filter. The solution was treated with DMF (18 mL) and partially concentrated until mainly DMF was left as a solvent (until 20 mbar), affording intermediate 55 as a solution in DMF (5.30 g, 6.87 mmol) as a dark red solution (18 mL), which was used in the next step without further purification. Quantitative yield was assumed. UPLC-MS (ESI+) calculated for C.sub.41H.sub.37NO.sub.13.sup.+ [M+H.sup.+] 752.23 found 752.52.

    Example a68. Synthesis of Compound 56

    [0579] To a round-bottom flask containing compound 55 (4.10 g, 5.46 mmol, 1.00 equiv.) in DMF (15 ml) was added additional DMF until a total of circa 94 mL was reached. Next, allyl (2-aminoethyl) carbamate (2.14 g, 2.72 Eq, 14.9 mmol) was added in dry DMF (9.0 ml) and the RM was placed in a water-bath. Next, HATU (2.18 g, 1.05 Eq, 5.73 mmol) was added, followed within one minute by the addition of DIPEA (2.12 g, 2.85 mL, 3.00 Eq, 16.4 mmol) and the resulting dark red solution was stirred at rt for circa 30 min. Next, additional HATU (455 mg, 1.20 mmol, 0.22 equiv.) in DMF (1.0 mL) was added, followed after another 55 minutes by a third batch of HATU (509 mg, 1.34 mmol, 0.25 equiv.). The RM was stirred at rt for another 5 minutes and was then partially concentrated in vacuo until a volume of 15 ml. The residue was then diluted with DCM (135 ml) and loaded onto a pre-wetted column. The residue was then purified by flash column chromatography over silicagel (0.fwdarw.20% MeOH/DCM). Fractions containing product were combined and concentrated, affording intermediate 56 (3.61 g, 4.07 mmol, 74.5%, 99% purity) as a dark red thick oil. UPLC-MS (ESI+) calculated for C.sub.47H.sub.48N.sub.3O.sub.14.sup.+ [M+H.sup.+] 878.31 found 878.71.

    Example a69. Synthesis of Compound 57

    [0580] To a dark red solution of compound 56 (3.61 g, 99% Wt, 1.00 Eq, 4.07 mmol) in a total volume of DMF (17.0 mL) was added triethylamine (2.06 g, 2.84 mL, 5.0 Eq, 20.3 mmol). The RM turned very dark red and was left to stir at rt for 18 hours. Next, Et.sub.2O (78 mL) was added (slowly) in one portion, while rapidly stirring. The stirring was stopped and the ether-layer was then decanted and the remaining dark solid was washed a few more times with Et.sub.2O (3100 ml). The solid was concentrated in vacuo, affording intermediate 57 (4.8 g, 81.9% purity) as a dark red solid that was used without further purification. UPLC-MS (ESI+) calculated for C.sub.32H.sub.38N.sub.3O.sub.12.sup.+ [M+H.sup.+] 656.25 found 656.60.

    Example a70. Synthesis of Compound 58

    ##STR00124##

    [0581] To intermediate 57 (4.0 g, 90 wt %, 5.5 mmol), was suspended in dry DMF (6.0 mL), followed by the addition of bis-iodo-sugar 7c (6.3 g, 16 mmol, 3 equiv.) and DiPEA (2.9 mL, 16 mmol, 3 equiv.). The resulting mixture was rotated at 45 C. for 45 minutes. Next, the remaining lumps were mostly broken up with a spatula and the suspension was stirred at 40 C. for 2 days. The RM was diluted with DCM (100 mL) and the resulting red solution was purified by flash column chromatography over silicagel (0% MeOH/DCM then, 2%.fwdarw.10% MeOH in DCM) to give compound 58 as a red residue (4.03 mol, 72%). LCMS (ESI+) calculated for C.sub.39H.sub.50N.sub.3O.sub.14.sup.+ (M+H.sup.+) 784.33 found 784.78.

    Example a71. Synthesis of Compound 59

    ##STR00125##

    [0582] A solution of compound 58 (4.21 g, 4.78 mmol, 89 wt %) in a mixture of DCM (130 mL) and MeOH (10 mL) was cooled by dry ice/acetone-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in DCM (580 mM, 9.0 mL, 5.22 mmol) was added dropwise over 5-10 minutes. After stirring for 17 minutes, a 2.sup.nd batch of mCPBA in DCM (580 mM, 10.0 mL, 5.80 mmol) was added over 5 minutes and the RM was stirred for another 9 minutes, before finally adding a 3rd batch of mCPBA in DCM (580 mM, 2.5 mL, 1.50 mmol). The reaction mixture was stirred for another 13 minutes at 78 C. and was then quenched with cold (78 C.) acetone (reagent grade, 43.9 mL) and the RM was stirred. After 90 min the cold bath was removed, and the RM was allowed to warm up to 0 C. over 40 minutes. The RM was diluted with DCM (450 mL) and saturated aqueous NaHCO.sub.3 solution (250 mL) and transferred to a separation funnel. The resulting biphasic system was separated, and the water-layer was extracted twice with DCM (150, 100 mL). The combined organic layers were then washed again with sat. aq. NaHCO.sub.3 solution (100 mL). The new water-layer was extracted with DCM (40 mL) and the combined organic layers were dried over Na.sub.2SO.sub.4 and filtered and then concentrated to give compound 59 (3.30 g, 93% purity, 80% yield, 3.84 mmol), which was used as such in the next step. LCMS (ESI+) calculated for C.sub.39H.sub.50N.sub.3O.sub.15.sup.+ (M+H.sup.+) 800.32 found 800.70.

    Example a72. Synthesis of Compound 60

    ##STR00126##

    [0583] To a solution of 59 (181 mg, 60 wt %, 136 mol, 1.00 equiv.) in a mixture of dry DCM (1.0 mL) and anhydrous acetonitrile (4.0 mL) was added potassium carbonate (167.8 mg, 1.21 mmol, 8.94 equiv.). The RM was then cooled to 0 C. with an ice-bath after which a solution of cyanuric chloride (62.6 mg, 2.88 mL, 118 mM, 339 mol, 2.5 equiv.) in anhydrous acetonitrile was added. After stirring for 70 minutes at 0 C. additional cyanuric chloride (863 L, 118 mM, 102 mol, 0.75 equiv.) in anhydrous acetonitrile was added. The RM was stirred at 0 C. for another 55 minutes and was then quenched with a solution of 3-aminopropane-1,2 diol (186 mg, 1.02 mL, 2 Molar, 2.04 mmol, 15 equiv.) in water. The ice bath was removed after 30 min and was then diluted with DCM (30 mL) and H.sub.2O (10 mL). The resulting bi-phasic system was separated. The water-layer was extracted twice more with DCM (10 mL, 2). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered over a phase-separator and concentrated in vacuo. The residue was taken up in DMF and purified by prep-HPLC (50%.fwdarw.70% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). The fractions containing pure product were combined and concentrated to give pure compound 60 (13.5 mg, 17.3 mol, 12.7% yield) as a red solid. In addition, fractions containing impure product were combined, affording impure 60 (11.4 mg, 63% pure, 9.20 mol, 6.7% yield). LCMS (ESI+) calculated for C.sub.39H.sub.48N.sub.3O.sub.14.sup.+ (M+H.sup.+) 782.82 found 782.64.

    Example a73. Synthesis of Compound 61

    ##STR00127##

    [0584] To a vial containing compound 60 (6.75 mg, 1 Eq, 8.63 mol) was added anhydrous DCM (300 L) and to the resulting red solution was added pyrrolidine (1.84 mg, 2.13 L, 3 Eq, 25.9 mol), generating a very dark red (nearly black) solution instantly. The mixture was mixed, followed by the addition of Pd(PPh.sub.3).sub.4 in dry DCM (1.50 mg, 64.8 L, 20 mmolar, 0.15 Eq, 1.30 mol). The dark red solution was again mixed and left at rt for 11 minutes. Additional 20 mmolar Pd(PPh.sub.3).sub.4 in dry DCM (64.8 L, 0.15 Eq, 1.30 mol) was added, followed after 13 and 12 minutes by a third (100 L, 0.23 Eq, 2.00 mol)) and fourth batch (64.8 L, 0.15 Eq, 1.30 mol) respectively. Finally, following the fourth addition the RM was stirred at rt for 47 minutes and was then diluted with DMF (300 L) and was then partially concentrated in vacuo until 30 mbar to give a dark red solution that was purified by prep-HPLC (20%.fwdarw.50% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 UM OBD, 30100 mm). The fractions containing pure product were combined and concentrated to give pure compound 61 (2.4 mg, 3.4 mol, 40% yield) as a red residue. In addition, fractions containing impure product were combined, affording impure 61 (3.3 mg, 53% pure, 2.5 mol, 29% yield). LCMS (ESI+) calculated for C.sub.35H.sub.44N.sub.3O.sub.12.sup.+ (M+H.sup.+) 698.29 found 698.56.

    Example a74. Synthesis of Compound BCN-HS-GGFG-OH (62)

    ##STR00128##

    [0585] A round-bottom flask containing a solution of BCN-OH 44 (150 mg, 92% Wt, 1 Eq, 919 mol) in Acetonitrile (10 mL) was cooled to 0 C. by ice. Next, Chlorosulfonyl isocyanate (137 mg, 83.9 L, 1.05 Eq, 965 mol) was added. The RM was stirred for 20 minutes and was then treated with triethylamine (279 mg, 384 L, 3 Eq, 2.76 mmol) followed by the addition of H-GlyGlyPheGly-OH.Math.TFA (496 mg, 1.2 Eq, 1.10 mmol) as a solid. Next, Water (1 mL) was added, and the RM was stirred vigorously, followed by the addition of additional triethylamine (186 mg, 256 L, 2 Eq, 1.84 mmol) was added to aid in solubility. The RM was stirred for circa 3 hours, generating a solution, which was diluted with DCM (20 mL) and water (20 mL) and brine (500 L). The resulting bi-phasic system was shaken and then separated. The water-layer was extracted with additional DCM (10 mL). The combined water layers were combined and EtOAc (20 mL) was added. Then 1 M aq. HCl was added till the water layer reached a pH of circa 4. The two layers were separated, and the water layer was extracted twice more with EtOAc (220 mL). All the EtOAc-based organic layers were combined and dried over Na.sub.2SO.sub.4, filtered and concentrated, affording compound 62 (419 mg, 708 mol, 77% yield) as a yellow oil. LCMS (ESI+) calculated for C.sub.26H.sub.34N.sub.5O.sub.9S.sup.+ (M+H.sup.+) 592.21 found 592.44.

    Example a75. Synthesis of Compound 63a

    ##STR00129##

    [0586] To a vial containing a dark red solution of compound 61 (2.4 mg, 1 Eq, 3.4 mol) in DMF (180 L) was added a solution of compound 62 (4.1 mg, 8.0 L, 866 mmolar, 2.0 Eq, 6.9 mol) in DMF, affording a clear red solution. Next, DIPEA (1.3 mg, 1.8 L, 3 Eq, 10 mol) was added, followed by a solution of HATU in DMF (1.4 mg, 7.3 L, 492 mmolar, 1.05 Eq, 3.6 mol) and the resulting mixture was mixed and left at rt for circa 75 minutes. The mixture was then purified by prep-HPLC (30%.fwdarw.70% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 10150 mm). The fractions containing pure product were combined and concentrated to give compound 63a (1.2 mg) as a red residue. LCMS (ESI+) calculated for C.sub.61H.sub.75N.sub.8O.sub.20S.sup.+ (M+H.sup.+) 1271.48 found 1271.95.

    Example a76. Synthesis of Compound 63b

    ##STR00130##

    [0587] Synthesis of BCN-HS-PEG2-OPNP has been described in WO2021144314A1, which is incorporated herein by reference. To a vial containing a dark red solution of compound 61 (4.85 mg, 1 Eq, 6.95 mol) in dry DCM (300 L) was added BCN-HS-PEG.sub.2-OPNP (4.37 mg, 92% Wt, 1.1 Eq, 7.65 mol) followed by triethylamine (2.11 mg, 2.91 L, 3 Eq, 20.9 mol). The resulting solution was mixed and left at rt for 4.5 hours and was then stored in the freezer for 1 day. The RM was then removed from the freezer and left at rt for another 4 hours before storing the mixture in the freezer for another 2 days. The material was removed from the freezer a final time and then purified by prep-HPLC (20%.fwdarw.50% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 10150 mm). The fractions containing product were combined and concentrated to give compound 63b (5.1 mg) as a bright red solid. LCMS (ESI+) calculated for C.sub.51H.sub.66N.sub.5O.sub.19S.sup.+ (M+H.sup.+) 1084.41 found 1084.91.

    ##STR00131##

    Example a77. Synthesis of Compound 64

    [0588] This compound was synthesized according to a literature procedure described in US20210030886A1.

    Example a78. Synthesis of Compound 65

    [0589] BCN-HS-C5-OH (64) (400 mg, 1 equiv. 1.12 mmol) was dissolved in dry DCM (4 mL) followed by addition of bis(4-nitrophenyl) carbonate (373 mg, 1.1 Eq, 1.23 mmol) and triethylamine (226 mg, 311 L, 2 Eq, 2.23 mmol) the yellow solution was stirred at rt for 220 minutes. The reaction mixture was concentrated in vacuo and then purified by flash column chromatography over silicagel (0%.fwdarw.10% EtOAc in DCM) to give product BCN-HS-C5-OPNP (65) (1.08 g, 7.24 mmol, 89% yield) as a pale-yellow oil. LCMS (ESI+) calculated for C.sub.23H.sub.33N.sub.4O.sub.9S.sup.+ (M+NH.sub.4.sup.+) 541.20 found 541.50.

    Example a79. Synthesis of Compound 63c

    ##STR00132##

    [0590] To a vial containing a dark red solution of compound 61 (4.85 mg, 1 Eq, 6.95 mol) in dry DCM (300 L) was added a solution of BCN-HS-C5-OPNP (65) (4.82 mg, 83% Wt, 1.1 Eq, 7.65 mol) in dry DCM (50 L) followed by triethylamine (2.11 mg, 2.91 L, 3 Eq, 20.9 mol). The resulting solution was mixed and left at rt for 3 days. The mixture was then diluted with DMF (250 L) and partially concentrated in vacuo to remove DCM. The resulting solution was then purified by prep-HPLC (20%.fwdarw.50% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30150 mm). The fractions containing product were combined and concentrated to give compound 63c (4.6 mg) as a bright red solid. LCMS (ESI+) calculated for C.sub.52H.sub.68N.sub.5018S.sup.+ (M+H.sup.+) 1082.43 found 1082.91.

    Example a80. Synthesis of Compound 66

    ##STR00133##

    [0591] Synthesis of BCN-HS-PEG2-OPNP has been described in WO2021144314A1, which is incorporated herein by reference. BCN-HS-PEG2-OPNP (10.2 mg, 1 Eq. 19.4 mol) was dissolved in dry DCM (400 L) and dry DMF (100 L) followed by addition of H-Glu(Fm)-OH (11.2 mg, 1.36 Eq. 26.5 mol) and DIPEA (15.1 mg, 20.3 L, 6 Eq, 116 mol). The resulting solution was mixed and left at rt for 4.5 hours and was then stored in the freezer for 3 days. The RM was then removed from the freezer and left at rt for another 3.5 hours. The RM was then directly loaded onto a pre-wetted column and was then purified using flash column chromatography over silicagel (0.fwdarw.20% MeOH in DCM). Fractions containing product were combined and concentrated in vacuo, affording compound 66 (11.2 mg, 97% purity, 15 mol, 79%) as a clear oil. .sup.1H NMR (400 MHZ, DMSO-d6) 7.92 (d, J=7.5 Hz, 2H), 7.67 (d, J=7.4 Hz, 2H), 7.44 (t, J=7.5 Hz, 2H), 7.36 (t, J=7.5 Hz, 1H), 4.45-4.30 (m, 2H), 4.27 (t, J=6.8 Hz, 1H), 4.13 (m, 1H), 3.96 (m, 2H), 3.88 (d, J=7.9 Hz, 2H), 3.85-3.77 (m, 1H), 3.53 (t, J=4.5 Hz, 2H), 3.50-3.44 (t, J=5.6 2H), 2.87 (m, 2H), 2.49-2.35 (m, 1H), 2.18 (m 1H), 2.00 (m, 1H), 1.50 (m, 3H), 1.34-1.19 (m, 4H), 0.83 (m, 3H). UPLC-MS (ESI+) calculated for C.sub.35H.sub.45N.sub.4O.sub.11S.sup.+ [M+NH.sub.4.sup.+] 729.28 found 729.64.

    Example a81. Synthesis of Compound 63d

    ##STR00134##

    [0592] To a vial containing BCN-HS-PEG2-Glu(Fm)-OH (66) (5.9 mg, 97 wt %, 1.1 Eq, 8.0 mol) in DCM (200 L) was added first DIPEA (2.9 mg, 4.0 L, 3 Eq, 23 mol) and then HATU (3.2 mg, 1.1 Eq, 8.4 mol) in 17 L of DMF. The RM was stirred for 5 minutes. This solution was added to a vial containing a dark red solution of compound 61 (5.3 mg, 1 Eq, 7.6 mol) in dry DCM (200 L). The resulting RM was mixed and left at rt for 50 minutes. Next, DMF (350 L) was added to the RM and the mixture was partially concentrated in vacuo to remove the DCM. The RM was stored in the freezer overnight. The RM was then removed from the freezer and triethylamine (7.7 mg, 11 L, 10 Eq, 76 mol) was added. The RM was stirred for 3 hours, followed by the addition of additional triethylamine (7.7 mg, 11 L, 10 Eq, 76 mol). The RM was stirred for another 4 hours. Next a third addition of triethylamine (7.7 mg, 11 L, 10 Eq, 76 mol) was added and the RM was left stirring for 4 hours. Then the RM was stored in the freezer overnight. The RM was then removed from the freezer and this solution was diluted to 700 L with additional DMF and purified by prep-HPLC (Column Xbridge prep C.sub.18, 5 m OBD, 30100 mm, 30%.fwdarw.95% MeCN in 10 mM aq. NH.sub.4HCO.sub.3). Fractions containing the product were combined and concentrated in vacuo. After the fractions were combined and completely concentrated, the red solid was redissolved twice in MeCN and then concentrated and once in DCM and concentrated to give compound 63d (3.9 mg, 3.2 mol, 42%) as a red solid. UPLC-MS (ESI+) calculated for C.sub.56H.sub.73N.sub.6O.sub.22S.sup.+ [M+H.sup.+] 1213.45 found 1213.99.

    ##STR00135## ##STR00136##

    Example a82. Synthesis of Compound 67

    [0593] To daunorubicin.Math.HCl (765.2 mg, 1 Eq, 1.357 mmol) was suspended in dry DMF (2.0 mL), followed by the addition of bis-iodo-sugar 7c (1.57 g, 807 L, 3.01 Eq, 4.09 mmol) and DiPEA (526 mg, 709 L, 3 Eq, 4.07 mmol). The flask was covered in aluminium foil and the suspension was stirred in the dark for 24 hours. Next, the RM was heated to 37 C. for another 20 hours and was then diluted with DCM (16 mL) and the resulting red solution was purified by flash column chromatography over silicagel (0%.fwdarw.7.5% MeOH in DCM) to give compound 67 as a red oil (662 mg, 1.01 mmol, 74.4%). LCMS (ESI+) calculated for C.sub.34H.sub.42NO.sub.12.sup.+ (M+H.sup.+) 656.27 found 656.45.

    Example a83. Synthesis of Compound 68

    [0594] A solution of compound 67 (662 mg, 1 Eq, 1.01 mmol) in DCM (25 mL) was cooled by dry ice/acetone-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in DCM (261 mg, 2.61 mL, 580 mmolar, 1.5 Eq, 1.51 mmol) was added dropwise over 5 minutes. After stirring for 21 minutes at 78 C. the reaction quenched with cold (78 C.) acetone (reagent grade, 8.00 mL) and the RM was stirred for another 30 minutes at-78 C. Next, the cold bath was removed, and the RM was allowed to warm up to rt. After the RM was allowed to warm to rt the mixture was diluted with DCM (55 mL). The resulting mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (15 mL, 2). The combined water-layers were extracted with DCM (20 mL, 4). Next, the combined organic layers were dried (Na.sub.2SO.sub.4) and filtered over a glass-filter and then concentrated in vacuo. The residue was taken up in DCM (100 mL) and H.sub.2O (30 mL). The bi-phasic system was shaken and then separated using a phase-separator. The resulting organic layer was concentrated in vacuo, affording to give compound 68 (450 mg, 0.61 mmol, 60%, 91% Purity), which was used as such in the next step. LCMS (ESI+) calculated for C.sub.34H.sub.42NO.sub.13.sup.+ (M+H.sup.+) 672.27.32 found 672.59.

    Example a84. Synthesis of Compound 69

    [0595] To a solution of 68 (450 mg, 91% Wt, 1 Eq, 610 mol) in a mixture of dry DCM (2.0 mL) anhydrous acetonitrile (3.0 mL) was added potassium carbonate (758 mg, 9 Eq, 5.49 mmol) was added. The RM was then cooled to 0 C. with an ice-bath after which a solution of Cyanuric chloride in dry MeCN (281 mg, 12.9 mL, 118 mmolar, 2.5 Eq, 1.52 mmol) was added slowly to the stirred solution. After complete addition additional dry DCM (3.0 mL) was added and the RM was stirred at 0 C. for 30 minutes, followed by the addition of another amount of DCM (6 mL). The resulting mixture was stirred for another 25 minutes and was then quenched with a solution of 3-amino-1,2-propanediol in H.sub.2O (870 mg, 15.7 Eq, 9.55 mmol) in H.sub.2O (circa 2.2 mL). The resulting mixture was stirred, and the ice bath was removed after 6 min, followed by the addition of DCM (75 mL) and H.sub.2O (10 mL). The resulting bi-phasic system was separated. The water-layer was extracted with DCM (20 mL). The combined organic layers were concentrated in vacuo and the residue was purified using flash column chromatography (0.fwdarw.10% MeOH in DCM). Fractions containing the product were concentrated to give compound 69 (246 mg, 0.32 mmol, 52%, 85% Purity) as a red residue. LCMS (ESI+) calculated for C.sub.34H.sub.40NO.sub.12.sup.+ (M+H.sup.+) 654.25 found 654.60.

    Example a85. Synthesis of Compound 70

    [0596] To a maleimidopropionic acid hydrazide.Math.HCl (10.9 mg, 1.9 Eq, 49.4 mol) was dissolved in Anhydrous MeOH (1.63 mL) and added to 69 (20.0 mg, 85 wt %, 1 Eq, 26.0 mol). Some solids remained therefore RM was swirled in the water-bath 40C till all solids dissolved. The RM was stirred in the dark at RT overnight. RM had 87% conversion to the desired product. RM was heated and stirred at 40 C. for 2 h. UPLC-MS analysis showed an increase in side-product formation. RM was stirred at RT for 4.5 h. As no further conversion was observed, Maleimidopropionic acid hydrazide.Math.HCl (1.14 mg, 0.2 Eq, 5.20 mol) was dissolved in 100 l dry MeOH and added to the RM. RM was stirred overnight. 89% conversion to the product was achieved and an increase in side-product formation. The RM was halted, and purification was started. RM was concentrated in vacuo. The crude was re-dissolved in 2 ml 2% MeOH in DCM and loaded on the column and purified using silica (2.fwdarw.40% MeOH in DCM). Fractions containing the product were combined, concentrated, and stored in the freezer. The product still had some impurities; therefore a second purification was performed. The product was re-dissolved in 1 ml in DCM to which 100 l DMF was added and loaded on the column. The vial it originated from was washed with 21 ml DCM and loaded onto the column and purified using silica purification (0.fwdarw.20% MeOH/DCM). Fractions containing the product were combined and concentrated, obtaining compound 66 (9.0 mg, 9.9 mol, 38%, 90% Purity) as a red residue. UPLC-MS (ESI+) calculated for C.sub.41H.sub.47N.sub.4O.sub.14.sup.+ [M+H.sup.+] 819.31 found 819.72.

    ##STR00137## ##STR00138## ##STR00139##

    Example a86. Synthesis of Compound 71

    [0597] To daunorubicin.Math.HCl (202.6 mg, 1 equiv., 359.2 mol) was suspended in dry DMF (1.2 mL), followed by the addition of bis-iodo-sugar 7b (442.9 mg, 3 Eq, 1.078 mmol) and DIPEA (139.3 mg, 188 L, 3 Eq, 1.078 mmol). The flask was covered in aluminium foil and the suspension was stirred in the dark at rt for 4 days, generating a dark red solution. Next, the RM was diluted with DCM (10 mL) and the resulting red solution was purified by flash column chromatography over silicagel (0%->15% MeOH in DCM) to give compound 71 as a red waxy oil (125.1 mg, 95% purity, 170 mol, 48% yield) and a 2.sup.nd batch with slightly less purity (48.2 mg, 88% purity, 62 mol, 17% yield) also as a red waxy oil. LCMS (ESI+) calculated for C.sub.33H.sub.39N.sub.4012.sup.+ (M+H.sup.+) 683.26 found 683.47.

    Example a87. Synthesis of Compound 72

    [0598] To a suspension of compound 71 (125.1 mg, 95% Wt, 1 Eq, 174.1 mol) in MeOH (800 L) was added a solution of triphenylphosphine in DCM (123.3 mg, 1.343 mL, 350 mmolar, 2.7 Eq, 470.0 mol) and water (400 L). The biphasic mixture was stirred 85 minutes at room temperature after which Fmoc-Val-Ala-PAB-OPNP (142.2 mg, 1.2 Eq, 208.9 mol) was added. After stirring the biphasic mixture for an additional 18 hours at room temperature, an extraction with DCM (22 mL) was carried out. The combined organic layers were dried over Na.sub.2SO.sub.4 and immediately purified by flash column chromatography over silicagel (0%.fwdarw.40% EtOAc in DCM (to remove excess Fmoc-Val-Ala-PAB-OPNP) followed by 0%.fwdarw.15% MeOH in DCM) to give compound 72 as a red solid (71.3 mg, 59.5 mol, 34.2%). LCMS (ESI+) calculated for C.sub.64H.sub.72N.sub.5O.sub.18.sup.+ (M+H.sup.+) 1198.49 found 1198.75.

    Example a88. Synthesis of Compound 73

    [0599] A solution of compound 72 (87.3 mg, 72.9 mol, 1.00 equiv.) in a mixture of anhydrous DCM (1.5 mL) and MeOH (200 L) was cooled with dry ice/acetone cooling-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in anhydrous DCM (13.8 mg, 138 L, 580 mmolar, 1.1 Eq, 80.1 mol) was added dropwise via the side of the flask. After stirring for 30 minutes additional mCPBA in anhydrous DCM (3.77 mg, 37.7 L, 580 mmolar, 0.3 Eq, 21.9 mol) was added dropwise via the side of the flask. The RM was stirred at 78 C. for another hour and was then quenched with an ice-cold solution of acetone (reagent grade, 2.5 mL) and the RM was stirred. After an hour the cold bath was removed, and the RM was allowed to warm up to room temperature over 105 minutes. Next, the RM was diluted with DCM (15 mL) and transferred to a separation funnel and washed twice with saturated aqueous NaHCO.sub.3 solution (10 mL, 2). The water layers were combined and extracted twice with DCM (20 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered through a membrane-filter, and concentrated in vacuo to give compound 73 (75.9 mg, 62.5 mol, 85.8%) as a red solid, which was used as such in the next step. LCMS (ESI+) calculated for C.sub.64H.sub.72N.sub.5O.sub.19.sup.+ (M+H.sup.+) 1214.48 found 1214.73.

    Example a89. Synthesis of Compound 74

    [0600] To a red solution of compound 73 (75.9 mg, 1 Eq, 62.5 mol) in a mixture of dry DCM (1.0 mL) anhydrous acetonitrile (9.0 mL) was added potassium carbonate (33.7 mg, 3.9 Eq, 244 mol). The RM was then cooled to 0 C. with an ice-bath after which a solution of cyanuric chloride (28.8 mg, 1.30 mL, 120 mmolar, 2.5 Eq, 156 mol) in anhydrous acetonitrile was added. After stirring for 155 minutes at 0 C. additional potassium carbonate (17.3 mg, 2 Eq, 125 mol) and cyanuric chloride (14.4 mg, 651 L, 120 mmolar, 1.25 Eq, 78.1 mol) in anhydrous acetonitrile were added. The RM was stirred at 0 C. for another 80 minutes and was then stored in the freezer for circa 18 hours. The RM was then removed from the freezer and placed in an ice-bath at 0 C. again, followed by a final addition of additional potassium carbonate (11.3 mg, 1.31 Eq, 81.8 mol) and freshly prepared cyanuric chloride (14.4 mg, 651 L, 120 mmolar, 1.25 Eq, 78.1 mol) in anhydrous acetonitrile. The RM was stirred at 0 C. for another 280 minutes and was then quenched with a solution of 3-aminopropane-1,2 diol (192.3 mg) in 4.58 mL water. The ice bath was removed after 30 min and the RM was then diluted with DCM (10 mL). The resulting bi-phasic system was separated. The water-layer was extracted with more DCM (15 mL, 3). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then partially concentrated in vacuo to a volume of circa 15 mL. The mixture was then diluted with a little bit DMF (300 L), and the resulting solution was purified by flash column chromatography over silicagel (0%.fwdarw.20% MeOH in DCM). The fractions containing product were combined and concentrated to give compound 74 (12.7 mg, 10.6 mol, 17.0%) as a red residue. LCMS (ESI+) calculated for C.sub.64H.sub.70N.sub.5O.sub.18.sup.+ (M+H.sup.+) 1196.47 found 1196.82.

    Example a90. Synthesis of Compound 75

    [0601] Synthesis of BCN-HS-PEG2-OPNP has been described in WO2021144314A1, which is incorporated herein by reference. To a solution of compound 74 (12.7 mg, 1 Eq, 10.6 mol) in DCM (3 mL) was added DMF (150 L) and the RM was concentrated to remove the DCM. Then, triethylamine (16.1 mg, 22.2 L, 15 Eq, 159 mol) was added followed by a solution of BCN-HS-PEG.sub.2-OPNP (6.70 mg, 143 L, 89 mmolar, 1.2 Eq, 12.7 mol) in DMF. The solution was mixed and then left at rt for 21 hours. The RM was then further diluted with DCM (1 mL) and purified by flash column chromatography over silicagel (0%.fwdarw.15% MeOH in DCM) to give impure compound 75. The material was then subjected to a purification by prep-HPLC (30%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). The fractions containing product were combined and concentrated to give compound 75 (2.3 mg). LCMS (ESI+) calculated for C.sub.65H.sub.82N.sub.7O.sub.23S.sup.+ (M+H.sup.+) 1360.52 found 1361.07.

    ##STR00140## ##STR00141##

    Example a91. Synthesis of Compound 76

    [0602] Daunorubicin, HCl (2.0 g, 1 Eq, 3.5 mmol) was dissolved in dry DMF (7.70 mL) after which (S)-1-azido-4-((2-iodo-1-(2-iodoethoxy)ethoxy)methyl)benzene 7e (5.1 g, 3.018 Eq, 11 mmol) and DIPEA (1.4 g, 1.9 mL, 3 Eq, 11 mmol) were added. The flask was covered in aluminium foil and the suspension was stirred in the dark at 40 C., generating a red solution over time. The RM was stirred at 40 C. for roughly 19 hours and analyzed. The RM was then heated at 40 C. while stirring for another 57 hours before it was stored in the freezer. The RM was diluted with DCM (100 mL) to a volume of circa 118 mL. This solution was then transferred onto the column. The residue was then flash column chromatography over silicagel (0-10% MeOH in DCM). Fractions containing product were combined and concentrated in vacuo to yield compound 76 (2.61 g, 3.406 mmol, 96%, 97.2% Purity) as dark red residue. UPLC-MS (ESI+) calculated for C.sub.38H.sub.41N.sub.4O.sub.12.sup.+ [M+H.sup.+] 745.27 found 745.63.

    Example a92. Synthesis of Compound 77

    [0603] Compound 76 (2.609 g, 97.22 wt %, 1 Eq, 3.406 mmol) was dissolved in DCM (80 mL). The mixture was cooled down by a dry-ice/acetone cooling-bath (78 C.). Then 3-chlorobenzoperoxoic acid (646.5 mg, 6.46 mL, 580 mmolar, 1.1 Eq, 3.75 mmol) was added dropwise added by using a microman pipette and was added via the side of the flask to prevent temperature spike. Meanwhile reagent-grade acetone was cooled in the dry ice/acetone cooling-bath. After 3 min a second addition of mCPBA in DCM (235.09 mg, 2.3489 mL, 580 mmolar, 0.4 Eq, 1.3624 mmol) was added. After 3 min a third addition of mCPBA in DCM (235.09 mg, 2.3489 mL, 580 mmolar, 0.4 Eq, 1.3624 mmol) was added. After another 3 min. the RM was quenched with 68.8 mL cold reagent grade acetone and left stirring in the cooling-bath. After 1 h the cold bath was removed, and the RM was allowed to warm up to 5 C. To the cold RM 160 mL sat. aq. NaHCO.sub.3 aqueous solution was added while stirring, followed by the addition of 200 mL DCM. The RM was then transferred to a separation funnel. The DCM layer was separated and washed twice with 160 mL sat. aq. NaHCO.sub.3 aqueous solution. The organic layer was dried over Na.sub.2SO.sub.4 and filtered through a membrane filter. The solution was concentrated under vacuo to give compound 77 (1.955 g, 2.229 mmol, 65.45%, 86.74% purity) was obtained. UPLC-MS (ESI+) calculated for C.sub.38H.sub.41N.sub.4O.sub.13.sup.+ [M+H.sup.+] 761.27 found 761.62.

    Example a93. Synthesis of Compound 78

    [0604] To compound 77 (0.978 g, 86.74 wt %, 1 Eq, 1.115 mmol) as a solution in mixture of dry acetonitrile (6 mL) and dry DCM (4 mL) was added potassium carbonate (616.2 mg, 4 Eq, 4.459 mmol) and the RM was cooled down with an ice bath (ice-bath 0 C.). Then cyanuric chloride (513.9 mg, 13.93 mL, 200 mmolar, 2.5 Eq, 2.787 mmol) was added at once and the mixture was stirred vigorously. After 2.5 h full the RM was quenched with 3-aminopropane-1,2 diol in H.sub.2O (1.249 g, 6.855 mL, 2 molar, 12.3 Eq, 13.71 mmol). After stirring for 10 min on ice the RM was stored in the freezer for 1 day. The RM was taken out of the freezer and then transferred to a separation funnel and 50 mL DCM was added. The resulting bi-phasic system was separated. The water layer was extracted four more times with 50 mL DCM (50 mL, 4). The combined organic layers were transferred to a round-bottom flask and concentrated. After concentrating it, a dark red residue was obtained. This was re-dissolved in 15 mL DCM and then loaded onto the column and purified by flash column chromatography over silicagel (0.fwdarw.10% MeOH in DCM). Fractions containing the product were combined and concentrated to give impure compound 78, which was subjected to a second purification by flash column chromatography over silicagel (0.fwdarw.10% MeOH in DCM). Fractions containing the product were combined and concentrated to give compound 78 (163.3 mg, 219.9 mol, 19.72%) as dark red residue. UPLC-MS (ESI+) calculated for C.sub.38H.sub.39N.sub.4O.sub.12.sup.+ [M+H.sup.+] 743.26 found 743.63.

    Example a94. Synthesis of Compound 79

    [0605] Compound 78 (50.0 mg, 1 Eq, 67.3 mol) was suspended in MeOH (290 L), followed by the addition of PPh3 in DCM (47.7 mg, 519 L, 350 mmolar, 2.7 Eq, 182 mol), generating a clear red solution. Next, H.sub.2O (145 L) was added. The resulting biphasic mixture was stirred at rt for 1 hour, followed by the addition of additional H.sub.2O (145 L). The RM was left to stir at rt for 1 day and was then heated to 40 C. and stirred for a few hours. Some of the DCM seemed to evaporate and 500 l DCM was added accordingly. The RM was then stirred at 35 C. for 1 day, followed by the addition of DMF (50 L). The resulting mixture was stirred at 35 C. for another 3 days. The reaction was then further diluted through the addition of DMF (50 L), and H2O (100 L) and the RM was stirred at 40 C. for another 2 days. The RM was then partially concentrated in vacuo (until 40 mbar), affording crude compound 79 as a solution in DMF that was used without further purification in the next step. UPLC-MS (ESI+) calculated for C.sub.38H.sub.41N.sub.2012+ [M+H.sup.+] 717.27 found 717.73.

    Example a95. Synthesis of Compound 80

    [0606] To BCN-HS-GGFG-OH (62) in DMF (5.4 mg, 91 L, 100 mmolar, 1.3 Eq, 9.1 mol) was added first DIPEA (2.7 mg, 3.6 L, 3 Eq, 21 mol) and then HATU (3.4 mg, 1.3 Eq, 9.1 mol). The RM was stirred for 5 minutes. Then, compound 79 in DMF (5.0 mg, 0.10 mL, 67. mmolar, 1.0 Eq, 7.0 mol) was added. The RM was stirred for 150 minutes. Because of partial conversion to the product, additional BCN-HS-GGFG-OH (62) in DMF (42 L, 100 mmolar, 0.6 Eq, 4.2 mol), DIPEA (0.90 mg, 1.2 L, 1 Eq, 7.0 mol) and HATU (1.6 mg, 0.6 Eq, 4.2 mol) and additional DMF (100 L), were pre-mixed for 5 min and added to the main RM, which was stirred for another 5 h. The RM was then diluted to 500 L of DMF and purified by Reverse-Phase Prep-HPLC (Column Xbridge prep C.sub.18, 5 m OBD, 30100 mm, 5%.fwdarw.95% MeCN in 10 mM aq. NH.sub.4HCO.sub.3). Fractions containing the product were combined and concentrated in vacuo to yield 80 (0.6 mg, 0.5 mol, 7%) as a red solid. UPLC-MS (ESI+) calculated for C.sub.64H.sub.72N.sub.7O.sub.20S.sup.+ [M+H.sup.+] 1290.45 found 1291.12.

    ##STR00142##

    Example a96. Synthesis of Compound 81

    [0607] To a round-bottom flask containing a solution of compound 6e (45.6 mg, 80 wt %, 48.1 mol, 1.00 equiv.) in DCM (1.5 mL) was added a solution of PPh3 in DCM (188 L, 690 mmolar, 130 mol, 2.7 equiv.) and H.sub.2O (800 L). The resulting bi-phasic mixture was stirred vigorously at rt for 3 days, followed by the addition of DMF (500 L) and additional DCM (500 L). The resulting mixture was stirred vigorously at rt for another 3 days and was then stored in the freezer. The mixture was removed from the freezer after 3 days and was then diluted with DCM (10 mL). The resulting bi-phasic system was separated, and the water-layer was extracted twice with DCM (10 mL, 2). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then concentrated in vacuo, affording crude amine intermediate (91.5 mg) and part of this material (11.7 mg) was used directly without further purification for the synthesis of compound 81; Compound 62 (11.0 mg, 66 wt %, 1.1 Eq, 12.3 mol) in DMF (200 L) was treated with DIPEA (4.33 mg, 5.84 L, 3 Eq, 33.5 mol) and HATU (4.46 mg, 1.05 Eq, 11.7 mol) and the resulting mixture was mixed and then added to a vial containing a solution of solution of crude amine intermediate (11.7 mg) in DMF (200 L). The resulting mixture was mixed and left at rt for circa 150 minutes. Next, additional compound 62 (11.0 mg, 66% Wt, 1.1 Eq, 12.3 mol) in DMF (100 L), DIPEA (4.33 mg, 5.84 L, 3 Eq, 33.5 mol) and HATU (4.46 mg, 1.05 Eq, 11.7 mol) were added to the RM. The resulting mixture was mixed and left at rt for another 110 minutes, before adding a final addition of additional compound 62 (11.0 mg, 66% Wt, 0.939 Eq, 10.5 mol) was dissolved in 100 L and DIPEA (4.33 mg, 5.84 L, 3 Eq, 33.5 mol) and HATU (4.46 mg, 1.05 Eq, 11.7 mol) to the RM. The RM was mixed and left at rt for another 40 minutes at rt and was then stored in the freezer for circa 16 hours. Next, the mixture was purified by prep-HPLC (30%.fwdarw.95% acetonitrile in 10 mM aq. NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 10150 mm). The fractions containing pure product were combined and concentrated to give compound 81 (4 mg) as a red solid. LCMS (ESI+) calculated for C.sub.64H.sub.72N.sub.7O.sub.21S.sup.+ (M+H.sup.+) 1306.45 found 1307.03.

    Example a97. Synthesis of Compound 82

    [0608] Synthesis of BCN-HS-PEG2-OPNP has been described in WO2021144314A1, which is incorporated herein by reference. To a round-bottom flask containing a solution of compound 6e (45.6 mg, 80 wt %, 48.1 mol, 1.00 equiv.) in DCM (1.5 mL) was added a solution of PPh3 in DCM (188 L, 690 mmolar, 130 mol, 2.7 equiv.) and H.sub.2O (800 L). The resulting bi-phasic mixture was stirred vigorously at rt for 3 days, followed by the addition of DMF (500 L) and additional DCM (500 L). The resulting mixture was stirred vigorously at rt for another 3 days and was then stored in the freezer. The mixture was removed from the freezer after 3 days and was then diluted with DCM (10 mL). The resulting bi-phasic system was separated, and the water-layer was extracted twice with DCM (10 mL, 2). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then concentrated in vacuo, affording crude amine intermediate (91.5 mg) and part of this material (11.7 mg) was used directly without further purification for the synthesis of compound 82. Crude amine intermediate (11.7 mg) was dissolved in dry DCM (400 L), followed by the addition of BCN-HS-PEG.sub.2-OPNP (7.75 mg, 91 wt %, 1.2 Eq, 13.4 mol) and triethylamine (3.39 mg, 4.67 L, 3 Eq, 33.5 mol). The resulting RM was mixed and left at rt for 23.5 hours, followed by the addition of HOBt (1.51 mg, 25.6 L, 436 mmolar, 1 Eq, 11.2 mol). The RM was left at rt for another 200 minutes and then DMAP (1.37 mg, 15.0 L, 744 mmolar, 1 Eq, 11.2 mol) was added. The RM was again mixed and left at rt for 5 hours. Finally, additional BCN-HS-PEG.sub.2-OPNP (11.7 mg, 2 Eq, 22.4 mol), triethylamine (3.39 mg, 4.67 L, 3 Eq, 33.5 mol) and DMAP (2.73 mg, 30.0 L, 744 mmolar, 2 Eq, 22.4 mol) were added and the resulting mixture was left at rt for 3 days. The RM was then diluted with DMF (200 L) and partially concentrated in vacuo to remove DCM. The resulting solution was purified by prep-HPLC (30%.fwdarw.95% acetonitrile in 10 mM aq. NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30150 mm). The fractions containing product were combined and concentrated to give compound 82 (2.1 mg) as a red solid. LCMS (ESI+) calculated for C.sub.54H.sub.63N.sub.4O.sub.20S.sup.+ (M+H.sup.+) 1119.38 found 1120.14.

    ##STR00143## ##STR00144##

    Example a98. Synthesis of Compound 83

    [0609] To a suspension of 6e (158 mg, 80% Wt, 1 Eq, 167 mol) in MeOH (2.0 mL) was added THF (2.0 mL), generating a clear solution. Next, water (500 L) was added, followed by a drop-wise addition of a solution of sodium periodate in water (200 mM, 1.67 mL, 333 mol) and the reaction mixture was stirred at rt for 24.5 hours in the dark and was then stored in the freezer for 3h 35 minutes. The mixture was removed from the freezer and DCM (10 mL) was added. The mixture was transferred to a separation funnel together with additional H.sub.2O (1 mL). The resulting bi-phasic system was mixed and separated, and the water-layer was extracted with DCM (10 mL, 2). The combined organic layers were filtered over a membrane-filter. The resulting solution was concentrated in vacuo, affording compound 83 (132 mg, 70% purity, 120 mol, 74%) as a red solid. LCMS (ESI+) calculated for C.sub.37H37N.sub.4O.sub.13.sup.+ (M+H.sup.+) 745.24 found 745.63.

    Example a99. Synthesis of Compound 84

    [0610] (9H-fluoren-9-yl) methyl (2-aminoethyl) carbamate, HCl (51.4 mg, 1.3 Eq, 161 mol) was dissolved in DMF (500 L) and then diluted with DCM (700 L), followed by the addition of DIPEA (48.1 mg, 64.8 L, 3 Eq, 372 mol). The resulting mixture was added to a vial containing compound 83 (132 mg, 70% Wt, 1 Eq, 124 mol). Finally, HATU (47.2 mg, 1 Eq, 124 mol) was added. The resulting mixture was mixed and left at room temperature for 40 minutes. Next, additional (9H-fluoren-9-yl) methyl (2-aminoethyl) carbamate, HCl (25.7 mg, 0.65 Eq, 80.6 mol) in a mixture of DMF (200 L) and DCM (200 L) together with DIPEA (24.1 mg, 32.4 L, 1.5 Eq, 186 mol) was added to the main RM, followed by additional HATU (23.6 mg, 0.5 Eq, 62.0 mol). The RM was mixed again and left for another 65 minutes before adding a final sequence of additional (9H-fluoren-9-yl) methyl (2-aminoethyl) carbamate, HCl (23.7 mg, 0.6 Eq, 74.4 mol) in a mixture of DMF (200 L) and DCM (200 L) together with DIPEA (16.0 mg, 21.6 L, 1 Eq, 124 mol) to the main RM, followed by additional HATU (18.9 mg, 0.4 Eq, 49.6 mol). The resulting mixture was then mixed and left at rt for another 20 minutes and then purified using flash column chromatography (0.fwdarw.30% MeOH in DCM). Fractions containing the product were concentrated to give compound 84 (123.1 mg, 80% purity, 98 mol, 79%) as a red waxy oil. LCMS (ESI+) calculated for C.sub.54H.sub.53N.sub.6O.sub.14.sup.+ (M+H.sup.+) 1009.36 found 1010.01.

    Example a100. Synthesis of Compound 85

    [0611] To a solution of compound 84 (123.1 mg, 80% Wt, 1 Eq, 97.60 mol) in DCM (1 mL) was added a solution of PPh3 in DCM (529 mM, 553.5 L, 292.8 mol, 3.00 equiv.) and H.sub.2O (600 L). The resulting bi-phasic mixture was stirred vigorously at rt for 3 days. Next, DMF (600 L) and additional H.sub.2O (300 L) was added, and the RM was stirred vigorously for another 3 days. Finally, the RM was diluted with DCM (10 mL) and the resulting bi-phasic system was separated. The water-layer was extracted with DCM (10 mL, 2). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and concentrated to give crude amine (205.8 mg) that was directly used without further purification in the next step. LCMS (ESI+) calculated for C.sub.54H.sub.55N.sub.4O.sub.14.sup.+ (M+H.sup.+) 983.37 found 983.73. To a solution of compound 62 (18 mg, 66 wt %, 1.3 Eq, 20 mol) in DMF (100 L) and DIPEA (5.9 mg, 8.0 L, 3 Eq, 46 mol) was added HATU (6.1 mg, 1.05 Eq, 16 mol). The mixture was mixed and after 3 minutes a solution of crude amine (15 mg, 1 Eq, 15 mol) in DCM (200 L) was added. The RM was mixed and left at rt for 1 hour. Next, additional compound 62 (9.2 mg, 66 wt %, 0.67 Eq, 10 mol) in DMF (100 L) and DIPEA (3.0 mg, 4.0 L, 1.5 Eq, 23 mol) were added, followed by additional HATU (2.9 mg, 0.5 Eq, 7.6 mol). The RM was mixed and left at rt for circa 30 minutes and was then diluted with DMF (200 L) and the mixture was then partially concentrated in vacuo to remove most of the DCM. The resulting solution was purified by prep-HPLC (30%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). The collected fractions were combined to give compound 85 (3.9 mg, 2.5 mol, 16% yield over 2 steps) as a red solid. LCMS (ESI+) calculated for C.sub.80H.sub.86N.sub.9O.sub.22S.sup.+ (M+H.sup.+) 1556.56 found 1557.07.

    Example a101. Synthesis of Compound 86

    [0612] To a solution of compound 85 (3.9 mg, 1 Eq, 2.5 mol) in DMF (200 L) was added triethylamine (2.5 mg, 3.5 L, 10 Eq, 25 mol). The resulting solution was mixed and left at rt for 16 hours, followed by the addition of additional triethylamine (2.5 mg, 3.5 L, 10 Eq, 25 mol). The RM was mixed and left at rt for another 3 hours, followed by the addition of a 3.sup.rd batch of triethylamine (2.5 mg, 3.5 L, 10 Eq, 25 mol)and after another 105 minutesa 4.sup.th batch of triethylamine (2.5 mg, 3.5 L, 10 Eq, 25 mol). Finally, the resulting RM was left at rt for 140 minutes and was then purified by prep-HPLC (30%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30100 mm). The collected fractions were combined to give compound 86 (0.7 mg, 0.5 mol, 20% yield) as a red solid. LCMS (ESI+) calculated for C.sub.65H.sub.76N.sub.9O.sub.20S.sup.+ (M+H.sup.+) 1334.49 found 1335.09.

    ##STR00145## ##STR00146## ##STR00147##

    Example a102. Synthesis of Compound 87

    [0613] To a solution of compound 3b (224 mg, 0.20 mmol) and triphenylphosphine (180 mg, 0.69 mmol) in DCM (2.86 mL) and MeOH (1.74 mL) was added water (1.29 mL) and the reaction mixture was stirred vigorously at room temperature for 18 h. The mixture was then concentrated to a dark brown/red solid and re-dissolved in DMF (2 mL). Fmoc-Gly-Gly-Phe-Gly-OH (136 mg, 0.24 mmol), DIPEA (65 mg, 88 L, 0.51 mmol,) and HATU (96 mg, 0.25 mmol) were added, and the reaction mixture stirred at room temperature for 30 min and then concentrated to a red/brown gum. The mixture was extracted into DCM/MeOH (9:1, 40 mL), washed with sat. aqueous brine solution (40 mL), dried through a phase separator and concentrated in vacuo. The residue was taken up in DCM (4 mL) and purified by flash column chromatography (1.fwdarw.15% MeOH/DCM) to afford compound 87 (273 mg, 94%) at 92% purity by LCMS as a dark red/brown solid that was used without further purification. UPLC-MS (ESI+) calculated for C.sub.69H.sub.83N.sub.6O.sub.19Si.sup.+ [M+H.sup.+] 1327.5 found 1327.9.

    Example a103. Synthesis of Compound 88

    [0614] To a cooled solution of compound 87 (147 mg, 0.11 mmol) in DCM (2.2 mL) and MeOH (0.43 mL) at 78 C. was added dropwise a solution of mCPBA (41 mg, 0.17 mmol, 70% purity) in DCM (1 mL) over 5 min and the reaction mixture was stirred at 78 C. for 5 min. The reaction mixture was then quenched with ice-cold acetone (3 mL) and the mixture was stirred at 78 C. for 20 min and then warmed to room temperature. The reaction mixture was diluted with DCM (20 mL) and washed with sat. aq. NaHCO.sub.3 solution (20 mL). The aqueous layer was re-extracted with DCM (25 mL) and the combined organic layers were dried through a phase separator and concentrated to give compound 88 (164 mg, 0.11 mmol, 96%) as a red solid at 87% purity by LCMS that was immediately redissolved in DCM (2 mL) and used in the next step without further purification. UPLC-MS (ESI+) calculated for C.sub.69H.sub.83N.sub.6O.sub.20Si.sup.+ [M+H.sup.+] 1343.5 found 1343.8.

    Example a104. Synthesis of Compound 89

    [0615] To a solution of compound 88 (164 mg, 0.12 mmol) in dry DCM (20 mL) and dry acetonitrile (45 mL) was added cyanuric chloride (32 mg, 0.17 mmol) and the reaction mixture was stirred for 1 h at room temperature. Cyanuric chloride (11 mg, 58 mol) in MeCN (0.5 mL) was added and stirred continued for 1 hour at room temperature. Next, additional cyanuric chloride (11 mg, 58 mol) in MeCN (0.5 mL) and DCM (4 mL) were added and stirring continued at room temperature for 3.5 h. The reaction was quenched by addition of a mixture of 3-aminopropane-1,2-diol (106 mg, 1.16 mmol, 90 L) in DCM (3 mL) and stirred at room temperature for 10 minutes before addition of MeOH (20 mL). The mixture was concentrated to a dark red gum that was extracted into DCM/MeOH (9:1, 25 mL) and insoluble residues were removed by filtration. The filtrate was concentrated to a red solid that was re-dissolved in DCM/MeOH (9:1, 4 mL) and purified by flash column chromatography (1.fwdarw.15% MeOH/DCM) to give compound 89 (24 mg, 89% purity) as a red solid. The solid was re-dissolved in DMF (0.5 mL) and MeCN (1 mL) and purified by flash reversed-phase chromatography (C.sub.18, 40.fwdarw.95% MeCN in aq. 10 mM NH.sub.4HCO.sub.3 solution). Fractions containing product were combined, concentrated to 10 ml water and extracted with DCM (210 mL). The combined organic layers were dried through a phase separator and concentrated to give compound 89 (11 mg, 7%) as a red solid. The aqueous layer was concentrated to dryness to afford additional compound 89 (5 mg, 3%) as a red solid. UPLC-MS (ESI+) calculated for C.sub.69H.sub.81N.sub.6O.sub.19Si.sup.+ [M+H.sup.+] 1325.5 found 1325.8.

    Example a105. Synthesis of Compound 91

    [0616] To a solution of compound 89 (15 mg, 11 mol) in DMF (1 mL) was added triethylamine (23 mg, 0.23 mmol, 32 L) and the reaction mixture was stirred at room temperature for 2 h. BCN-OSu (5 mg, 17 mol) was added, and the reaction mixture stirred at room temperature for 18 h. The reaction mixture was concentrated to yield a red residue that was triturated with diethyl ether (34 mL, decanted off) and dried to give compound 91 (39 mg, quant.) as a red gum/solid that was used without purification in the next step. UPLC-MS (ESI+) calculated for C.sub.65H.sub.83N.sub.6O.sub.19Si.sup.+ [M+H.sup.+] 1279.5 found 1279.8.

    Example a106. Synthesis of Compound 92

    [0617] To a cooled solution of compound 91 (39 mg, 11.3 mol) in dry DMF (0.2 mL) and dry THF (0.6 mL) at 10 C. was added triethylammonium acetate (9 mg, 56 mol) and stirring was continued at 10 C. for 5 min. Tetrabutylammonium fluoride solution in THF (1 M, 56 L, 56 mol) was added and the resulting solution was stirred at 10 C. for 20 min. Additional tetrabutylammonium fluoride solution in THF (1 M, 56 L, 56 mol) was added, resulting in a dark green solution that turned to red after two minutes, and stirring continued at 10 C. for 90 minutes. The reaction mixture was quenched by addition of ice-cold water (5 mL) and diluted with DCM (25 mL). Sat. aqueous brine (30 mL) was added, and layers were separated. The water-layer was extracted with DCM (10 mL) and the combined organic layers were dried through a phase separator and concentrated to 0.3 mL. The mixture was diluted with DCM (1 mL) and purified by flash column chromatography (0.5.fwdarw.15% MeOH/DCM) to give impure compound 92, which was subjected to a 2.sup.nd purification by flash column chromatography (0.fwdarw.15% MeOH/DCM) to give compound 92 (2.0 mg) as a red solid. UPLC-MS (ESI+) calculated for C.sub.59H.sub.69N.sub.6O.sub.19.sup.+ [M+H.sup.+] 1165.5 found 1165.4.

    ##STR00148##

    Example a107. Synthesis of (1-O-(cyclopropyl)--D-arabinopyranose (94)

    [0618] Arabinosyl bromide 10 (3000 mg, 8.85 mmol, 1.0 equiv.) was dissolved in dry Et.sub.2O (36 mL, 0.25 M), this gave a suspension. Cyclopropanol (800 L, 12.40 mmol, 1.4 equiv.) was added, followed by Ag.sub.2O (2050 mg, 8.85 mmol, 1.0 equiv.). The mixture was stirred for 24 h in the dark. TLC analysis (1:9 v/v EtOAc-DCM) and 1H-NMR showed not full conversion (50% conversion) and the reaction was additionally stirred at rt for 24 h. TLC analysis (1:9 v/v EtOAc-DCM) and .sup.1H-NMR showed not full conversion (60% conversion). Cyclopropanol (400 L, 6.20 mmol, 0.7 equiv.) and Ag.sub.2O (1025 mg, 4.43 mmol, 0.5 equiv.) were added and the reaction was additionally stirred at rt for 24 h. TLC analysis (1:9 v/v EtOAc-DCM) and 1H-NMR showed full conversion and the reaction was filtered over Celite, the Celite pad was washed with Et.sub.2O and the ether was removed by rotary evaporation. No further work-up was done and the obtained product 93 was used as such in the next step. The crude mixture of compound 93 (2800 mg, 8.85 mmol, 1.0 equiv.) was dissolved in MeOH (20 mL, 0.45 M), followed by addition of NaOMe (5.4 M in MeOH, 660 L, 3.56 mmol, 0.4 equiv.). After stirring for 2 h at room temperature, TLC analysis (1:9 v/v MeOH-EtOAc) showed full conversion. The reaction mixture was neutralized with a few drops of HCl solution (1 M, 2.5 mL, 2.5 mmol) whereby it changed from a turbid reaction mixture into a clear solution. The mixture was concentrated. The crude product (2.1 g) was dissolved in MeOH (10 mL), applied onto a silica, and purified by flash column chromatography (0.fwdarw.10% MeOH in EtOAc, column pre-conditioned with PE) to obtain the pure compound 94 (520 mg, 2.73 mmol, yield 31% over 2 steps) as a white solid. .sup.1H NMR (300 MHZ, CD.sub.3OD) : 4.9-4.7 (brs, OH, overlap with water), 4.29-4.24 (m, 1H), 3.92-3.78 (m, 2H), 3.72-3.63 (m, 1H), 3.61-3.45 (m, 3H), 0.83-0.70 (m, 1H), 0.62-0.49 (m, 2H), 0.49-0.38 (m, 1H). .sup.13C NMR (101 MHZ, CD3OD) : 105.1, 74.3, 72.2, 69.7, 67.2, 53.0, 6.4, 5.4.

    Example a108. Synthesis of (R)-2-cyclopropyloxy-2-(2-hydroxyethoxy)ethan-1-ol (95)

    [0619] Compound 94 (520 mg, 2.73 mmol, 1.0 equiv.) was dissolved in water (6 mL) and cooled down by a salt ice-bath (5 C.). In the dark, NaOAc (290 mg, 3.5 mmol, 1.3 equiv.) dissolved in water (2 mL) was added followed by NalO.sub.4 (1.46 g, 6.8 mmol, 2.5 equiv.) in portions (15 min). Additional water (3 mL) was added till a final concentration of 0.25 M was reached. The reaction mixture was stirred on ice for 15 minutes after which it was removed. TLC analysis (1:9 v/v MeOH-EtOAc) after 2 h showed full conversion of the starting compound 94 and the reaction mixture was cooled down by a salt ice-bath (5 C.) and NaBH.sub.4 (310 mg, 8.2 mmol, 3.0 equiv.) was added in portions (15 min, colour of the reaction mixture changed from dark red to colourless). TLC analysis (1:9 v/v MeOH-EtOAc) after an hour showed complete conversion of the intermediate aldehyde. EtOAc (30 mL) was added, and the organic layer was separated. The aqueous layer was extracted with EtOAc (30 mL) five times. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated, to obtain product 95 (395 mg, 2.43 mmol, 89%) as a clear orange oil. .sup.1H NMR (300 MHz, CDCl.sub.3) : 4.73 (dd, J=5.9, 3.8 Hz, 1H), 3.99-3.89 (m, 1H), 3.82 (t, J=4.3 Hz, 2H), 3.79-3.56 (m, 3H), 3.56-3.47 (m, 1H), 3.2-2.8 (brs, 2H), 0.72-0.48 (m, 4H). .sup.13C NMR (101 MHZ, CDCl.sub.3) : 103.3, 69.2, 62.7, 61.7, 51.4, 6.4, 5.7.

    Example a109. Synthesis of (R)-(2-iodo-1-(2-iodoethoxy)ethoxy)cyclopropane (7h)

    [0620] Diol 95 (395 mg, 2.43 mmol, 1.0 equiv.) was dissolved in dry THF (13 mL, 0.19 M) and cooled down by a salt ice-bath (5 C.). Imidazole (1.16 g, 17.04 mmol, 7.0 equiv.) was added at once followed by PPh3 (1.91 g, 7.28 mmol, 3.0 equiv.) in portions (10 min). Finally, 12 (1.85 g, 7.29 mmol, 3.0 equiv.) was added in portions (10 min). The mixture was stirred overnight in the dark (salt ice-bath was not removed and the reaction mixture was allowed to slowly warm up to room temperature). After dilution with EtOAc (25 mL), the organic layer was washed with Na.sub.2S.sub.2O.sub.3 (10% aq., 20 mL). The organic layer was separated, and the aqueous layer was extracted with EtOAc (220 mL). The organic layers were combined, washed with brine (20 mL), and dried over Na.sub.2SO.sub.4. The mixture was concentrated, the crude product containing PPh.sub.3/PPh.sub.3O (3.7 g) were dissolved in DCM (4 mL), applied onto a silica, and purified using column chromatography (0.fwdarw.15% EtOAc in PE) to obtain pure compound 7h (540 mg, 1.41 mmol, 58%) as a light-yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3) : 4.72 (t, J=5.4 Hz, 1H), 4.00-3.78 (m, 2H), 3.59-3.48 (m, 1H), 3.34-3.18 (m, 4H), 0.79-0.44 (m, 4H). 13C NMR (101 MHZ, CDCl.sub.3) (ppm) 102.9, 67.9, 50.8, 6.3, 5.4, 5.2, 2.5.

    ##STR00149## ##STR00150##

    Example a110. Synthesis of Compound 96

    [0621] Intermediate 57 (389.7 mg, 81.9 wt %, 1 Eq, 486.8 mol) was suspended in dry DMF (600 L) and then heated and to 43 C. for 10 minutes to obtain a nearly clear solution. Next, bis-iodo-sugar 7h (464.9 mg, 2.5 Eq, 1.217 mmol) in dry DMF (200 L) and DIPEA (188.8 mg, 254 L, 3 Eq, 1.460 mmol) were added. The resulting mixture was stirred in the dark at 38 C. for 2 days, followed by another day at rt. The RM was then diluted with DCM (12 mL) and the resulting red solution was purified by flash column chromatography over silicagel (0.fwdarw.10% MeOH in DCM) to give compound 96 as a red foam (344.5 mg, 89% purity, 0.39 mmol, 81%). LCMS (ESI+) calculated for C.sub.39H.sub.48N.sub.3O.sub.14.sup.+ (M+H.sup.+) 782.32 found 782.64.

    Example a111. Synthesis of Compound 97

    [0622] A solution of compound 96 (344.5 mg, 89 wt %, 1 Eq, 392.2 mol) in a mixture of DCM (10 mL) was cooled by a dry ice/acetone-bath to a temperature of 78 C. The mixture was vigorously stirred after which a freshly made stock solution of mCPBA in DCM (845.2 L, 580 mmolar, 1.25 Eq, 490.2 mol) was added dropwise via the side of the flask. After stirring for 14 minutes, a 2.sup.nd batch of mCPBA in DCM (135.2 L, 580 mmolar, 0.2 Eq, 78.43 mol) was added dropwise via the side of the flask and the RM was stirred for another 17 minutes. The RM was then quenched with cold (78 C.) acetone (reagent-grade, 8.0 mL) and the RM was stirred at 78 C. After 1 hour the cooling-bath was removed, and the RM was allowed to warm up to rt over 35 minutes. The RM was diluted with DCM (30 mL) and saturated aqueous NaHCO.sub.3 solution (15 mL) and transferred to a separation funnel. The resulting biphasic system was separated. The organic layer was dried over Na.sub.2SO.sub.4, filtered, and then concentrated to give compound 97 (334.2 mg, 88% purity, 0.37 mmol, 94% yield), which was used as such in the next step. LCMS (ESI+) calculated for C.sub.39H.sub.48N.sub.3O.sub.15.sup.+ (M+H.sup.+) 798.31 found 798.70.

    Example a112. Synthesis of Compound 98

    [0623] To a solution of 97 (334.2 mg, 88 wt %, 1 Eq, 368.6 mol) in a mixture of dry DCM (3.0 mL) and anhydrous acetonitrile (4.0 mL) was added potassium carbonate (203.8 mg, 4 Eq, 1.475 mmol). The RM was then cooled to 0 C. with an ice-bath after which a solution of cyanuric chloride (4.2 mL, 200 mmolar, 2.3 Eq, 0.84 mmol) in anhydrous acetonitrile was added. After stirring for 70 minutes at 0 C. the RM was quenched with a solution of 3-aminopropane-1,2 diol in H.sub.2O (2.267 mL, 2 molar, 12.3 Eq, 4.534 mmol). The ice bath was removed after 15 min and was then allowed to warm to rt. The resulting bi-phasic system was then separated. The resulting water layer was extracted with DCM (10 mL). The combined organic layers were concentrated in vacuo and the residue was purified by flash column chromatography over silicagel (0.fwdarw.20% MeOH in DCM) to give compound 98 (8.2 mg, 11 mol, 2.9%) as a red solid. LCMS (ESI+) calculated for C.sub.39H.sub.46N.sub.3O.sub.14.sup.+ (M+H.sup.+) 780.30 found 780.64.

    Example a113. Synthesis of Compound 99

    [0624] To a vial containing compound 98 (8.2 mg, 83.5 wt %, 1 Eq, 8.8 mol) was added anhydrous DCM (300 L) and to the resulting red solution was added pyrrolidine (1.9 mg, 2.2 L, 3 Eq, 26 mol), followed by the addition of Pd(PPh.sub.3).sub.4 in dry DCM (1.50 mg, 64.8 L, 20 mmolar, 0.15 Eq, 1.30 mol). The dark red solution was mixed and left at rt for 16 minutes. Additional 20 mmolar Pd(PPh.sub.3).sub.4 in dry DCM (1.5 mg, 66 L, 20 mmolar, 0.15 Eq, 1.3 mol). was added, followed after 45 and 20 minutes by a third (1.5 mg, 66 L, 20 mmolar, 0.15 Eq, 1.3 mol)) and fourth (1.5 mg, 66 UL, 20 mmolar, 0.15 Eq, 1.3 mol)) batch of Pd(PPh.sub.3).sub.4 in dry DCM respectively. Finally, following the fourth addition, the RM was stirred at rt for 40 minutes and was then transferred to a separation funnel together with sat. aq. NH.sub.4Cl (1 mL). The mixture was shaken, and the resulting bi-phasic system was separated. The water-layer was extracted twice with DCM (2 mL, 2). To the combined organic layers was added DMF (250 L) and the resulting solution was partially concentrated in vacuo until all the DCM was removed. The resulting solution was purified by prep-HPLC (20%.fwdarw.50% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30150 mm). The fractions containing the product were combined and concentrated to give compound 99 (0.6 mg) as a red solid. LCMS (ESI+) calculated for C.sub.35H.sub.42N.sub.3O.sub.12.sup.+ (M+H.sup.+) 696.28 found 696.56.

    Example a114. Synthesis of Compound 100

    [0625] To compound 99 (0.6 mg) in DMF was added BCN-HS-PEG.sub.2-OPNP (0.6 mg, 9 L, 131 mmolar, 1 mol) followed by triethylamine (0.3 mg, 0.4 L, 3 mol). The resulting RM was mixed and left at rt for 4.5 hours to give compound 100 as a red solution that was not isolated. LCMS (ESI+) calculated for C.sub.51H.sub.64N.sub.5O.sub.19S.sup.+ (M+H.sup.+) 1082.39 found 1082.84.

    ##STR00151## ##STR00152## ##STR00153##

    Example a115. Synthesis of Compound 101

    [0626] To a round-bottom flask of N,N-Dimethylethane-1,2-diamine (3.02 g, 1.65 Eq, 34.2 mmol) in ethanol (148 mL) was added a solution of allyl (2,5-dioxopyrrolidin-1-yl) carbonate (4.13 g, 1 Eq, 20.7 mmol) in 30 mL ethanol dropwise over 15 min. The RM was stirred overnight at rt. The RM was then stored in the freezer for 3 days. Next, the RM was taken out of the freezer and allowed to warm to rt. And was then concentrated in vacuo and then re-dissolved in 200 ml water. The pH was adjusted to pH 3 by addition of aqueous HCl (1 Molar) and extracted with DCM (100 mL, 4) to remove any bis-reacted amine. This organic layer was discarded. The aqueous layer was adjusted to a pH of 14 with the addition of aqueous NaOH (1 Molar) and extracted with DCM (100 mL, 4). The combined organic layers were washed with aqueous NaOH (2 Molar, 200 mL, 2) and dried over Na.sub.2SO.sub.4, filtrated and concentrated to give impure compound 101. The impure material was re-dissolved in 100 ml water, generating a milky white suspension with yellow solids. The aqueous layer was adjusted to a pH=3 with the addition of aqueous HCl (1 Molar). The solution became less milky, but still not clear. The aqueous layer was washed with MTBE (350 mL), followed by washing steps with Et.sub.2O (50 mL, 3). Finally, the aqueous layer was washed with DCM (50 mL, 3). Next, the aqueous was adjusted to a pH 14 with the addition of aqueous NaOH (1 Molar) and was then extracted with DCM (50 mL, 4). The combined organic layers were then washed with aqueous NaOH (2 Molar, 100 mL, 2), dried over Na.sub.2SO.sub.4, filtrated, and concentrated to obtain a compound 101 (1.01 g, 5.87 mmol, 28.3%) as a clear oil. UPLC-MS (ESI+) calculated for C.sub.8H.sub.17N.sub.2O.sub.2.sup.+ [M+H.sup.+] 173.13 found 173.29.

    Example a116. Synthesis of Compound 102

    [0627] To a round-bottom flask containing compound 55 in DMF (1.61 g, 36.9 mL, 58 mmolar, 1 equiv., 2.14 mmol) was added a solution of allyl methyl (2-(methylamino) ethyl) carbamate (101) (1.00 g, 5.82 mmol, 2.72 equiv.) in dry DMF (3.7 mL). The RM was then placed in a water-bath and HATU (854 mg, 1.05 equiv., 2.25 mmol) was added, followed within one minute by the addition of DIPEA (830 mg, 1.12 mL, 3 equiv., 6.42 mmol) and the resulting dark red solution was stirred at rt for 10 min. The RM was then partially concentrated in vacuo until a volume of 6 ml. The residue was then diluted with DCM (80 mL) and loaded onto a pre-wetted column. The residue was then purified using flash column chromatography over silicagel (0.fwdarw.20% MeOH in DCM). Fractions containing product were combined and concentrated in vacuo, affording compound 102 (1.44 g, 88.1% purity, 1.40 mmol, 65.6%) as a dark red thick oil. UPLC-MS (ESI+) calculated for C.sub.49H.sub.52N.sub.3O.sub.14.sup.+ [M+H.sup.+] 906.34 found 906.90.

    Example a117. Synthesis of Compound 103

    [0628] To a dark red solution of compound 102 (1.44 g, 88.1 wt %, 1.40 mmol, 1 equiv.) in a total volume of DMF (5.85 mL) was added triethylamine (979 L, 7.02 mmol, 5.0 equiv.). The RM turned very dark red (near black) and was left to stir at rt for circa 18 hours. The RM was treated with Et.sub.2O (78 mL) slowly in one go while stirring rapidly. The ether-layer was decanted off and the residue was washed a few more times with Et.sub.2O (330 ml). The residue was concentrated in vacuo to give compound 103 (619 mg, 60.5% purity, 547.0 mol, 39.0%) as a dark red solid, which was used without further purification. UPLC-MS (ESI+) calculated for C.sub.34H.sub.42N.sub.3O.sub.12.sup.+ [M+H.sup.+] 684.28 found 684.65.

    Example a118. Synthesis of Compound 104

    [0629] Compound 103 (598.6 mg, 60.5 wt %, 529.3 mol, 1.00 equiv.) was dissolved in dry DMF (2.41 mL) after which (R)-2-(2-iodo-1-(2-iodoethoxy)ethoxy) propane (7c) (613.5 mg, 1.598 mmol, 3.02 Eq,) was added. The vial was covered in aluminium foil and the reaction was stirred in the dark for 6 days at 40 C. Next, the RM was diluted with DCM (17 mL) and the resulting red solution was purified by flash column chromatography over silicagel (0%.fwdarw.5% MeOH in DCM). The fractions containing pure product were combined and concentrated to give pure compound 104 (110.9 mg, 213.9 mol, 40.4%) as a dark red residue. In addition, fractions containing impure product were combined, affording impure compound 104 (149.3 mg, 89.7% purity, 165 mol, 31.2%). LCMS (ESI+) calculated for C.sub.41H.sub.53N.sub.3O.sub.14.sup.+ (M+H.sup.+) 812.36 found 812.83.

    Example a119. Synthesis of Compound 105

    [0630] Compound 104 (149.3 mg, 89.7 wt %, 165.0 L, 1.00 equiv.) was dissolved in dry DCM (3.0 mL) and dry MeOH (1.0 mL). The mixture was cooled down by a dry ice/acetone cooling-bath (78 C.). Then mCPBA (31.31 mg, 312.8 L, 580 mmolar, 1.1 Eq, 181.4 mol) was dropwise added by using a microman pipette. The RM was stirred vigorously for 5 min. Meanwhile, reagent-grade acetone was cooled in the dry ice/acetone cooling-bath. The RM was then quenched with cold reagent-grade acetone (3.33 mL, 275 Eq, 45.4 mmol) and left stirring in the cold bath for 90 minutes. The dry ice bath was then removed, and the RM was allowed to warm up to room temperature. This was followed by the addition of 20 mL DCM. The RM was then transferred to a separation funnel and washed twice with 15 mL sat. NaHCO.sub.3 aq. solution. The organic layer was dried over Na.sub.2SO.sub.4 and filtered through a membrane filter. The solution was concentrated under vacuo to afford compound 105 (136.3 mg, 82.8% purity, 136 mol, 82.6%) as a red solid. LCMS (ESI+) calculated for C.sub.41H.sub.54N.sub.3O.sub.15.sup.+ (M+H.sup.+) 828.35 found 828.75.

    Example a120. Synthesis of Compound 106

    [0631] To a solution of 105 (136.3 mg, 82.8 wt %, 1.0 Eq, 136.3 mol) in dry MeCN (2.5 mL) and dry DCM (1.0 mL) was added potassium carbonate (75.36 mg, 4.00 Eq, 545.3 mol) and the RM was cooled down with an ice bath (ice bath 0 C.). Then cyanuric chloride in dry MeCN (62.84 mg, 3.408 mL, 100 mmolar, 2.5 Eq, 340.8 mol) was added at once and the mixture was stirred vigorously for 1 h. The RM was then quenched with 3-aminopropane-1,2 diol in H.sub.2O (152.8 mg, 838.4 L, 2 molar, 12.3 Eq, 1.677 mmol). A red-purple precipitate was formed on the sides of the flask. The RM was transferred to a separation funnel, 20 mL of DCM and 20 ml of water were added. Both layers were separated. The organic layer was dried over Na.sub.2SO.sub.4, filtered, and concentrated in vacuo to afford a dark red solid. The residue was redissolved in DCM and purified using flash column chromatography over silicagel (0.fwdarw.10% MeOH in DCM). Fractions containing the product were concentrated in vacuo to afford impure compound 106. The material was subjected to a 2.sup.nd purification using flash column chromatography over silicagel (0.fwdarw.10% MeOH in DCM). Fractions containing the product were concentrated in vacuo to yield compound 106 (26.3 mg, 55% purity, 18 mol, 13%) as a dark red solid. LCMS (ESI+) calculated for C.sub.41H.sub.52N.sub.3O.sub.14.sup.+ (M+H.sup.+) 810.34 found 810.69.

    Example a121. Synthesis of Compound 107

    [0632] Compound 106 (26.3 mg, 55 wt %, 1.0 Eq, 17.9 mol) was dissolved in degassed anhydrous DCM (700 L, degassed for 10 min with N.sub.2). To this solution was added pyrrolidine (3.81 mg, 4.40 L, 3 Eq, 53.6 mol), a dark red solution was then obtained, followed by Pd(PPh.sub.3).sub.4 in degassed anhydrous DCM (3.10 mg, 134 L, 20 mmolar, 0.15 Eq, 2.68 mol). The RM was stirred for 25 min, followed by the addition of additional Pd(PPh.sub.3).sub.4 in degassed anhydrous DCM (3.10 mg, 134 L, 20 mmolar, 0.15 Eq, 2.68 mol). The RM was stirred for another 1 h. Next, pyrrolidine (3.81 mg, 4.40 L, 3 Eq, 53.6 mol) was added, followed after 2 minutes by a 3.sup.rd batch of Pd(PPh.sub.3).sub.4 (3.10 mg, 0.15 Eq, 2.68 mol) as a solid. The RM was stirred for 90 minutes. Finally, a fourth batch of Pd(PPh.sub.3).sub.4 (3.10 mg, 0.15 Eq, 2.68 mol) was added as a solid. The RM was stirred for another 3.5 h. The RM was then transferred to a separation funnel and 10 mL sat. aq. NH.sub.4Cl were added. The resulting bi-phasic system was separated, and the water layer was extracted once with DCM (10 mL). The second organic layer was lighter red, but the water layer stayed dark red. No DP was observed in the water layer. The combined organic layers were dried over Na.sub.2SO.sub.4 and filtered. Next, 300 L DMF was added, and the solution was partially concentrated until all the DCM was gone. This solution was diluted to 800 L with additional DMF and purified by prep-HPLC (Column Xbridge prep C.sub.18, 5 m OBD, 30100 mm, 20%.fwdarw.50% MeCN in 10 mM aq. NH.sub.4HCO.sub.3). Fractions containing the product were combined and concentrated in vacuo. After the fractions were combined and completely concentrated, the red solid was redissolved twice in MeCN and then concentrated and once in DCM and concentrated to yield compound 107 (3.9 mg, 5.4 mol, 30%) as a dark red solid. LCMS (ESI+) calculated for C.sub.37H.sub.48N.sub.3O.sub.12.sup.+ (M+H.sup.+) 726.32 found 726.76.

    Example a122. Synthesis of Compound 108

    [0633] To BCN-HS-GGFG-OH (62) (6.3 mg, 66 wt %, 1.3 Eq, 7.0 mol) was added first DIPEA (2.1 mg, 2.8 L, 3 Eq, 16 mol) and then HATU (2.7 mg, 1.3 Eq, 7.0 mol) in 100 L of DMF. The RM was stirred for 5 minutes. Then, intermediate 107 in 400 L of DMF (3.9 mg, 1.0 Eq, 5.4 mol) was added. The RM was stirred for 1 h. Because of partial conversion, BCN-HS-GGFG-OH (62) (4.1 mg, 1.3 Eq, 7.0 mol) in DMF (150 L), DIPEA (6.1 mg, 3 Eq, 16 mol) and HATU (2.7 mg, 1.3 Eq, 7.0 mol) were pre-mixed for 5 min and added to the main RM, which was stirred for another 2 h. The RM was then diluted to 800 L with additional DMF and purified by Prep-HPLC (Column Xbridge prep C.sub.18, 5 m OBD, 30100 mm, 30%.fwdarw.95% MeCN in 10 mM aq. NH.sub.4HCO.sub.3). The fraction containing the product was concentrated. After the fraction was concentrated, the red solid was redissolved twice in MeCN and then concentrated and once in DCM and concentrated to yield compound 108 (1.2 mg, 0.92 mol, 17%) as a red solid. LCMS (ESI+) calculated for C.sub.63H.sub.79N.sub.8O.sub.20S.sup.+ (M+H.sup.+) 1299.51 found 1300.15.

    ##STR00154##

    Example a123. Synthesis of Compound 110

    [0634] In a vial, amino-PEG2-t-Boc-hydrazide 109 (41.0 mg, 1 Eq, 141 mol) and sodium carbonate (16.6 mg, 1.11 Eq, 157 mol) were dissolved in 1,4-dioxane (1.00 mL) and water (1.00 mL). The RM was stirred at room temperature for 10 minutes, followed by the addition of Fmoc-chloride (37.8 mg, 1.04 Eq, 146 mol). The RM was stirred at rt for 4.5 hours. Then additional sodium carbonate (11.6 mg, 0.778 Eq, 109 mol) and Fmoc-chloride (7.1 mg, 0.20 Eq, 27 mol) were added and left stirring for 19 hours. The pH was adjusted to pH=3 with 4 M aq. HCl and the aqueous phase was extracted thrice with ethyl acetate (35 mL). The organic phases were combined and washed twice with saturated brine (220 mL), dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was then dissolved in DCM (1.0 mL) and then purified using flash column chromatography over silicagel (0.fwdarw.40% aceton in DCM). Fractions containing product were combined and concentrated in vacuo, affording compound 110 (43.4 mg, 84.5 mol, 60.0%) as a clear oil. LCMS (ESI+) calculated for C.sub.27H.sub.36N.sub.3O.sub.7.sup.+ (M+H.sup.+) 514.25 found 514.65.

    Example a124. Synthesis of Compound 111

    [0635] To a solution of compound 110 (43.4 mg, 1 Eq, 84.5 mol) in DCM (1.50 mL) was added 4 M HCl in 1,4-dioxane (46.2 mg, 317 L, 15 Eq, 1.27 mmol) and the RM was stirred at rt for 3 hours. Then, additional 4 M HCl in 1,4-dioxane (61.6 mg, 423 L, 20 Eq, 1.69 mmol) was added and left stirring for 2 hours. The RM was concentrated in vacuo and thrice co-evaporated with toluene, affording compound 111 (35.7 mg, 79.3 mol, 93.9%) as a clear oil. Compound 111 was then dissolved in anhydrous MeOH (1 mL) and used without further purification in the next step. LCMS (ESI+) calculated for C.sub.22H.sub.28N.sub.3O.sub.5.sup.+ (M+H.sup.+) 414.20 found 414.53.

    Example a125. Synthesis of Compound 112

    [0636] 250 L of compound 111 in MeOH solution (8.9 mg, 19.8 mol) was loaded onto a cartridge (PL-HCO3 MP SPE, 500 mg/6 ml tube) and flushed thrice with anhydrous MeOH. The filtrate was concentrated in vacuo, affording an acid-free version of compound 111 (6.9 mg, 2.3 Eq, 15 mol). This was then dissolved in anhydrous MeOH (0.250 mL) and added to a vial containing a mixture of compound 69 (5.1 mg, 85% Wt, 1.0 Eq, 6.6 mol) in anhydrous MeOH (0.250 mL). The resulting suspension was swirled in a 40 C. water-bath for a few minutes until everything dissolved. The RM was stirred for 90 minutes, followed by the addition of acetic acid (0.40 mg, 0.38 L, 1 Eq, 6.6 mol) and the RM was left stirring for 42 hours. In parallel, another batch of compound 111 in MeOH solution (250 L, 8.9 mg, 19.8 mol) was subjected to the procedure described above, but with a reaction time of 18 hours, following the addition of compound 69. Both reaction-mixtures were combined and then diluted with DMF (500 L). The resulting mixture was partially concentrated in vacuo to evaporate the majority of MeOH to give a solution of crude 112 in mainly DMF that was used in the next step without further purification. LCMS (ESI+) calculated for C.sub.56H.sub.65N.sub.4O.sub.16.sup.+ (M+H.sup.+) 1049.44 found 1049.90.

    Example a126. Synthesis of Compound 113

    [0637] To a vial containing a dark red solution of compound 112, (14 mg, 1 Eq. 13 mol) in DMF (500 L) was added triethylamine (14 mg, 19 L, 10 Eq, 0.13 mmol) and the RM was stirred for 3 hours at rt. Then, additional triethylamine (14 mg, 19 L, 10 Eq, 0.13 mmol) was added and the RM was heated in a 40 C. water-bath for 2.5 hours. This solution was diluted to 800 L with additional DMF and purified by prep-HPLC (Column Xbridge prep C.sub.18, 5 m OBD, 30150 mm, 30%.fwdarw.95% MeCN in 10 mM aq. NH.sub.4HCO.sub.3). Fractions containing the product were combined and concentrated in vacuo. After the fractions were combined and completely concentrated, the red solid was redissolved twice in MeCN and then concentrated and once in DCM and concentrated to yield compound 113 (3.6 mg, 4.4 mol, 33%) LCMS (ESI+) calculated for C.sub.41H.sub.55N.sub.4O.sub.14.sup.+ (M+H.sup.+) 827.37 found 827.86.

    Example a127. Synthesis of Compound 114

    [0638] To a vial containing a dark red solution of compound 113 (3.6 mg, 1 Eq, 4.4 mol) in DMF (0.300 mL), was added a solution of BCN-OSu (1.5 mg, 347 mmolar, 1.2 Eq, 5.2 mol) in DMF (15 L), followed by the addition of triethylamine (1.3 mg, 1.8 L, 3 Eq, 13 mol). The RM stirred for 75 minutes at rt. This solution was diluted to 800 L with additional DMF and purified by prep-HPLC (Column Xbridge prep C.sub.18, 5 m OBD, 30150 mm, 30%.fwdarw.95% MeCN in 10 mM aq. NH.sub.4HCO.sub.3). Fractions containing the product were combined and concentrated in vacuo. After the fractions were combined and completely concentrated, the red solid was redissolved twice in MeCN and then concentrated and once in DCM and concentrated to yield compound 114 (0.9 mg, 0.9 mol, 20%) LCMS (ESI+) calculated for C.sub.52H.sub.67N.sub.4O.sub.16.sup.+ (M+H.sup.+) 1003.45 found 1003.94.

    ##STR00155##

    Example a128. Synthesis of Compound 115

    [0639] This compound was synthesized according to a literature procedure described by Weterings et al. in Chemical Science, Volume 11, Issues 33, Pages 9011-9016.

    Example a129. Synthesis of Compound 116

    [0640] To a vial containing compound 115 (168.2 mg, 1.0 Eq, 509.0 mol) in DCM (3.00 mL) was added bis(4-nitrophenyl) carbonate (309.7 mg, 2 Eq, 1.018 mmol) and triethylamine (154.5 mg, 213 L, 3.0 Eq, 1.527 mmol). The solution was stirred for 2 h at rt. The RM was then purified using flash column chromatography over silicagel (0.fwdarw.30% aceton in DCM). Fractions containing product were combined and concentrated in vacuo, affording compound 116 (262.5 mg, 77% purity 0.41 mmol, 80%) that was used without further purification in the next step. .sup.1H NMR (400 MHZ, DMSO-d6) 8.11-8.01 (m, 2H), 6.78 (m, 2H), 4.04 (t, J=4.8 Hz, 2H), 3.88 (d, J=13.9 Hz, 2H), 3.62 (d, J=13.9 Hz, 2H), 3.54 (t, J=4.8 Hz, 2H), 3.47 (m, 1H), 3.38 (m, 2H), 3.34-3.25 (m, 2H), 3.15-3.02 (m, 2H), 2.86-2.95 (m, 1H), 1.34 (s, 5H), 1.25-1.14 (m, 6H). LCMS (ESI+) calculated for C.sub.22H.sub.30N.sub.3O.sub.8S.sup.+ (M+H.sup.+) 496.17 found 496.16.

    Example a130. Synthesis of Compound 117

    [0641] To vial containing compound 61 (4.5 mg, 1 Eq, 6.4 mol) was added a solution of intermediate 116 (6.4 mg, 2 Eq, 13 mol) in DMF (100 L), followed by the addition of triethylamine (2.0 mg, 2.7 L, 3 Eq, 19 mol) and additional DMF (50 L). The resulting solution was mixed and left at rt for circa 1 hour and was then purified by prep-HPLC (30%.fwdarw.95% acetonitrile in 10 mM NH.sub.4HCO.sub.3 in water, column Xbridge prep C.sub.18, 5 M OBD, 30150 mm). The fractions containing product were combined and concentrated to give compound 117 (1.6 mg) as a red solid. LCMS (ESI+) calculated for C.sub.51H.sub.68N.sub.5O.sub.17S.sup.+ (M+H.sup.+) 1054.43 found 1054.93.

    B. Conjugation of Linker-Payloads to Antibodies

    Example b1. Conjugation of Azide-Modified Trastuzumab with Compound 9g to Obtain Conjugate Trast-9g

    [0642] A bioconjugate according to the invention was prepared by conjugation of compound 9g as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (145.6 L, 3.0 mg, 20.6 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (34.4 L), DMF (8.8 L) compound 9g (11.3 L, 14.22 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25742 Da, approximately 80% of total Fc/2 fragment, calculated mass 25740 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25514 Da, approximately 20% of total Fc/2 fragment, calculated mass 25511 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b2. Conjugation of Azide-Modified Trastuzumab with Compound 9d to Obtain Conjugate Trast-9d

    [0643] A bioconjugate according to the invention was prepared by conjugation of compound 9d as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (73.1 L, 3.0 mg, 41.1 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (46.9 L), sodium deoxycholate (20 L 110 mM), PG (40.3 L) and compound 9d (19.7 L, 10.14 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25817 Da, approximately 80% of total Fc/2 fragment, calculated mass 25815 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25514 Da, approximately 20% of total Fc/2 fragment, calculated mass 25511 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b3. Conjugation of Azide-Modified Trastuzumab with Compound 9c to Obtain Conjugate Trast-9c

    [0644] A bioconjugate according to the invention was prepared by conjugation of compound 9c as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (145.6 L, 3.0 mg, 20.6 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (14.4 L), DMF (8.7 L) and compound 9c (31.3 L, 3.83 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25770 Da, approximately 80% of total Fc/2 fragment, calculated mass 25768 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25517 Da, approximately 20% of total Fc/2 fragment, calculated mass 25511 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b4. Conjugation of Azide-Modified Trastuzumab with Compound 9f to Obtain Conjugate Trast-9f

    [0645] A bioconjugate according to the invention was prepared by conjugation of compound 9f as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (728.2 L, 15 mg, 20.6 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (71.8 L), sodium deoxycholate (71.8 L 110 mM), DMF (51.2 L) and compound 9f (48.8 L, 8.2 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25755 Da, approximately 80% of total Fc/2 fragment, calculated mass 25752 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25514 Da, approximately 20% of total Fc/2 fragment, calculated mass 25509 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b5. Conjugation of Azide-Modified Trastuzumab with Compound 36 to Obtain Conjugate Trast-36

    [0646] A bioconjugate according to the invention was prepared by conjugation of compound 36 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (145.6 L, 3 mg, 20.6 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (454.4 L), sodium deoxycholate (80 L 110 mM) and compound 36 (120 L, 0.64 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25744 Da, approximately 60% of total Fc/2 fragment, calculated mass 25741 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25329 Da, approximately 40% of total Fc/2 fragment, calculated mass 25327 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b6. Conjugation of Azide-Modified Trastuzumab with Compound 47 to Obtain Conjugate Trast-47

    [0647] A bioconjugate according to the invention was prepared by conjugation of compound 47 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (485.4 L, 10.0 mg, 20.6 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (124.1 L), sodium deoxycholate (76.2 L 110 mM) and compound 47 (76.2 L, 3.5 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25622 Da, approximately 80% of total Fc/2 fragment, calculated mass 25620 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25382 Da, approximately 20% of total Fc/2 fragment, calculated mass 25378 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b7. Conjugation of Azide-Modified Trastuzumab with Compound 53 to Obtain Conjugate Trast-53

    [0648] A bioconjugate according to the invention was prepared by conjugation of compound 53 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (447.82 L, 9.225 mg, 20.6 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (352.2 L), sodium deoxycholate (100 L 110 mM) and compound 53 (100 L, 2.46 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25475 Da, approximately 90% of total Fc/2 fragment, calculated mass 25473 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25234 Da, approximately 10% of total Fc/2 fragment, calculated mass 25230 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b8. Conjugation of Azide-Modified Trastuzumab with Compound 39 to Obtain Conjugate Trast-39

    [0649] A bioconjugate according to the invention was prepared by conjugation of compound 39 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (109.2 L, 2.25 mg, 20.6 mg/ml in TBS pH 7.4), prepared according to WO2016170186, was added TBS pH 7.4 (370.8 L), sodium deoxycholate (60 L 110 mM) and compound 39 (60 L, 1.5 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25653 Da, approximately 70% of total Fc/2 fragment, calculated mass 25652 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25240 Da, approximately 30% of total Fc/2 fragment, calculated mass 25238 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b9. Transient Expression and Purification of Trast (HC-E152C,S375C)

    [0650] A trastuzumab variant with heavy chain comprising E152C and S375C mutations, (identified by SEQ ID NO: 2), and non-modified light chain (identified by SEQ ID NO: 3) was transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) at 500 mL scale. The supernatant was purified using a XK 16/20 column packed with 25 mL protein A sepharose. In a single run 500 mL supernatant was loaded onto the column followed by washing with at least 10 column volumes of 20 mM Tris pH 7.5, 150 mM NaCl. Retained protein was eluted with 0.1 M sodium acetate pH 2.7. The eluted trast (HC-E152C,S375C) was immediately neutralized with 2.5 M Tris-HCl pH 7.2 and dialyzed against PBS pH 7.4. Next the IgG was concentrated to 20.76 mg/ml using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and stored at 80 C. prior to further use.

    Example b10. Reduction and Re-Oxidation of Trast (HC-E152C,S375C)

    [0651] Trast (HC-E152C,S375C) (20 mg, 20.76 mg/mL in PBS pH 7.4) was incubated with EDTA pH 8.0 (54 L, 200 mM in MQ) and DTT (54 L, 200 mM in MQ) for 2 hours at 37 C. The reduced antibody was buffer exchanged to PBS pH 7.4 using a HiTrap desalting column (Cytiva, 25 mL columns connected in series) on an 100F NGC system (Bio-Rad). Next, the reduced antibody (17.8 mg, 6.13 mg/mL in PBS pH 7.4) was incubated with EDTA pH 8.0 (145 L, 200 mM in MQ) and dehydroascorbic acid (119 L, 10 mM in MQ: DMSO (9:1)) for 3 hours at room temperature. The re-oxidized antibody was buffer exchanged to PBS pH 7.4 using a HiTrap desalting column (Cytiva, 25 mL columns connected in series) on an 100F NGC system (Bio-Rad). LC-MS analysis of the fabricator-digested sample showed one major product for the Fc/2-fragment (observed mass 25249 Da, approximately 60% of total Fc/2 fragment), corresponding to the Fc/2 fragment with G0F-glycoform and free Cys.sub.375, and one minor product (observed mass 25411 Da, approximately 40% of total Fc/2 fragment), corresponding to the Fc/2 fragment with G0F-glycoform and free Cys.sub.375. LC-MS analysis of the fabricator-digested sample showed one major product for the Fab-fragment (observed mass 97639 Da, approximately 70% of total Fab-fragment), corresponding to the Fab-fragment with both Cys.sub.152-residues in the free reduced form. Re-oxidized trast (HC-E152C,S375C) was stored at 80 C. prior to further use.

    Example b11. Conjugation of Re-Oxidized Trast (HC-E152C,S375C) with Compound 70 to Obtain Conjugate Trast (HC-E152C,S375C)-70

    [0652] A bioconjugate according to the invention was prepared by conjugation of compound 70 as linker-conjugate to reduced and re-oxidized trast (HC-E152C,S375C) as biomolecule. To a solution of reduced and re-oxidized trast (HC-E152C,S375C) (60 L, 232 g, 3.87 mg/ml in PBS pH 7.4), prepared as described above, was added compound 70 (9.4 L, 1 mM solution in DMF). The reaction was incubated at rt for 1 hour followed by buffer exchange using spinfiltration (Amicon Ultra-0.5, Ultracel-10 Membrane, Millipore), 3 with 400 L PBS pH 7.4. Conjugation to Cys.sub.375 was evaluated using a fabricator-digested sample via analysis of the Fc/2-fragment. LC-MS analysis showed two products corresponding to the conjugated Fc-2 fragment with G0F and G1F glycan (observed masses 26067 and 26231 Da, approximately 15% and 10% of total Fc/2-fragment, respectively), and two products corresponding to the conjugated Fc-2 fragment with G0F and G1F glycan and the conjugated Fc-2 fragment with subsequent hydrolysis or fragmentation of the hydrazone (observed masses 25433 and 25595 Da, approximately 30% and 20% of total Fc/2-fragment, respectively). Conjugation to Cys.sub.152 was evaluated using a fabricator-digested sample followed by reduction via analysis of the Fd-fragment. LC-MS analysis showed one major product corresponding to the conjugated Fd-fragment (observed mass 26201 Da, approximately 60% of total Fd-fragment), and one minor product corresponding to the conjugated Fd fragment with hydrolysis or fragmentation of the hydrazone (observed mass 25565 Da, approximately 40% of total Fd-fragment).

    Example b12. Conjugation of Azide-Modified Trastuzumab with Compound 75 to Obtain Conjugate Trast-75

    [0653] A bioconjugate according to the invention was prepared by conjugation of compound 75 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (652 L, 15.0 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (148 L), sodium deoxycholate (100 L 110 mM) and compound 75 (100 L, 6 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25726 Da, approximately 60% of total Fc/2 fragment, calculated mass 25725 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25327 Da, approximately 20% of total Fc/2 fragment, calculated mass 25328 Da), corresponding to the conjugated Fc/2 fragment containing a fragmentation product of PNU.

    Example b13. Conjugation of Azide-Modified Trastuzumab with Compound 63a to Obtain Conjugate Trast-63a

    [0654] A bioconjugate according to the invention was prepared by conjugation of compound 63a as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (87 L, 2.0 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (73 L), sodium deoxycholate (20 L 110 mM) and compound 63a (20 L, 4 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25636 Da, approximately 80% of total Fc/2 fragment, calculated mass 25636 Da), corresponding to the conjugated Fc/2 fragment.

    Example b14. Conjugation of Azide-Modified Palavizumab with Compound 63a to Obtain Conjugate Palav-63a

    [0655] A bioconjugate according to the invention was prepared by conjugation of compound 63a as linker-conjugate to palavizumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of palavizumab-(6-N.sub.3-GalNAc).sub.2 (19.6 L, 0.5 mg, 25.6 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (20.4 L), sodium deoxycholate (5 L 110 mM) and compound 63a (5 L, 4 mM solution in DMF). The reaction was incubated at rt overnight. LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25636 Da, approximately 80% of total Fc/2 fragment, calculated mass 25636 Da), corresponding to the

    Example b15. Conjugation of Azide-Modified Rituximab with Compound 63a to Obtain Conjugate Rit-63a

    [0656] A bioconjugate according to the invention was prepared by conjugation of compound 63a as linker-conjugate to rituximab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of rituximab-(6-N.sub.3-GalNAc).sub.2 (20.4 L, 0.5 mg, 24.5 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (19.6 L), sodium deoxycholate (5 L 110 mM) and compound 63a (5 L, 4 mM solution in DMF). The reaction was incubated at rt overnight. LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25604 Da, approximately 80% of total Fc/2 fragment, calculated mass 25604 Da), corresponding to the conjugated Fc/2 fragment.

    Example b16. Conjugation of Azide-Modified Trastuzumab with Compound 63b to Obtain Conjugate Trast-63b

    [0657] A bioconjugate according to the invention was prepared by conjugation of compound 63b as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (87 L, 2.0 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (73 L), sodium deoxycholate (20 L 110 mM) and compound 63b (20 L, 4 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25449 Da, approximately 70% of total Fc/2 fragment, calculated mass 25449 Da), corresponding to the conjugated Fc/2 fragment.

    Example b17. Conjugation of Azide-Modified Trastuzumab with Compound 63c to Obtain Conjugate Trast-63c

    [0658] A bioconjugate according to the invention was prepared by conjugation of compound 63c as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (5.0 L), sodium deoxycholate (3.3 L 110 mM) and compound 63c (3.3 L, 4 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25446 Da, approximately 80% of total Fc/2 fragment, calculated mass 25447 Da), corresponding to the conjugated Fc/2 fragment.

    Example b18. Conjugation of Azide-Modified Trastuzumab with Compound 80 to Obtain Conjugate Trast-80

    [0659] A bioconjugate according to the invention was prepared by conjugation of compound 80 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (5.0 L), sodium deoxycholate (3.3 L 110 mM) and compound 80 (3.3 L, 8 mM solution in DMF). The reaction was incubated at rt overnight. LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25656 Da, approximately 60% of total Fc/2 fragment, calculated mass 25655 Da), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 24366 Da, approximately 25% of total Fc/2 fragment, calculated mass 24365 Da), corresponding to the remaining azido-modified Fc/2 fragment.

    Example b19. Conjugation of Azide-Modified Trastuzumab with Compound 81 to Obtain Conjugate Trast-81

    [0660] A bioconjugate according to the invention was prepared by conjugation of compound 81 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (5.0 L), sodium deoxycholate (3.3 L 110 mM) and compound 81 (3.3 L, 8 mM solution in DMF). The reaction was incubated at rt overnight. LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25671 Da, approximately 50% of total Fc/2 fragment, calculated mass 25671 Da), corresponding to the conjugated Fc/2 fragment, and various minor products (observed masses 25256 Da, 25530 Da and 26246 Da, approximately 40% of total Fc/2 fragment).

    Example b20. Conjugation of Azide-Modified Trastuzumab with Compound 108 to Obtain Conjugate Trast-108

    [0661] A bioconjugate according to the invention was prepared by conjugation of compound 108 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (5.0 L), sodium deoxycholate (3.3 L 110 mM) and compound 108 (3.3 L, 6 mM solution in DMF). The reaction was incubated at rt for 4 hours. LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25664 Da, approximately 80% of total Fc/2 fragment, calculated mass 25664 Da), corresponding to the conjugated Fc/2 fragment.

    Example b21. Conjugation of Azide-Modified Trastuzumab with Compound 117 to Obtain Conjugate Trast-117

    [0662] A bioconjugate according to the invention was prepared by conjugation of compound 117 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (5.0 L), sodium deoxycholate (3.3 L 110 mM) and compound 117 (3.3 L, 3 mM solution in DMF). The reaction was incubated at rt for 5 minutes. LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25491 Da, approximately 80% of total Fc/2 fragment, calculated mass 25419 Da), corresponding to the

    Example b22. Conjugation of Azide-Modified Trastuzumab with Compound 86 to Obtain Conjugate Trast-86

    [0663] A bioconjugate according to the invention was prepared by conjugation of compound 86 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (8.3 L), sodium deoxycholate (5 L 110 mM) and compound 86 (15 L, 2.7 mM solution in PG). The reaction was incubated at rt for 4 hours. LC-MS analysis of the fabricator-digested sample showed one major product (observed mass 25698 Da, approximately 50% of total Fc/2 fragment, calculated mass 25699 Da), corresponding to the conjugated Fc/2 fragment, and one major product (observed mass 25255 Da, approximately 50% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment with fragmentation of PNU.

    Example b23. Conjugation of Azide-Modified Trastuzumab with Compound 63d to Obtain Conjugate Trast-63d

    [0664] A bioconjugate according to the invention was prepared by conjugation of compound 63d as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (18.3 L), sodium deoxycholate (5.0 L 110 mM) and compound 63d (5 L, 8 mM solution in DMF). The reaction was incubated at rt for 4 hours. Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25577 Da, approximately 70% of total Fc/2 fragment, calculated mass 25578 Da), corresponding to the conjugated Fc/2 fragment.

    Example b24. Conjugation of Azide-Modified Trastuzumab with Compound 114 to Obtain Conjugate Trast-114

    [0665] A bioconjugate according to the invention was prepared by conjugation of compound 114 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (8.3 L), sodium deoxycholate (5.0 L 110 mM) and compound 114 (15.0 L, 2.7 mM solution in PG). The reaction was incubated at rt for 4 hours. Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25367 Da, approximately 40% of total Fc/2 fragment, calculated mass 25368 Da), corresponding to the conjugated Fc/2 fragment, and one major products (observed mass 25112 Da, approximately 40% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment with fragmentation of PNU.

    Example b25. Conjugation of Azide-Modified Trastuzumab with Compound 92 to Obtain Conjugate Trast-92

    [0666] A bioconjugate according to the invention was prepared by conjugation of compound 92 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (8.3 L), sodium deoxycholate (5.0 L 110 mM) and compound 92 (15 L, 2.7 mM solution in PG). The reaction was incubated at rt for 4 hours. Mass spectral analysis of the fabricator-digested sample showed one major products (observed mass 25529 Da, approximately 50% of total Fc/2 fragment, calculated mass 25530 Da), corresponding to the conjugated Fc/2 fragment, and one major products (observed mass 25114 Da, approximately 50% of total Fc/2 fragment, calculated mass 25118 Da), corresponding to the conjugated Fc/2 fragment with fragmentation of PNU.

    Example b26 MV0323. Conjugation of Azide-Modified Trastuzumab with Compound 92 to Obtain Conjugate Trast-92

    [0667] A bioconjugate according to the invention was prepared by conjugation of compound 92 as linker-conjugate to trastuzumab-(6-N.sub.3-GalNAc).sub.2 as biomolecule. To a solution of trastuzumab-(6-N.sub.3-GalNAc).sub.2 (21.7 L, 0.5 mg, 23.0 mg/ml in TBS pH 7.5), prepared according to WO2016170186, was added TBS pH 7.5 (8.3 L), sodium deoxycholate (5.0 L 110 mM) and compound 92 (15 UL, 0.9 mM solution in PG). The reaction was incubated at rt overnight. Mass spectral analysis of the fabricator-digested sample showed one minor product (observed mass 25482 Da, approximately 25% of total Fc/2 fragment, calculated mass 25484 Da), corresponding to the conjugated Fc/2 fragment, and two other products (observed masses 24723 Da and 25067 Da, approximately 50% and 25% of total Fc/2 fragment, respectively), corresponding to fragmentation products of the conjugated Fc/2 fragment.

    [0668] C. In vitro evaluation

    Example c1. In Vitro Studies Compounds 6a, 6b, 6c, 6d and 6e

    [0669] SK-OV-3, NCI-N87, JIMT-1 and MDA-MB-231 cells were plated in 96-well plates (5000 cells/well) in RPMI-1640 GlutaMAX (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, 150 L/well) and incubated overnight in a humidified atmosphere at 37 C. and 5% CO2. Compounds 6a, 6b, 6c, 6d and 6e were added in triplo in a square root of 10-fold dilution series to obtain a final concentration ranging from 1 pM to 10 nM (for SK-OV-3 and NCI-N87) or 10 pM to 100 nM (for JIMT-1 and MDA-MB-231) and a final volume of 200 L/well. The cells were incubated for 3 days in a humidified atmosphere at 37 C. and 5% CO2. The culture medium was replaced by 0.01 mg/mL resazurin (Sigma Aldrich) in RPMI-1640 GlutaMAX supplemented with 10% FBS (200 L/well). After 4 hours in a humidified atmosphere at 37 C. and 5% CO.sub.2 the fluorescence was detected with a fluorescence plate reader (Infinite M1000 Tecan) at 560 nm excitation and 590 nm emission. The relative fluorescent units (RFU) were normalized to cell viability percentage by setting wells without cells at 0% viability and wells with untreated cells at 100% viability. Viability was plotted using Graphpad prism software (see FIG. 20). IC.sub.50 values calculated by non-linear regression using Graphpad prism software and are shown in the table below. For compound 6a and 6b no accurate IC50 value could be calculated in JIMT-1 and MDA-MB-231 cells since no plateau was reached at the lowest tested concentration.

    TABLE-US-00001 TABLE 1 IC.sub.50 values of free payloads in various HER2 positive and HER2 negative cell lines. MDA-MB- SK-OV-3 NCI-N87 JIMT-1 231 (HER2 3+) (HER2 3+) (HER2 1+) (HER2) Compound 6a 28 pM 85 pM No IC.sub.50 No IC.sub.50 Compound 6b 99 pM 224 pM No IC.sub.50 No IC.sub.50 Compound 6c 187 pM 416 pM 159 pM 174 pM Compound 6d 217 pM 339 pM 259 pM 169 pM Compound 6e 845 pM 795 pM 520 pM 705 pM

    Example c2. In Vitro Studies with Trast-9g, Trast-9d, Trast-9c and Trast-36

    [0670] SK-OV-3 (Her2 3+), NCI-N87 (Her2 3+), JIMT-1 (Her2 1+) and MDA-MB-231 (Her2) cells were plated in 96-well plates (5000 cells/well) in RPMI-1640 GlutaMAX (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, 150 L/well) and incubated overnight in a humidified atmosphere at 37 C. and 5% CO2. ADCs were added in triplo in a square root of 10-fold dilution series to obtain a final concentration ranging from 1 pM to 10 nM and a final volume of 200 L/well. The cells were incubated for 5 days in a humidified atmosphere at 37 C. and 5% CO2. The culture medium was replaced by 0.01 mg/mL resazurin (Sigma Aldrich) in RPMI-1640 GlutaMAX supplemented with 10% FBS (200 L/well). After 4 hours in a humidified atmosphere at 37 C. and 5% CO.sub.2 the fluorescence was detected with a fluorescence plate reader (Infinite M1000 Tecan) at 560 nm excitation and 590 nm emission. The relative fluorescent units (RFU) were normalized to cell viability percentage by setting wells without cells at 0% viability and wells with untreated cells at 100% viability. Viability was plotted using Graphpad prism software (see FIG. 17). IC50 values for ADCs on SKOV-3, N87 and JIMT-1 were calculated by non-linear regression using Graphpad prism software and are shown in the table below.

    TABLE-US-00002 TABLE 2 IC.sub.50 values of ADCs in various HER2 positive and HER2 negative cell lines. SK-OV-3 NCI-N87 JIMT-1 MDA-MB-231 (HER2 3+) (HER2 3+) (HER2 1+) (HER2) trast-9g 0.097 nM 0.039 nM 0.046 nM No IC.sub.50 trast-9d 0.287 nM 0.207 nM 0.133 nM trast-9c 0.211 nM 0.179 nM 0.145 nM trast-36 0.222 nM 0.086 nM 0.015 nM

    Example c3. In Vitro Studies with Trast-9g, Trast-63a, Trast-63b and Trast-75

    [0671] BT-474 (Her2 3+), SK-OV-3 (Her2 3+) and MDA-MB-231 (Her2-) cells were plated in 96-well plates (5000 cells/well) in RPMI-1640 GlutaMAX (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, 150 L/well) and incubated overnight in a humidified atmosphere at 37 C. and 5% CO2. ADCs were added in triplo in a square root of 10-fold dilution series to obtain a final concentration ranging from 1 pM to 10 nM and a final volume of 200 L/well. The cells were incubated for 5 days in a humidified atmosphere at 37 C. and 5% CO2. The culture medium was replaced by 0.01 mg/mL resazurin (Sigma Aldrich) in RPMI-1640 GlutaMAX supplemented with 10% FBS (200 L/well). After 4 hours in a humidified atmosphere at 37 C. and 5% CO2 the fluorescence was detected with a fluorescence plate reader (Infinite M1000 Tecan) at 560 nm excitation and 590 nm emission. The relative fluorescent units (RFU) were normalized to cell viability percentage by setting wells without cells at 0% viability and wells with untreated cells at 100% viability. Viability was plotted using Graphpad prism software (see FIG. 21). IC50 values for ADCs on SKOV-3, N87 and JIMT-1 were calculated by non-linear regression using Graphpad prism software and are shown in the table below. For trast-6b no accurate IC50 value could be calculated in SK-OV3 cells since not more than 50% of the cells died at the highest tested concentration.

    TABLE-US-00003 TABLE 3 IC.sub.50 values of ADCs in various HER2 positive and HER2 negative cell lines. BT-474 SK-OV-3 MDA-MB-231 (HER2 3+) (HER2 3+) (HER2) trast-9g 43 pM 57 pM No IC.sub.50 trast-75 219 pM 149 pM trast-63a 196 pM 224 pM trast-63b 390 pM No IC.sub.50

    D. In Vitro Evaluation

    Example d1. In Vivo Tolerability in CD-1 Mice

    [0672] Nave female CD-1 mice (n=3), obtained from Beijing Vital River Laboratory Animal Technology Co, were treated with vehicle or a single dose of antibody-drug conjugate trast-9g (5 mg/kg), trast-9d (at 20 mg/kg), trast-36 (20 mg/kg), trast-47 (20 mg/kg) or trast-9c (40 mg/kg). After dosing, animals were checked daily for morbidity and mortality for 25 days. During routine monitoring, the animals were checked for any effects of treatments on behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail (depicted in (FIGS. 18A and 18B).

    Example d2. In Vivo Efficacy in JIMT-1 CDX Model

    [0673] Female NOD/SCID mice (5- to 8-week-old at study initiation, obtained from GemPharmatech Co. Ltd., China) were inoculated subcutaneously it the right front flank region with 510.sup.6 JIMT-1 human breast cancer cells in 0.1 ml of PBS for tumor development. When the tumor volume was in the range of 100 to 150 mm.sup.3, groups of five mice were injected i.v. with either vehicle, trast-9g (at 0.3 or 1 mg/kg), trast-9d (at 3.0 or 5.0 mg/kg), trast-36 (at 0.6 or 2.0 mg/kg), trast-47 (at 0.6 or 2.0 mg/kg) and trast-9c (at 2.0 mg/kg). In all cases a single dose was administered on day 0. Tumor volume and body weight was measured twice per week after randomization (FIGS. 19A, 19B and 19C).

    Sequence List

    TABLE-US-00004 SequenceidentificationofGGFGcleavablelinker (SEQ.IDNO:1): GGFG Sequenceidentificationoftrastuzumab(HC-E152C, S375C)heavychain(SEQ.IDNO:2): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequenceidentificationoftrastuzumab(HC-E152C, S375C)lightchain(SEQ.IDNO:3): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC