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BIS(2-HALOACETAMIDO)-COMPOUNDS FOR USE AS LINKING AGENTS AND RESULTANT PRODUCTS WHICH COMPRISE ANTIBODIES, HALF-ANTIBODIES AND ANTIBODY FRAGMENTS

20220249687 · 2022-08-11

    Inventors

    Cpc classification

    International classification

    Abstract

    Bis(2-haloacetamido)-compounds for use as linkers to chemically cross-linking multiple thiol groups, and particularly, although not exclusively, the thiol groups of cysteine amino acids in peptide chains are described, along with their use as linking agents and resultant products which comprise antibodies, half-antibodies and antibody fragments having thiol groups bonded to said linkers (e.g. antibody-protein conjugates and antibody-drug conjugates), and methods of making said conjugates and products. (Formula I)

    ##STR00001##

    Claims

    1. A linker compound of Formula (I) or a salt thereof: ##STR00043## wherein each X is independently F, Cl, Br, or I; R.sup.1 is H, COOR.sup.A, CONH.sub.2, CONHR.sup.A, CONR.sup.A.sub.2, CONHL, or CONR.sup.AL; L, if present, is a chain terminating in a reactive group R.sup.3; each R.sup.A, if present, is independently selected from C.sub.1-4alkyl; n is 0, 1, 2, or 3; and each R.sup.2, if present, is independently selected from F, Cl, Me, CF.sub.3, OMe, and OCF.sub.3 or R.sup.2 is a group as defined for R.sup.1.

    2. The compound of claim 1 wherein L is a polyether or polythioether terminating in R.sup.3 and R.sup.3 is selected from N.sub.3, a group comprising a C≡C bond, a protected amine, a leaving group and a moiety of Formula (H): ##STR00044## wherein each X is independently F, Cl, Br, or I; n is 0, 1, 2, or 3; and each R.sup.2, if present, is independently selected from F, Cl, Me, CF.sub.3, OMe, and OCF.sub.3 or R.sup.2 is a group as defined for R.sup.1 in claim 1.

    3. The compound of claim 1 wherein n is 0.

    4. The compound of claim 1, wherein X is Br or I.

    5. The compound of claim 1, wherein the compound is of Formula (IIa), (IIb) or (IIc): ##STR00045## wherein X is Br or I; and R.sup.1 is H, COOMe, CONH.sub.2, or CONHL.

    6. The compound of claim 1, wherein the compound is of Formula (IVa), (IVb) or (IVc) ##STR00046## optionally wherein: each X is Br; or each X is I; or two X one arylene are Br and two X or the other arylene are I.

    7. The compound of claim 1, wherein the compound is selected from Examples (1) to (18): ##STR00047## ##STR00048## ##STR00049##

    8. A half-antibody comprising a linker residue bridging intra- the heavy chain-heavy chain cysteine residues of the hinge region, wherein the linker residue is a moiety as defined in claim 1 with two X displaced by thiol groups.

    9. A mono-functionalized antibody comprising a linker residue bridging the heavy and light chains, wherein the linker residue is a moiety as defined in claim 7, with two X displaced by thiol groups.

    10. A thio bridged fab antibody fragment comprising a linker residue which is a moiety as defined in claim 1 with two X displaced by thiol groups.

    11. The thio bridged fab antibody fragment of claim 10, wherein the linker residue is a moiety of Formula (IXa), (IXb), (IXc) or (IXd): ##STR00050## wherein X is Br or I; and where m is selected from 3, 4, 5, 6, 7, 8, 9, and 10 and custom-character represents a point of attachment to the protein chain.

    12. A method for producing a mono-functionalized antibody comprising a linker residue bridging the heavy and light chains, wherein the linker residue is a moiety of Formula (Ia) with two X displaced by thiol groups, the method comprising treating a fully reduced antibody with about 1 to 1.1 equivalents of a linker compound of Formula Ia: ##STR00051## wherein X is Br; preferably wherein n is 0.

    13. A method for producing a mono-functionalized antibody comprising a linker residue bridging the heavy and light chains, wherein the linker residue is a moiety as defined in claim 1 with two X displaced by thiol groups, the method comprising: (i) providing a partially reduced antibody by reducing an antibody using about 1 to 1.1 equivalent of a reducing agent; then (ii) treating said partially reduced antibody with said linker compound.

    14. . A method of producing a half-antibody comprising a linker residue bridging intra- the heavy chain-heavy chain cysteine residues of the hinge region and a linker residue bridging the light and heavy chains, wherein the linker residue is a moiety as defined in claim 1 with two X displaced by thiol groups, the method comprising treating a fully reduced antibody with at least 4 equivalents of said linker compound.

    15. A method of producing a half-antibody wherein the linker residue is a moiety of Formula (Ib) bridging intra-HC-HC cysteine residues of the hinge region, the method comprising treating a fully reduced antibody with about 2 to 2.2 equivalents of a linker compound of Formula (Ib): ##STR00052## wherein X is I; preferably wherein n is 0 and/or preferably wherein the R.sup.1 group and two haloacetamide groups are arranged in a 1,3,5 configuration; optionally wherein the method comprises, after treatment of the fully reduced antibody with about 2 to 2.2 equivalents of a linker compound of Formula (Ib), treatment with a further linker compound according to the invention.

    16. A method of producing a hetero-bi-functionalised half-antibody having a first linker residue bridging intra- the heavy chain-heavy chain cysteine residues of the hinge region and a second linker residue bridging the light and heavy chains, wherein each linker residue is a moiety as defined in claim 1 with two X displaced by thiol groups, the method comprising: (i) treating a partially reduced antibody with said first linker compound to produce a first conjugate; then (ii) further reducing said first conjugate to produce a reduced conjugate; then (iii) treating said reduced conjugate with said second linker compound, wherein the first and second linker compounds are different, to produce said hetero-bi-functionalised half-antibody conjugate.

    17. The method of claim 12, wherein at least one of the linker compounds includes L and the method includes a step of attaching a further moiety selected from an antibody, half-antibody, antibody fragment, protein, polypeptide, drug or a fluorescent or radio label.

    18. A method of producing a Fab-Mab or Fab-protein conjugate, the method comprising treating a reduced or partially reduced antibody or a protein with a thio bridged fab antibody fragment according to claim 10.

    19. The method of claim 12, wherein R.sup.1 is H, COOMe, CONH.sub.2, or CONHL and n is 0.

    20. The half-antibody of claim 8, wherein R.sup.1 is H, COOMe, CONH.sub.2, or CONHL and n is 0.

    Description

    SUMMARY OF THE FIGURES

    [0158] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

    [0159] FIG. 1 shows representative diagrams of certain conjugate products according to the invention, where the labels L1 and L2 denote head groups rebridging or intra bridging at the disulfides and the black curve indicates a spacer. Native disulfides are indicated by a black line joining black dots.

    [0160] FIG. 2 shows a comparison of the aqueous stability of methyl ester derivatised 3,4- and 3,5-linker compounds.

    [0161] FIG. 3 shows a comparison between the aqueous stability of ester and amide derivatised aryl bis-haloacetamide linkers.

    [0162] FIG. 4 shows the percentage remaining of the bis-haloacetamide derivatives 14-17 in the presence of glutathione (2.2 equiv.) in aqueous phosphate buffer (100 mM, pH 7.5).

    [0163] FIG. 5 shows SDS-PAGE analysis of cross-linking of fully reduced Tmab with excess of linker compounds 2, 5, 8 and 12 under varying stoichiometry. For key see Example 4.

    [0164] FIG. 6 shows SDS-PAGE analysis of cross-linking of fully reduced Tmab with excess of linker compounds 2, 5, 8 and 12 at varying pH. For key see Example 5.

    [0165] FIG. 7 shows SDS-PAGE analysis of cross-linking of fully reduced Tmab with linker compounds 3, 6, 10 and 13. For key see Example 6.

    [0166] FIG. 8 shows SDS-PAGE analysis of cross-linking of fully reduced Tmab with linker compounds 14, 15, 16 and 17. For key see Example 7.

    [0167] FIG. 9 shows SDS-PAGE analysis of cross-linking of fully reduced Rituximab with linker compounds 3, 6, 9 and 12. For key see Example 8.

    [0168] FIG. 10 shows SDS-PAGE analysis of cross-linking of partially reduced Tmab with compounds 2, 5, 9 and 12. For key see Example 9.

    [0169] FIG. 11 shows SDS-PAGE analysis of bifunctional cross-linking of using sequential method with linker compounds 5 and 15 (a) and a schematic representation of the product (b). For key see Example 9.

    [0170] FIG. 12 shows (a) a schematic representation of a thio-bridged Fab derivative, (b) SDS-PAGE analysis of conjugation of partially reduced Tmab with Ipilimumab-fAb (for key see Example 10), and (c) a schematic representation of a tri-specific mAb according to the invention. For key see Example 12.

    [0171] FIG. 13 shows (a) SDS-PAGE analysis of cross-linking of fully reduced Tmab with compounds 15 and 17, (b) characterization of the reaction products from 15 using mass spectroscopy, and (c) characterization of the reaction products from 17 using mass spectroscopy. For key see Example 11.

    [0172] FIG. 14 shows confocal microscopy of spheroid models.

    DETAILED DESCRIPTION OF THE INVENTION

    [0173] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

    [0174] Compound Stability

    [0175] The linker compounds of the invention have desirable stability in water, and do not readily undergo hydrolysis.

    [0176] Significant differences are observed in the aqueous stability of the methyl bis(2-haloacetamido)-benzoate compounds stored in phosphate buffer at pH=7. In general, the 3,5-compounds (meta arrangement) were much more stable than the 2,3- and 3,4-compounds (ortho arrangement). Interestingly, stability did not directly correlate with the halide leaving group within each group of compounds. For 3,4-substituted benzoates, stability was in the order I<Br<Cl (most stable). For 3,5-substituted benzoates, stability was in the order Br<Cl<I (most stable).

    [0177] Interestingly, when the aryl bears an amido group (that is, R.sup.1 is an amide) the iodo is more stable than the corresponding bromo compound, reversing the usual trend for halide stability.

    [0178] For compounds having R.sup.1 amide groups, the stability of the ortho and corresponding meta compounds was more similar. Exchanging the ester functional group for an amide functional group reversed the trend for halide stability (iodo- more stable than bromo-) and 2,3-modified were of similar stability to 1,3-modified. The presence of group L improves solubility.

    [0179] Unsubstituted compounds (R.sup.1 is H) showed the same stability trend as the ester derivatives, but were generally less stable. Without wishing to be bound by any particular theory, the inventors the inventors believe that the presence of an inductively electron-donating/mesomerically electron-withdrawing group R.sup.1 may increase stability. Consequently, in some compounds, conjugates and method described, R.sup.1 is not H.

    [0180] All are sufficiently stable for use.

    [0181] Reactivity Towards Thiols

    [0182] Relative reactivities of the compounds were evaluated towards the small thiol containing peptide glutathione (pH=7, RT). The bis-(2-chloroacetamides) show significantly slower reaction rates than bromo- or iodo-derivatives. The 3,4-bis-Cl compound (4) was found to react significantly faster than the 3,5-bis-Cl compound (1). Without wishing to be bound by any particular theory, the inventors speculate that the difference in reactivity and stability of the ortho and meta compounds may be attributed to differences in the 3D structures of each compound. Methyl 3,5-bis(2-chloroacetamido)benzoate (1) is predicted to have a planar conformation. Thus, steric hindrance could slow the backside attack of thiolate at the carbon attached to halogen (through an S.sub.N2 mechanism). This might explain the greater reactivity of ortho compounds like methyl 3,4-bis(2-chloroacetamido)benzoate (4). Compound (4) is not planar and might afford a more accessible carbon for backside attack.

    [0183] Accordingly, in some compounds, conjugates and method described herein, the linker compound may be selected such that it is not a compound of Formula (VIb).

    [0184] Although the chloro-derivatives do react with thiols and are viable linking compounds, the examples described herein primarily use bromo- and iodo-acetamides as the reaction rates are quicker. In other words, preferably X is Br or I.

    [0185] All are sufficiently reactive to be used in the methods of the invention.

    [0186] Selectivity of Compounds

    [0187] For ester and amide substituted compounds and unsubstituted arylene compounds, there is a different pattern of thiol bridging for the ortho- and meta-substituted linker compounds when reacted with Trastuzumab.

    [0188] When the linker compound is provided in excess the reaction produces a half-antibody product with two linkers attached, one bridging intra-heavy chain (H-H) disulphide. The inventors observed that linker compounds having a meta (ie. 1,3-bis-(2-haloacetamido)-) arrangement produce more side products, with more LC-LC and inter H-H produced. Accordingly, where the antibody is fully reduced and the linker is provided in excess (4 or more equivalents) it may be preferable to use an ortho linker compound of Formula (Ia).

    [0189] However, the inventors have observed that while ortho give less ‘side-products’ (LC-LC, inter-HC), the meta compounds have preference for the intra-HC attachment and the production of half-antibodies. Accordingly, when a fully reduced antibody is treated with fewer than 4 equivalents of linker it is preferable to use a meta compound (that is, a linker compound of Formula (Ib)) for the production of half-antibody conjugates.

    [0190] The inventors have also observed that, regardless of regiochemical structure, aryl bis(2-iodoacetamido)-compounds (X is I) show greater preference for intra-HC linking than the corresponding aryl bis(2-bromoacetamido)-compounds (X is Br). Accordingly, for the preferential production of half-antibody conjugates ((1) and (3) in FIG. 1) compounds having X is I are preferred.

    [0191] For the production of HC-LC conjugates (that is, antibodies having a linker bridging a heavy chain and light chain) the inventors have observed that ortho compounds show greater selectively. Accordingly, where a fully reduced antibody is treated with fewer than 4 equivalents of linker compound it is preferable to use an ortho compound (that is, a linker compound of Formula (Ia)) for the production of monofunctionalised HC-LC antibody conjugates (that is, one linker bridging the heavy and light chains).

    [0192] The inventors have also observed that, regardless of regiochemical structure, aryl bis(2-bromoacetamido)-compounds (X is Br) show greater preference for HC-LC linking than the corresponding aryl bis(2-iodoacetamido)-compounds (X is I). Accordingly, for the preferential production of HC-LC antibody conjugates ((2) in FIG. 1) compounds having X is Br are preferred.

    [0193] Where stoichiometric and near-stoichiometric (slight excess) amounts of the reducing agent are used, the linker location in the conjugate may be determined by the regioselectivity of the disulfide bridge reduction.

    [0194] The inventors have observed that compounds undergo selective reduction of a single HC-LC disulfide bridge, producing one HC-LC conjugate per mAb. For reactions using such a partially reduced antibody, unsubstituted arylene linkers were shown to proceed better. Unsubstituted arylene linkers may be preferred for the production of HC-LC conjugates when less than 4 equivalents of the reducing agent are used.

    [0195] Example 11 provides a good illustration of this regioselectivity. Fully reduced Tmab (4 disulfides reduced) was treated with only two equivalents of linker compound. The 3,4-compound (X═I) shows high preference for LC-HC, with almost no intra-HC bridging. The 3,5-compound (X═I) shows high preference for intra-HC with almost all HC having one linker, but also forms HC-LC conjugate with 2 linkers (which means the initial intra-HC product with one linker can then further react with another linker to attach the LC). No HC-LC product was observed containing only one linker molecule.

    [0196] The inventors believe that these preferences extend to other mAbs, with examples relating to rituximab described herein.

    [0197] It will therefore be recognised that by exploiting the selectivity of the linker compounds it is possible to achieve site-selective monofunctionalisation of mAbs such as Trastuzumab by treating the fully reduced mAb with a limited amount (less than 4 equivalents) of any linker compound of the invention. The invention further offers the potential to achieve hetero-bi-functionalisation of mAbs, either through sequential reduction and treatment with linker compounds or by using mixtures of linker compounds having different site selectivity to contemporaneously hetero-bi-functionalise mAbs.

    [0198] Stability of Conjugates

    [0199] Regio-chemistry of the diacetamido compounds has little difference on the stability of the conjugates (antibodies, half-antibodies and antibody fragments comprising linkers of the invention). The 3,4-linked conjugate is slightly more stable than 2,3- and 3,5-linked. All di-acetamido conjugation products were observed to be significantly more stable than the conjugation product of maleimide with glutathione. All conjugates show similar stability when stored in the presence of dithiothreitol (DTT) to samples stored without DTT.

    [0200] Definitions

    [0201] Antibody

    [0202] The term antibody is well understood in the art, and is synonymous with immunoglobulin (Ig). There are five types of antibodies in humans, IgG, IgA, IgM, IgE, and IgD. While the term antibody is intended to cover all types, it will be recognised that in this specification the antibody type will most commonly be an IgG. The inventors observe common features amongst IgG isotypes, for example they have tested IgG1 and IgG4 and observe similar selectivity for the linker compounds and products. It will also be apparent to the skilled person that the term antibody in intended to encompass monoclonal antibodies. The examples use the monoclonal antibodies Trastuzumab, Ipilimumab, and Rituximab. It should be understood that while these monoclonal antibodies may be preferred in some instances, the invention is not intended to be limited to them.

    [0203] As used herein, and unless context dictates otherwise, the term antibody refers to entire antibody, so for a monomeric form both heavy chains and both light chains in their Y-arrangement.

    [0204] Half-Antibody

    [0205] The term half-antibody is understood in the art and used herein to describe a heavy-light chain pair.

    [0206] Antibody Fragment

    [0207] As used herein, unless otherwise specified, the term antibody fragment refers to an antigen-binding fragment that can be generated from the variable region of the antibody. The term antibody fragment is therefore intended to encompass F(ab′).sub.2, Fab, Fab′ and Fv antibody fragments. It will be appreciated that the antibody fragment will be selected depending on purpose, but that selecting and linking the appropriate antibody fragment is within the ability of the skilled person. An especially preferred fragment is a Fab fragment.

    [0208] In some embodiments the invention is directed to tri-specific antibodies. In this way specific Fab-Mab conjugates can be produced (see (4) and (8) in FIG. 1).

    [0209] Antibody Conjugate

    [0210] The term “conjugate” as used herein refers to the presence of a linker according to the invention in product. The linker is attached to an antibody, half-antibody or antibody fragment. The linker may be attached may rebridge two chains, or may link heavy chain disulfides intra- rather than inter.

    [0211] The linker may attach to a further moiety, for example an antibody, half-antibody, antibody fragment, protein, polypeptide, drug or other compound (e.g. a fluorescent or radio label). For example, linker compounds like linker compound 18 includes two bis(haloacetamido) headgroups which can rebridge thiol bridges and so can be used for the formation of, for example, a mAb protein conjugate (See Example 10), while linker compounds 14, 15, 16, and 17 include an azide which may be used for attaching a further moiety through, for example, click chemistry or amide coupling.

    [0212] Where a conjugate as described herein is linked to a further moiety, the term “conjugate product” is used. Examples of conjugate products include fAb-mAb conjugates, antibody-drug conjugates, mAb-protein conjuagtes and conjugates bearing labels (e.g. a mAb-fluorophore conjugate).

    [0213] Protein or Polypeptide

    [0214] The linkers of the invention may be used to produce, for example, mAb-protein conjugates. It is recognised that the term protein encompasses an antibody, half antibody or antibody fragment, but as used herein the term is intended to encompass non-antibody proteins. The term polypeptide refers to a short chain of amino acids. This can be conjugated via, for example, the azide moiety of linker compounds 14, 15, 16, and 17 (via reduction and peptide coupling) or introduced directly through use of compound 18. Numerous polypeptide drugs are known in the art and may be useful in the methods and products of the present invention.

    [0215] Abbreviations

    [0216] It will be appreciated that capitalization of antibody nomenclature varies in the art. The following definitions are not intended to be limited to the combination of upper and lower cases letters used in the following list.

    [0217] Ab—antibody

    [0218] Mab/mAb—monoclonal antibody

    [0219] TCEP—Tris(2-carboxyethyl)phosphine (often supplied as the HCl salt).

    [0220] Tmab/TmAb—Trastuzumab

    [0221] IFab/IfAb—Fab fragment of Ipilimumab

    [0222] Imab/ImAb—Ipilimumab

    [0223] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

    [0224] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

    [0225] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

    [0226] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

    [0227] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

    [0228] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example+/−10%.

    EXAMPLES

    Example 1

    General synthesis of methyl 3,5-bis(2-haloacetamido)benzoates

    [0229] The methyl 3,5-bis(2-haloacetamido)benzoate linking compounds of the present invention can be made according to the following general synthesis, wherein X is Br or Cl as appropriate.

    ##STR00019##

    [0230] Compound 1

    [0231] 2-Chloroacetyl chloride (1.35 g, 12.04 mmol, 2 eq.) was added dropwise to a cooled solution of methyl 3,5-diaminobenzoate (1 g, 6.02 mmol) in DCM. The mixture was allowed to warm and stirred at room temperature for 2 hrs. The resultant solution was washed with water, saturated ammonium chloride solution, dried over MgSO.sub.4 and the solvent was evaporated under reduced pressure to give the acetamide (1) as pale yellow solid (1.7 g, 89%). .sup.1H NMR (CDCl.sub.3, 400 MHz): δ=8.31 (s, 2H, NH), 8.20 (s, 1H, Ar), 7.93 (s, 2H, Ar), 4.16 (s, 2H, 2x CH.sub.2), 3.88 (s, 3H, Me). .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 165.82, 164.10, 137.58, 131.88, 117.45, 115.56, 52.45, 42.71. ESI-HRMS: Expected for C.sub.12H.sub.12Cl.sub.2N.sub.2O.sub.4Na (M+Na.sup.+)=m/z 341.0066. Found: m/z 341.0077.

    [0232] Compound 2

    [0233] Methyl 3,5-diaminobenzoate (1.0 g, 6.02 mmol) was dissolved in DMF (10 mL) and bromoacetyl bromide (2.4 eq., 1.26 mL, 14.45 mmol) was added dropwise while stirring over a 10-minute period. Solution was left stirring for 24 hrs and then made up to 150 mL with ethyl acetate and washed with water (4×30 mL) and brine (30 mL) and dried over MgSO.sub.4. The solvent was evaporated under vacuum with pale orange crystals being obtained (2) (1.86 g, 76% yield). .sup.1H NMR, 400 MHz, CDCl.sub.3: δ 9.48 (s, 1H), 8.1 (s, 1H), 7.9 (s, 2H) 3.88 (s, 4H), 3.77 ppm (s, 3H); .sup.13C NMR, 500 MHz, CDCl.sub.3: δ 166.3, 165.0, 138.8, 131.2, 116.5, 115.1, 52.1, 26.8 ppm; HRMS-ESI: calcd for C.sub.12H.sub.12N.sub.2O.sub.4Br2+: 406.9237; found:406.9261.

    [0234] Compound 3

    [0235] KI (1.56 g, 9.43 mmol, 2 eq.) was added to a solution of methyl 3,5-bis(2-chloroacetamido) benzoate (1) (1 g, 3.14 mmol) in dry acetone (20 ml). The mixture was refluxed for 3 hrs. The resultant mixture was filtrated and the solvent was evaporated under reduced pressure to give the acetamide (3) as yellow solid (1 g, 63%). .sup.1H NMR (CD.sub.3COCD.sub.3, 500 MHz): δ 9.91 (s, 1H), 8.20 (s, 1H), 8.16 (s, 2H), 4 (s, 4H), 3.89 (s, 3H). .sup.13C NMR (CD.sub.3COCD.sub.3, 126 MHz): δ 166.82, 166.09, 140.01, 127.53, 131.52, 115.56, 114.00, 51.85, −0.00. HRMS: Expected for C.sub.12H.sub.13I.sub.2N.sub.2O.sub.4 (M+H+)=m/z 502.8969, Found: m/z 502.8959.

    Synthesis of methyl 3,4-bis(2-haloacetamido)benzoates

    [0236] The methyl 3,4-bis(2-haloacetamido)benzoate linking compounds of the present invention can be made according to the following general synthesis, wherein X is Br or Cl as appropriate.

    ##STR00020##

    [0237] Compound 4

    [0238] 2-Chloroacetyl chloride (1.35 g, 12.04 mmol, 2 eq.) was added dropwise to a cooled solution of methyl 3,4-diaminobenzoate (1 g, 6.02 mmol) in DCM. The mixture was allowed to warm and stirred at room temperature for 2 hrs. The resultant solution was washed with water, saturated ammonium chloride solution, dried over MgSO.sub.4 and the solvent was evaporated under reduced pressure to give the acetamide (X) as bale yellow solid (1.5 g, 79%). .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 8.84 (s, 1H, NH), 8.54 (s, 1H, NH), 8.04 (d, J=2 Hz, 1H, Ar), 7.94 (dd, J=8.4, 2 Hz, 1H, Ar), 7.75 (d, J=8.4 Hz, 1H, Ar), 4.19 (d, J=12 Hz, 4H, 2 x CH2), 3.88 (s, 3H, Me). .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 165.82, 164.10, 137.58, 131.88, 117.45, 115.56, 52.45, 42.71. ESI-HRMS: Expected for C.sub.12H.sub.12Cl.sub.2N.sub.2O.sub.4Na (M+Na+)=m/z 341.0066. Found: m/z 341.0086.

    [0239] Compound 5

    [0240] 2-bromoacetyl bromide (2.91 g, 14.44 mmol, 2.4 eq.) was added dropwise to a solution of methyl 3,4-diaminobenzoate (1.0g, 6.02 mmol, 1.0 eq.) in dimethylformamide (DMF) at RT. The reaction mixture was kept under stirring at RT for 24 hours. Approximately 150 mL of ethyl acetate was added to the obtained solution which was washed out with distilled water for 3 times, saturated ammonium chloride for 1 time, dried over MgSO4 and the remaining solvent was evaporated under reduced pressure to afford Methyl 3,4-bis(2-bromoacetamido)benzoate (5) as a yellowish powder (2.40 g, 98%). Further recrystallization with DCM was carried.

    [0241] .sup.1H NMR (500 MHz, Chloroform-d) δ 8.75 (s, 1H, NH), 8.47 (s, 1H, NH), 8.05 (s, 1H, Ar), 7.98 (d, J=8 Hz, 1H, Ar), 7.77 (d, J=8 Hz, 1H, Ar), 4.07 (s, 2H, CH.sub.2), 4.04 (s, 2H, CH.sub.2), 3.91 (s, 3H, CH.sub.3). .sup.13C NMR (100 MHz, CDCl.sub.3) δ 167.79, 167.15, 165.31, 134.72, 128.82, 127.21, 126.40, 124.76, 52.43, 29.11. Esi-HRMS: Expected for C.sub.12H.sub.12N.sub.2O.sub.4Br.sub.2 (M+H.sup.+)=m/z 404.9091, Found: m/z 404.9130.

    [0242] Compound 6

    [0243] KI (1.56 g, 9.43 mmol, 2 eq.) was added to a solution of methyl 3,4-bis(2-chloroacetamido) benzoate (4) (1 g, 3.14 mmol) in dry acetone (20 ml). The mixture was refluxed for 3 hrs. The resultant mixture was filtrated and the solvent was evaporated under reduced pressure to give the acetamide (6) as yellow solid (1 g, 63%). .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 9.73 (s, 1H), 9.61 (s, 1H), 8.02 (1H), 7.80 (d, J=8.5 Hz, 1H), 7.65 (d, J=8.5 Hz, 1H), 3.86 (s, 3H), 3.86 (s, 4H). .sup.13C NMR (CDCl.sub.3, 126 MHz): δ 167.79, 167.15, 165.94, 127.53, 127.36, 126.65, 124.22, 52.11, −0.74, −1.0. HRMS: Expected for C.sub.12H.sub.13I.sub.2N.sub.2O.sub.4 (M+H+)=m/z 502.8969, Found: m/z 502.8959.

    [0244] Compound 7

    [0245] 2,3-diaminobenzoic acid (1.0 g, 6.57 mmol, 1 eq.) was dissolved into 10 ml of anhydrous methanol (MeOH) and acidified to pH=1 through the addition of four drops of concentrated H.sub.2SO.sub.4. Reaction was heated to 50° C. and kept under stirring and reflux for 72 hours. The product was subsequently neutralized with sodium carbonate, and concentrated under reduced pressure. The remaining solution was then washed out with distilled water for 3 times, saturated ammonium chloride for 1 time, dried out with MgSO.sub.4, and excess of solvent was evaporated under reduced pressure to afford Methyl 2,3-diaminobenzoate as a dark brown solid (0.70g, 64%). 2-bromoacetyl bromide (0.58 g, 2.88 mmol, 2.4 eq.) was then added dropwise to a solution of methyl 2,3-diaminobenzoate (0.2 g, 1.20 mmol, 1.0 eq.) in anhydrous DCM at RT. Reaction flask was kept under stirring at RT for 24 hours. The resultant solution was then washed out with distilled water for 3 times, concentrated ammonium chloride for 1 time, dried out with MgSO.sub.4, and excess of solvent was evaporated under reduced pressure to afford methyl 2,3-bis(2-bromoacetamido)benzoate (7) as a yellowish solid (0.38 g, 78%). .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 10.70 (s, 1H, NH), 9.07 (s, 1H, NH), 7.92 (d, J=8 Hz, 2H, Ar), 7.38 (t, J=8 Hz, 1H, Ar), 4.09 (s, 2H, CH2), 3.99 (s, 2H, CH2), 3.95 (s, 3H, CH2). .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 167.33, 166.17, 164.80, 131.99, 128.79, 126.38, 122.61, 52.89, 29.28, 28.61. ESI-HRMS: Expected for C.sub.12H.sub.12N.sub.2O.sub.4Br.sub.2 (M+H+)=m/z 406.9237, Found: m/z 406.9251.

    [0246] Compound 8

    [0247] 2-Chloroacetyl chloride (9.3 g, 84 mmol, 6 equiv.) was added dropwise to a cooled aqueous NaOH (0.55 M) solution of benzene-1,3-diamine (1.5 g, 14 mmol). The mixture was allowed to warm and stirred at room temperature overnight. The obtained precipitate was filtered, washed with water 5-6 times and completely dried under high vacuum to give the acetamide (8) as a white solid (0.97 g, 27%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ 10.34 (s, 2H, NH), 7.96 (t, J=2.0 Hz, 1H, Ar), 7.37-7.25 (m, 3H, Ar), 4.25 (s, 4H, 2 x CH2). .sup.13C NMR (DMSO-d.sub.6, 100 MHz): δ 164.61, 138.82, 129.14, 114.81, 110.36, 45.53. ESI-HRMS: Expected for C.sub.10H.sub.10Cl.sub.2N.sub.2O.sub.2Na (M+Na.sup.+)=m/z 283.0012. Found: m/z 283.0049.

    [0248] Compound 9

    [0249] 2-bromoacetyl bromide (2.05 g, 10.2 mmol, 2.2 equiv.) was added dropwise to a cooled solution of benzene-1,3-diamine (0.5 g, 4.6 mmol) and TEA (1.35 g, 13.3 mmol, 2.2 equiv.) in DCM. The obtained precipitate was filtered and washed with water 5-6 times before washing with ether. The solid compound was dried under high vacuum to give the acetamide (9) as a pale yellow solid (2.48 g, 77%). .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ 10.42 (s, 2H, 2 X NH), 7.96 (t, J=2.0 1H, Ar), 7.37-7.24 (m 3H, Ar), 4.04 (s, 4H, 2 X CH2). .sup.13C NMR (DMSO-d.sub.6, 126 MHz): δ 164.79, 138.95, 129.14, 114.72, 110.17, 30.36. ESI-HRMS: Expected for C.sub.10H.sub.10Br.sub.2N.sub.2O.sub.2Na (M+Na.sup.+)=m/z 370.9001. Found: m/z 370.9039.

    [0250] Compound 10

    [0251] KI (1.3 g, 7.7 mmol, 4 equiv.) was added to a solution of N,N′-(1,3-phenylene)bis(2-chloroacetamide) (8) (0.50 g, 1.9 mmol) in dry acetone (20 mL). The mixture was refluxed for 3 hs. The obtained mixture was filtrated and the solvent was evaporated under reduced pressure to give the acetamide (10) as a yellow solid (0.63 g, 74%). .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ 10.36 (s, 2H, NH), 7.92 (t, J=2.0 Hz, 1H, Ar), 7.33-7.22 (m, 3H, Ar), 3.83 (s, 4H, 2 X CH2). .sup.13C NMR (DMSO-d.sub.6, 126 MHz): δ 166.56, 139.18, 129.10, 114.36, 109.86, 1.58. HRMS: Expected for C.sub.10C.sub.10I.sub.2N.sub.2O.sub.2Na (M+Na.sup.+)=m/z 466.8724, Found: m/z 466.8758. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time, 8.89 minutes, purity, 93.4%.

    [0252] Compound 11

    [0253] 2-Chloroacetyl chloride (9.3 g, 84 mmol, 6 equiv.) was added dropwise to a cooled aqueous NaOH (0.55 M) solution of benzene-1,2-diamine (1.5 g, 14 mmol). The mixture was allowed to warm and stirred at room temperature overnight. The obtained precipitate was filtered, washed with water 5-6 times and the obtained solid compound obtained was completely dried under high vacuum to give the acetamide (11) as a white solid (1.8 g, 51%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ 9.69 (s, 2H, NH), 7.55 (dd, J=7.4, 3.7 Hz, 2H, Ar), 7.23 (dd, J=6.1, 3.5, 2 Hz, 2H, Ar), 4.34 (s, 4H, 2 x CH2). .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 165.12, 130.17, 125.61, 125.08, 43.19. ESI-HRMS: Expected for C.sub.10H.sub.10Cl.sub.2N.sub.2O.sub.2Na (M+Na.sup.+)=m/z 283.0012. Found: m/z 283.0010.

    [0254] Compound 12

    [0255] 2-bromoacetyl bromide (2.05g, 10.2 mmol, 2.2 equiv.) was added dropwise to a cooled solution of benzene-1,2-diamine (0.50 g, 4.6 mmol) and TEA (1.03 g, 10.2 mmol, 2.2 equiv.) in DCM. The obtained precipitate was filtered and washed with water 5-6 times before washing with ether. The solid compound was dried under vacuum to give the acetamide (12) as a yellow solid (1.87 g, 58%). .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ 9.71 (s, 2H, NH), 753 (dd, J=7.5, 3.7 Hz, 2H, Ar), 7.23 (dd, J=6.0, 3.5 Hz 2H, Ar), 4.13 (s, 4H, 2 X CH2). .sup.13C NMR (DMSO-d.sub.6, 126 MHz): δ 165.14, 130.27, 125.56, 124.93, 30.14. ESI-HRMS: Expected for C.sub.10H.sub.10Br.sub.2N.sub.2O.sub.2Na (M+Na.sup.+)=m/z 370.9001. Found: m/z 370.9012.

    [0256] Compound 13

    [0257] KI (1.3 g, 7.7 mmol, 4 equiv.) was added to a solution of N,N′-(1,3-phenylene)bis(2-chloroacetamide) (11) (0.50 g, 1.9 mmol) in dry acetone (20 mL). The mixture was refluxed for 3 hs. The obtained mixture was filtrated and the solvent was evaporated under reduced pressure to give the acetamide (13) as a yellow solid (0.63 g, 45%). .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ 9.63 (s, 2H, NH), 7.49 (dd, J=7.4, 3.7 Hz, 2H, Ar), 7.20 (dd, J=6.1, 3.5 Hz, 2H, Ar), 3.90 (s, 4H, 2 X CH2). .sup.13C NMR (DMSO-d.sub.6, 126 MHz): δ 166.92, 130.42, 125.35, 124.70, 1.49. HRMS: Expected for C.sub.10H.sub.10I.sub.2N.sub.2O.sub.2Na (M+Na.sup.+)=m/z 466.8724. Found: m/z 466.8770.

    [0258] Compound 14

    Synthesis of 3,5-bis(2-chloroacetamido)benzoic acid (19)

    [0259] ##STR00021##

    [0260] 2-Chloroacetyl chloride (1.62 g, 14.5 mmol, 2.2 equiv.) was added dropwise to a cooled solution of 3,4-diaminobenzoate (1.0 g, 6.6 mmol) in THF. The mixture was allowed to warm and stirred at room temperature for 2 hs. The obtained precipitate was filtered, washed with water 5-6 times and then completely dried under high vacuum to give the acetamide (19) as a bale yellow solid (1.8 g, 90%). .sup.1H NMR (CD.sub.3COCD.sub.3, 400 MHz): δ 9.64 (s, 2H, NH), 8.24 (d, J=78.3 Hz, 3H, Ar), 4.30 (s, 4H, CH.sub.2). .sup.13C NMR (CD.sub.3COCD.sub.3, 101 MHz) δ 166.91, 140.07, 132.58, 117.11, 115.48, 44.07. ESI-HRMS: Expected for C.sub.11H.sub.9Cl.sub.2N.sub.2O.sub.4 (M−H.sup.+)=m/z 302.9939. Found: m/z 302.9956.

    Synthesis of 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-chloroacetamido)benzoate (20)

    [0261] ##STR00022##

    [0262] A solution of EDC.HCl (0.63 g, 3.3 mmol, 1.1 equiv.) in DMF (5 mL) was added to a stirred solution of acid (19) (1 g, 3 mmol) and N-hydroxysuccinimide (0.38 g, 3.3 mmol, 1.1 equiv.) in THF at room temperature. The reaction was then stirred at room temperature for 2 hs before being concentrated under reduced pressure. The obtained residue was dissolved in EtOAc and washed with water, dried over MgSO.sub.4 and the solvent was evaporated under reduced pressure giving a foamy solid. The crude was further purified by precipitation (EtOAc/petroleum ether) to give acetamide the (20) a yellow solid (0.75 g, 57%). .sup.1H NMR (CD.sub.3COCD.sub.3, 400 MHz,): δ 9.78 (s, 2H, NH), 8.34 (d, J=64.1 Hz, 3H, Ar), 4.32 (s, 4H, 2 x ClCH.sub.2CO), 2.99 (s, 4H, COCH2CH2CO). .sup.13C NMR (CD.sub.3COCD.sub.3, 101 MHz): δ 170.86, 170.43, 170.37, 165.95, 162.44, 140.81, 127.32, 117.21, 117.15, 44.06, 26.40. ESI-HRMS: Expected for C.sub.15H.sub.13Cl.sub.2N.sub.3O.sub.6Na (M+Na.sup.+)=m/z 424.0074. Found: m/z 424.0106.

    2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (22)

    [0263] ##STR00023##

    [0264] A solution of Ph.sub.3P (0.54 g, 2.1 mol, 1 equiv.) in ether (5 mL) was added dropwise to aqueous HCl (5%, 5 mL) solution of tri-PEG azide (21) (0.50 g, 2.1 mmol), the mixture was left stirring for 24 hs. Then, the ether was removed under reduced pressure, and the aqueous layer was extracted with DCM until Ph.sub.3P oxide was not detected in the aqueous layer. The aqueous layer was adjusted to pH=12, and azide-linked amine was extracted from the aqueous layer with DCM. The combined DCM solution was evaporated under reduced pressure to give the azide-linked amine (22) as a light yellow liquid (0.25 g, 56%). .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 3.65-3.53 (m, 11 H), 3.44 (td, J=5.2, 1.3 Hz, 2H), 3.41-3.22 (m, 3H), 2.79 (td, J=5.3, 1.4 Hz, 2H). .sup.13C NMR (CDCl.sub.3, 101 MHz): δ 73.41, 70.65, 70.60, 70.58, 70.23, 69.95, 50.63, 41.75. ESI-HRMS: Expected for C.sub.8H.sub.19N.sub.4O.sub.3 (M+H.sup.+)=m/z 219.1452. Found: m/z 219.1464.

    N,N′-(5((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-chloroacetamide) (23)

    [0265] ##STR00024##

    [0266] 2-(2-azidoethoxy)ethan-1-amine (22) (0.34 g, 1.6 mmol, 1.3 equiv.) was added to an anhydrous solution of activated ester (20) (0.50 g, 1.2 mmol) in THF. The reaction mixture was then stirred at room temperature for 1 h before being concentrated under reduced pressure. The obtained residue was dissolved in DCM and washed with water, dried over MgSO.sub.4 and the solvent was evaporated under reduced pressure. The crude was further purified by silica gel chromatography: (5% to 20% MeOH/DCM) to give azide-linked acetamide (23) as a white solid (0.39 g, 62%). .sup.1H NMR (CDCl.sub.3, 400 MHz,): δ 8.78 (s, 2H, NHCH), 8.02 (s, 1H, Ar), 7.68 (s, 2H, Ar), 7.20 (d, J=8.8 Hz, 1H, CONHCH2), 4.13 (s, 4H, 2 x ClCH.sub.2CO), 3.75-3.41 (m, 14H), 3.27 (t, J=5.0 Hz, 2H, CH.sub.2CH.sub.2N.sub.3). .sup.13C NMR (CDCl.sub.3, 101 MHz): δ 166.77, 164.69, 137.88, 135.96, 115.04, 114.31, 70.57, 70.51, 70.47, 70.24, 69.84, 69.55, 50.57, 42.96, 40.08. ESI-HRMS: Expected for C.sub.15H.sub.13Cl.sub.2N.sub.3O.sub.6Na (M+Na.sup.+)=m/z 424.0074. Found: m/z 424.0106. HPLC: column: HiQ Sit HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 6.54 minutes, purity: 99.3%.

    N,N′-(5-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-bromoacetamide) (14)

    [0267] ##STR00025##

    [0268] KBr (1.4 g, 12 mmol, 6 equiv.) was added to a solution of N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-chloroacetamide) (23) (1.0 g, 2.0 mmol) in dry acetone (20 mL). The mixture was refluxed for 4 days. The obtained mixture was filtrated and the solvent was evaporated under reduced pressure. The crude was further purified by silica gel chromatography: (50% to 70% acetone/chloroform) to give the azide-linked acetamide 14 as a yellow solid (0.6 g, 51%). .sup.1H NMR (CDCl.sub.3, 500 MHz,): δ 8.79 (s, 2H, 2 x NHCH), 7.96 (s, 1H, Ar), 7.67 (s, 2H, Ar), 7.10 (s, 1H, NHCH), 3.95 (s, 4H, BrCH.sub.2CO), 3.84-3.44 (m, 14H), 3.27 (t, J=5.0 Hz, 2H, CHCH.sub.2N.sub.3). .sup.13C NMR (CDCl.sub.3, 126 MHz): δ 167.04, 164.62, 138.22, 135.84, 114.87, 114.14, 70.67, 70.59, 70.55, 70.36, 69.94, 69.59, 50.64, 40.24, 29.42. ESI-HRMS: Expected for C.sub.19H.sub.27Br.sub.2N.sub.6O.sub.6 (M+H.sup.+)=m/z 593.0353. Found: m/z 593.0344. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 40% MeCN: 60% water. Detection at 280 nm. Retention time: 6.52 minutes, purity: 99.4%.

    [0269] Compound 15

    N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-iodoacetamide) (15)

    [0270] ##STR00026##

    [0271] KI (1.0 g, 8.0 mmol, 4 equiv.) was added to a solution of N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyI)-1,3-phenylene)bis(2-chloroacetamide) (23) (1.0 g, 2.0 mmol) in dry acetone (20 mL). The mixture was refluxed for 3 hs. The obtained mixture was filtrated and the solvent was evaporated under reduced pressure. The crude was further purified by silica gel chromatography: (50% to 70% acetone/chloroform) to give azide-linked acetamide (15) as a yellow solid (1.19 g, 88%). .sup.1H NMR (CD.sub.3COCD.sub.3, 500 MHz): δ 9.98 (s, 2H, 2 x NHCH), 7.93 (d, J=3.7 Hz, 3H, Ar), 7.69 (d, J=5.3 Hz, 1H, CONHCH2), 3.95 (s, 4H, ICH.sub.2CO), 3.60-3.11 (m, 16H). .sup.13C NMR (CD.sub.3COCD.sub.3, 126 MHz): δ 170.49, 167.51, 140.48, 137.24, 114.46, 113.41, 71.14, 70.58, 51.42, 40.42, 1.34. ESI-HRMS: Expected for C.sub.19H.sub.27Cl.sub.2N.sub.6O.sub.6 (M+H.sup.+)=m/z 505.1364. Found: m/z 505.141600. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 10.31 minutes, purity: 97.8%.

    [0272] Compound 16

    3,4-bis(2-chloroacetamido)benzoic acid (24)

    [0273] ##STR00027##

    [0274] 2-chloroacetyl chloride (1.62 g, 14.5 mmol, 2.2 equiv.) was added dropwise to a cooled solution of 3,4-diaminobenzoate (1.0 g, 6.6 mmol) in THF. The mixture was allowed to warm and stirred at room temperature for 2 hs. The obtained precipitate was filtered, washed with water 5-6 times and the obtained solid compound was completely dried under high vacuum to give the acetamide (24) as a white solid (1.8 g, 90%). .sup.1H NMR (DMF-ch, 500 MHz): δ 10.09 (d, J=20.4 Hz, 2H, NH), 8.30 (d, J=1.9 Hz, 1H, Ar), 8.00-7.83 (m, 2H, Ar), 4.45 (d, J=7.7 Hz, 4H, CH.sub.2). .sup.13C NMR (126 MHz, DMF-d.sub.7): δ 166.86, 166.02, 165.84, 135.31, 129.94, 128.03, 127.15, 127.00, 124.12, 43.64, 43.56. ESI-HRMS: Expected for C.sub.11H.sub.11Cl.sub.2N.sub.2O.sub.4Na (M+Na.sup.+)=m/z 305.0090. Found: m/z 305.0092. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 4.38 minutes, purity: 93%.

    2,5-dioxopyrrolidin-1-yl 3,4-bis(2-chloroacetamido)benzoate (25)

    [0275] ##STR00028##

    [0276] A solution of EDC.HCl (0.63 g, 3.3 mmol, 1.1 equiv.) in DMF (5 mL) was added to a stirred solution of acid (24) (1 g, 3 mmol) and N-hydroxysuccinimide (0.38 g, 3.3 mmol, 1.1 equiv.) in THF at room temperature. The reaction mixture was then stirred at room temperature for 2 hs before being concentrated under reduced pressure. The obtained residue was dissolved in EtOAc and washed with water, dried over MgSO.sub.4 and the solvent was evaporated under reduced pressure giving foamy solid. The crude was further purified by precipitation (EtOAc/petroleum ether) to give the acetamide (25) as a yellow solid (0.6 g, 45%). .sup.1H NMR (CD.sub.3COCD.sub.3, 400 MHz): δ 9.51 (d, J=16.2 Hz, 2H, NH), 8.34 (s, 1H, Ar), 8.18-7.89 (m, 2H, Ar), 4.38 (s, 4H, 2 x ClCH.sub.2CO), 2.98 (s, 4H, COCH2CH2CO). .sup.13C NMR (CD.sub.3COCD.sub.3, 100 MHz): δ 170.48, 166.85, 166.31, 161.95, 138.26, 130.66, 129.04, 128.65, 125.21, 122.83, 43.88, 26.39. ESI-HRMS: Expected for C.sub.15H.sub.13Cl.sub.2N.sub.3O.sub.6Na (M+Na.sup.+)=m/z 424.0074. Found: m/z 424.0094.

    N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-chloroacetamide) (26)

    [0277] ##STR00029##

    [0278] 2-(2-azidoethoxy)ethan-1-amine (22) (0.34 g, 1.6 mmol, 1.3 equiv.) was added to an anhydrous solution of activated ester (25) (0.50 g, 1.2 mmol) in THF. The reaction mixture was then stirred at room temperature for 1 h before being concentrated under reduced pressure. The obtained residue was dissolved in DCM and washed with water, dried over MgSO.sub.4 and the solvent was evaporated under reduced pressure. The crude was further purified by silica gel chromatography: (5% to 20% MeOH/DCM) to give the azide-linked acetamide (26) as a white solid (0.25 g, 40%). .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 9.05 (s, 2H, 2 x NHCH), 7.66 (s, 1H, Ar), 7.58-7.40 (m, 2H, Ar), 7.14 (t, J=5.4 Hz, 1 H, CONHCH.sub.2), 4.15 (d, J=10.6 Hz, 4H, 2 x CICH.sub.2CO), 3.69-3.46 (m, 14H), 3.26 (t, J=5.0 Hz, 2H, CH.sub.2CH.sub.2N.sub.3). .sup.13C NMR (CDCl.sub.3, 126 MHz): δ 166.29, 166.09, 165.49, 132.96, 132.74, 129.15, 125.57, 125.10, 124.86, 70.63, 70.60, 70.51, 70.24, 69.94, 69.65, 50.63, 42.91, 42.68, 40.01. ESI-HRMS: Expected for C.sub.19H.sub.27Cl.sub.2N.sub.6O.sub.6 (M+H.sup.+)=m/z 505.1364. Found: m/z 505.1365. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 6.76 minutes, purity: 99.8%.

    N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-bromoacetamide) (16)

    [0279] ##STR00030##

    [0280] KBr (1.4 g, 12 mmol, 6 equiv.) was added to a solution of N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-chloroacetamide) (26) (1.0 g, 2.0 mmol) in dry acetone (20 mL). The mixture was refluxed for 4 days. The obtained mixture was filtrated and the solvent was evaporated under reduced pressure. The crude was further purified by silica gel chromatography: (50% to 70% acetone/chloroform) to give the azide-linked acetamide 16 as a white solid (0.5 g, 43%). .sup.1H NMR (CDCl.sub.3, 500 MHz,): δ 9.02 (s, 1H, NHCH), 8.96 (s, 1H, NHCH), 7.79 (d, J=2.0 Hz, 1H, Ar), 7.70-7.60 (m, 2H, Ar), 7.15 (s, 1H, CONHCH2), 4.09 (d, J=12.2 Hz, 4H, BrCH.sub.2CO), 3.78-3.50 (m, 14H), 3.37 (t, J=5.0 Hz, 2H, CHCH.sub.2N.sub.3). .sup.13C NMR (CDCl.sub.3, 126 MHz): δ 166.31, 165.79, 165.13, 133.28, 132.55, 132.28, 125.68, 125.14, 124.79, 70.64, 70.62, 70.52, 70.26, 69.96, 69.63, 50.65, 40.10, 29.71, 29.09, 28.71. ESI-HRMS: Expected for C.sub.19H.sub.27Br.sub.2N.sub.6O.sub.6 (M+H.sup.+)=m/z 593.0353. Found: m/z 593.0344. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 7.62 minutes, purity: 99.1%.

    [0281] Compound 17

    N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide)(17)

    [0282] ##STR00031##

    [0283] KI (1.0 g, 8.0 mmol, 4 equiv.) was added to a solution of N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-chloroacetamide) (26) (1.0 g, 2.0 mmol) in dry acetone (20 mL). The mixture was refluxed for 3 hs. The obtained mixture was filtrated and the solvent was evaporated under reduced pressure. The crude was further purified by silica gel chromatography: (50% to 70% acetone/chloroform) to give the azide-linked acetamide (17) as a yellow solid (1 g, 73%). .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 9.30 (s, 1 H, 2 x NHCH), 9.17 (s, 1H, NH), 7.61 (s, 1H, Ar), 7.41 (s, 1H, CONHCH2), 7.27 (s, 2H, Ar), 3.93 (d, J=10.8 Hz, 4H, 2 x ICH.sub.2CO), 3.75-3.48 (m, 14H), 3.36 (t, J=5.0 Hz, 2H, CH.sub.2CH.sub.2N.sub.3). .sup.13C NMR (CDCl.sub.3, 126 MHz): δ 170.97, 168.13, 166.49, 129.71, 125.31, 125.01, 70.48, 70.31, 70.24, 69.73, 50.60, 40.09, −0.33, −0.53. ESI-HRMS: Expected for C.sub.19H.sub.27I.sub.2N.sub.8O.sub.6 (M+H.sup.+)=m/z 689.0076. Found: m/z 689.0108. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 9.10 minutes, purity: 96.2%.

    [0284] Compound 18

    3,6,9,12,15,18,21-heptaoxatricosane-1,23-ditosylate (27)

    [0285] ##STR00032##

    [0286] 4-Toluenesulphonyl chloride (1.33 g, 7.02 mmol, 2.6 equiv.) was added to a solution of anhydrous pyridine (0.51 g, 6.5 mmol, 2.4 equiv.) and 3,6,9,12,15,18,21-heptaoxatricosane-1,23-diol (1.0 g, 2.7 mmol) in anhydrous DCM (10 mL) and the mixture left to stir under N.sub.2 overnight at room temperature. The solution was then concentrated under reduced pressure and subject to standard work-up (EtOAc). The resultant residue was then purified by silica gel chromatography (40 to 80% EtOAc/petroleum ether) to give 3,6,9,12,15,18,21-heptaoxatricosane-1,23-ditosylate (27) as a colourless oil (1.1 g, 60%). .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 7.81-7.68 (m, 4H, Ar), 7.34-7.26 (m, 4H, Ar), 4.23-4.01 (m, 4H, 2x SO2OCH.sub.2), 3.70-3.48 (m, 28H), 2.39 (d, J=1.5 Hz, 6H, CHCCH.sub.3). .sup.13C NMR (CDCl.sub.3, 101 MHz): δ 144.74, 132.87, 129.77, 127.92, 70.67, 70.54, 70.49, 70.44, 69.20, 68.60, 21.60. ESI-HRMS: Expected for C.sub.30H.sub.47O.sub.13S.sub.2 (M+H.sup.+)=m/z 679.2453. Found: m/z 679.2448.

    1,23-diazido-3,6,9,12,15,18,21-heptaoxatricosane (28)

    [0287] ##STR00033##

    [0288] Sodium azide (2 g, 30 mmol, 10 equiv.) was added to a solution of the ditosylate (27) (2.0 g, 3.0 mmol) in DMF and the mixture allowed to stir at 80° C. overnight. The mixture was then concentrated under reduced pressure to remove DMF and the product extracted using ethyl acetate (3×20 mL). The organic extract was then washed with saturated brine solution, dried over MgSO.sub.4, concentrated to give diazide (28) as a colourless oil (1 g, 80.6%). .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 3.66-3.54 (m, 28H), 3.36-3.28 (m, 4H, 2x N3CH2). .sup.13C NMR (CDCl.sub.3, 101 MHz): δ .sup.13C NMR δ 70.62, 69.96, 50.60. ESI-HRMS: Expected for C.sub.16H.sub.33O.sub.7N.sub.6 (M+H.sup.+)=m/z 421.2405. Found: m/z 421.2466.

    1,23-diamino-3,6,9,12,15,18,21-heptaoxatricosane (29)

    [0289] ##STR00034##

    [0290] Triphenylphosphine (1.87 g, 7.14 mmol, 3 equiv.) was added portion-wise to a stirred solution of diazide (28) (1.0 g, 2.4 mmol) in dry THF. The reaction was stirred at room temperature overnight. Water (50 mL) was added and the reaction was left stirring at room temperature overnight. The THF was evaporated under reduced pressure and the reaction was filtrated and the filtrate was then washed with DCM (3×100 mL) to remove phosphine oxide. The filtrate was evaporated under reduced pressure to yield diamino-PEG (29) as yellow oil (0.50 g, 57%). .sup.1H NMR (CDCl.sub.3, 400 MHz,): δ 3.58 (dd, J=2.4, 1.1 Hz, 24H), 3.44 (t, J=5.2 Hz, 4H, 2x NH.sub.2CH.sub.2CH.sub.2O), 2.80 (t, J=5.2 Hz, 4H, 2x NH.sub.2CH.sub.2CH.sub.2O), 1.67 (s, 4H, 2x NH.sub.2). .sup.13C NMR (CDCl.sub.3, 101 MHz,): δ 73.27, 70.48, 70.18, 41.68. ESI-HRMS: Expected for C.sub.16H.sub.37O.sub.7N.sub.2 (M+H.sup.+)=m/z 369.2600. Found: m/z 369.2850.

    N,N′,N″,N′″-((5,8,11,14,17,20,23-heptaoxa-2,26-diazaheptacosanedioyl)bis(benzene-4,1,2-triyl))tetrakis(2-chloroacetamide) (30)

    [0291] ##STR00035##

    [0292] 1,23-diamino-3,6,9,12,15,18,21-heptaoxatricosane (29) (150 mg, 0.407 mmol) was added to an anhydrous solution of activated ester (25) (409 mg, 1.02 mmol, 2.5 equiv.) in anhydrous THF. The reaction mixture was then stirred at room temperature for 2 hs before being concentrated under reduced pressure. The crude was purified by silica gel chromatography: (5% to 20% MeOH/DCM) to give the acetamide (30) as a white solid (340 mg, 89%). .sup.1H NMR (DMSO-d.sub.6, 500 MHz,): δ 9.81 (d, J=25.2 Hz, 4H), 8.54 (t, J=5.6 Hz, 2H), 7.98 (s, 2H), 7.73 (d, J=1.4 Hz, 4H), 4.35 (d, J=9.3 Hz, 8H), 3.64-3.45 (m, 28H), 3.41 (q, J=5.9 Hz, 4H)..sup.13C NMR (DMSO-d.sub.6, 126 MHz): δ 172.72, 165.38, 165.20, 165.17, 133.26, 131.12, 129.13, 124.98, 124.59, 123.79, 69.71, 69.56, 68.83, 43.28, 43.21. ESI-HRMS: Expected for C.sub.38H.sub.53Cl.sub.4N.sub.6O.sub.13 (M+H.sup.+)=m/z 941.2419. Found: m/z 941.2451. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 5.83 minutes, purity: 98%.

    N,N′,N″,N′″-((5,8,11,14,17,20,23-heptaoxa-2,26-diazaheptacosanedioyl)bis(benzene-4,1,2-triyl))tetrakis(2-lodoacetamide) (18)

    [0293] ##STR00036##

    [0294] KI (176 mg, 1.06 mmol, 10 equiv.) was added to a solution of N,N′,N″,N′″-((5,8,11,14,17,20,23-heptaoxa-2,26-diazaheptacosanedioyl)bis(benzene-4,1,2-triyl))tetrakis(2-chloroacetamide) (30) (100 mg, 0.106 mmol) in mixture of dry acetone (400 mL). The mixture was refluxed for 3 hs. The resultant mixture was filtrated and the solvent was evaporated under reduced pressure. The crude was purified by silica gel chromatography: (50% to 70% Acetone/Chloroform) to give acetamide (18) as a white solid (130 mg, 93.5%). .sup.1H NMR (DMSO-d.sub.6, 500 MHz,): δ 9.62 (d, J=16.8 Hz, 4H), 8.31 (d, J=4.9 Hz, 3H), 7.98 (s, 2H), 7.84-7.59 (m, 4H), 3.98 (d, J=7.0 Hz, 8H), 3.75-3.30 (m, 32H). .sup.13C NMR (DMSO-d.sub.6, 126 MHz): δ 167.18, 167.09, 165.31, 133.53, 130.78, 129.30, 124.57, 124.29, 123.34, 69.64, 68.87, 1.67, 1.59. ESI-HRMS: Expected for C.sub.38H.sub.52I.sub.4N.sub.6O.sub.13Na.sub.1 (M+Na.sup.+)=m/z 1330.9669. Found: m/z 1330.9765. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (0.75 mL/minu), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 9.55 minutes, purity: 98%.

    Example 2

    Aqueous Stability

    [0295] The stabilities of ester and amide functionalised linker compounds were determined and compared.

    [0296] In a first experiment, the aqueous stability of linker compounds 1-6 over 4 days was observed. Stability was determined in phosphate buffer (100 mM, pH 7.5) in the presence of 10% DMF-d7 using .sup.1H NMR solvent suppression method. The data show that di-haloacetamides that are ortho-substituted (1,2-) have lower aqueous stability than those that are meta-substituted (1,3-). See FIG. 2.

    [0297] A comparison of the aqueous stability of aryl bis-haloacetamide linkers having ester and amide functionalisation was then made by determining the percentage remaining of the bis-haloacetamide derivatives (2, 3, 5, 6) and 14-16) over 4 days. Stability was again appraised in phosphate buffer (100 mM, pH 7.5) with final concentration of 10% DMF-d7 using solvent suppression method. See FIG. 3.

    [0298] Surprisingly, the inventors observed that exchanging the ester functional group for an amide functional group reversed the trend for halide stability (iodo- more stable than bromo-) and 3,4-modified were of similar stability to 3,5-modified.

    Example 3

    Reactivity with Amino Acid Thiol Side Chains

    [0299] The reactivity of the linker compounds of the present invention was tested by reaction with glutathione. The following representative reactions are described. Analogous reactions were performed with amide functionalised linker compounds.

    Reactivity of methyl 3,5-bis(2-haloacetamido)benzoates with glutathione

    [0300] ##STR00037##

    [0301] Glutathione (290 mg, 0.943 mmol, 3 equiv.) was dissolved in a solution of aqueous sodium phosphate buffer (100 mM, pH 7.5, 8 mL). Methyl 3,5-bis(2-chloroacetamido)benzoate (1) (100 mg, 0.314 mmol) was dissolved in THF (2 mL), added gradually to glutathione aqueous solution and held overnight at room temperature. The reaction was subsequently concentrated down under reduced pressure and purified by C-18 chromatography (100% H.sub.2O to 20% MeCN/H.sub.2O) to give the product 4.17 as a sticky solid (0.20 mg, 74%). .sup.1H NMR (D.sub.2O, 500 MHz): δ 7.73 (s, 1H, Ar), 7.56 (s, 2H, Ar), 4.57-4.54 (m, 2H, 2 x NHCHCO), 3.79 (s, 3H, OMe), 3.73-3.63 (m, 6H, 2 x NHCH.sub.2COOH, CH2CHNH.sub.2), 3.40 (s, 4H, 2 x SCH.sub.2CO), 3.13-2.89 (m, 4H, 2 x SCH.sub.2CH), 2.43 (t, J=8 Hz, 4H, 2 x CH.sub.2CH.sub.2CO), 2.06-2.01 (m, 4H, 2 x CH.sub.2CH.sub.2CO). .sup.13C NMR (126 MHz, D.sub.2O): δ 176.17, 174.81, 174.00, 171.69, 170.39, 167.65, 137.89, 130.43, 117.32, 54.11, 52.92, 52.89, 43.42, 36.24, 33.77, 31.37, 26.19. HRMS: Expected for C.sub.32H.sub.44O.sub.16S.sub.2N.sub.8Na (M+Na+)=m/z 883.2209 Found: m/z 883.2187. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (1 mL/min), 10% MeCN: 90% water: 0.1% TFA. Detection at 214 nm. Retention time: 36.39 minutes, purity: 99%.

    Reactivity of methyl 3,4-bis(2-haloacetamido)benzoates with glutathione

    [0302] ##STR00038##

    [0303] Glutathione (290 mg, 0.943 mmol, 3 equiv.) was dissolved in a solution of aqueous sodium phosphate buffer (100 mM, pH 7.5, 8 mL). Methyl 3,4-bis(2-chloroacetamido)benzoate (4) (100 mg, 0.314 mmol) was dissolved in THF (2 mL), added gradually to glutathione aqueous solution and held overnight at room temperature. The reaction was subsequently concentrated down under reduced pressure and purified by C-18 chromatography (100% H.sub.2O to 20% MeCN/H.sub.2O) to give the product (32) as a sticky solid (0.17 mg, 63%). .sup.1H NMR (500 MHz, D.sub.2O): δ 8.00 (s, 1H, Ar), 7.93 (dd, J=8.5, 2 Hz, 1H, Ar), 7.63 (d, J=8.5 Hz, 1H, Ar), 4.70-4.56 (m, 2H, 2 x NHCHCO), 3.86 (s, 3H, OMe), 3.76-3.65 (m, 6H, 2 x NHCH.sub.2COOH, CH2CHNH.sub.2), 3.52-3.45 (m, 4H, 2 x SCH.sub.2CO), 3.15-2.90 (m, 4H, 2 x SCH.sub.2CH), 2.47-2.43 (m, 4H, 2 x CH.sub.2CH.sub.2CO), 2.07-2.02 (m, 4H, 2 x CH.sub.2CH.sub.2CO). .sup.13C NMR (126 MHz, D.sub.2O): δ 176.11, 174.88, 173.88, 171.63, 171.22, 167.95, 135.56, 128.79, 128.22, 128.04, 125.81, 54.08, 52.90, 52.81, 43.35, 35.72, 35.52, 33.85, 33.79, 31.36, 26.09. HRMS: Expected for C.sub.32H.sub.44O.sub.16S.sub.2N.sub.8Na (M+Na.sup.+)=m/z 883.2209. Found: m/z 883.2187. HPLC: column: HiQ Sil HS (150×4.60 mm). Mobile phase: isocratic: (1 mL/min), 10% MeCN: 90% water: 0.1% TFA. Detection at 214 nm. Retention time: 14.25 minutes, purity: 99%.

    Reactivity of methyl 2,3-bis(2-bromoacetamido)benzoate with glutathione

    [0304] ##STR00039##

    [0305] Glutathione (0.11g, 3.66mmol, 3eq.) was dissolved into 6mL of phosphate buffer 0.1 M pH=7 and added to 4 ml of Methyl 2,3-bis(2-bromoacetamido)benzoate (7) (0.05 g, 1.22 mmol, 1 eq.) solution in THF. The mixture was kept under stirring at RT for 24 hours. Reverse phase column (C18) was used to purify the obtained product and the fractions obtained with 10% Acetonitrile/water afforded the product (0.05 g, 48%).

    [0306] Comparison of Amide Functionalised Compounds

    [0307] The inventors compared the reactivity of various linker compounds of the invention by determining the percentage remaining of the bis-haloacetamide derivatives 14-17 in the presence of glutathione (2.2 equiv.) in aqueous phosphate buffer (100 mM, pH 7.5). See FIG. 4.

    [0308] They observed that, despite no difference between 3,4- and 3,5-amide modified derivatives for hydrolysis (reaction with water), there is a significant difference between their reaction with the thiol of glutathione. The 3,4-modified derivatives react faster than 3,5-modified compounds with thiols.

    [0309] The ester compounds reacted so quickly a reaction rate could not be determined.

    Example 4

    Reaction of Bromo Acetamide Linkers with Trastuzumab

    [0310] Linker compounds 2, 5, 9 and 12 were reacted with Trastuzumab under varying stoichiometries and the reaction products analysed using SDS-PAGE.

    ##STR00040##

    [0311] The reactions used fully reduced Tmab (0.034 mM) in Tris.HCI buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 7.5) with bis-bromoacetamide linkers. Tmab (0.033 mM) was reduced with 5 equiv. of TCEP for 2 hs at room temperature.

    [0312] The analysis and attributed products are shown in FIG. 5.

    [0313] L: protein ladder,

    [0314] lane 1: Tmab incubated with 5 equiv. of methyl 3,4-bis(2-bromoacetamido)benzoate (5),

    [0315] lane 2: Tmab incubated with 8 equiv. of methyl 3,4-bis(2-bromoacetamido)benzoate (5),

    [0316] lane 3: Tmab incubated with 5 equiv. of methyl 3,5-bis(2-bromoacetamido)benzoate (2),

    [0317] lane 4: Tmab incubated with 8 equiv. of methyl 3,5-bis(2-bromoacetamido)benzoate (2),

    [0318] lane 5: Tmab incubated with 5 equiv. of N,N′-(1,2-phenylene)bis(2-bromoacetamide) (12),

    [0319] lane 6: Tmab incubated with 8 equiv. of N,N′-(1,2-phenylene)bis(2-bromoacetamide) (12),

    [0320] lane 7: Tmab incubated with 5 equiv. of N,N′-(1,3-phenylene)bis(2-bromoacetamide) (9),

    [0321] lane 8: Tmab incubated with 8 equiv. of N,N′-(1,3-phenylene)bis(2-bromoacetamide) (9).

    [0322] Key: HC: heavy chain, LC: light chain, LC-LC: light chain homodimers, HC-HC: heavy chain homodimers. Protein samples were resolved by reducing SDS-PAGE (4-12% gel).

    [0323] The inventors observed a different pattern of thiol bridging between the ortho-(1 ,2- and 3,4-) and meta-(1,3- and 3,5-) substituted linking compounds. When excess (5 or 8 equivalents) of linker is used, the meta-substituted linkers are less selective as more LC-LC (47 kDa) and HC-HC (106 kDa) are produced. The major product for all reactions is the HALF-ANTIBODY conjugate, HC-LC (75 kDa) with 2 linkers attached.

    [0324] The product of reaction with linker compound 5 (labelled HC-LC) was analysed by mass spectrometry. Cross-linked Tmab with methyl 3,4-bis(2-bromoacetamido) benzoate (5) showed a major peak at 74,528.03 Da, and expansion of the spectrum at the 74 KDa region showed major peaks at 74,528.03 Da and 74,689.24 Da (glycoforms). This mass spectrometric analysis of the crude reaction product confirms the attachment of two linkers, one linker bridging intra-HC-HC cysteine residues of the hinge region.

    [0325] FIG. 1(1) shows a schematic representation of the half-antibody produced with two rebridged disulfide bonds, intra-chain heavy-heavy and heavy-light chains (labelled HC-LC). FIG. 1(5) is chemically identical to the structure shown in FIG. 1(1), but represents the physical state in which the half-antibody may be observed. In size-exclusion chromatography, the product will appear to have a molecular weight of 150 kDa, but each covalent bonded structure has a weight of 75 kDa (half-antibody) as represented by FIG. 1(1).

    [0326] Analysis of the reaction product of reaction with unfunctionalised linker compound 12 confirmed attachment of two linkers, with one linker again bridging intra-HC-HC cysteine residues of the hinge region.

    [0327] Deconvoluted spectrum protein MS of cross-linked Tmab with N,N′-(1,2-phenylene)bis(2-bromoacetamide) (12). The MS spectrum of cross-linked Tmab showed a major peak at 74,572.57 Da, while expansion of the spectrum at 74 KDa region showed major peaks at 74,572.57 Da and 74,410.91 Da.

    Example 5

    Influence of pH on selectivity of bis-(2-bromoacetamide)-linkers

    [0328] The influence of pH on reaction with bis-(2-bromoacetamide) linking compounds was investigated by conducting an SDS-PAGE analysis of cross-linking of fully reduced Tmab (0.033 mM) in Tris.HCI buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 6, 7.5, or 8) incubated with bis-haloacetamide linkers. Tmab (0.033 mM) was reduced with 5 equiv. of TCEP for 2 h at room temperature.

    [0329] The analysis and attributed products are shown in FIG. 6.

    [0330] L: protein ladder,

    [0331] Lane 1, 5 and 9: Tmab incubated with 5 equiv. of methyl 3,4-bis(2-bromoacetamido)benzoate (5),

    [0332] Lane 2, 6 and 10: Tmab incubated with 5 equiv. of methyl 3,5-bis(2-bromoacetamido)benzoate (2),

    [0333] Lane 3, 7 and 11: Tmab incubated with 5 equiv. of N,N′-(1,2-phenylene)bis(2-bromoacetamide) (12),

    [0334] Lane 4, 8 and 12: Tmab incubated with 5 equiv. of N,N′-(1,3-phenylene)bis(2-bromoacetamide) (9).

    [0335] HC: heavy chain, LC: light chain, LC-LC: light chain homodimers, HC-HC: heavy chain homodimers. Protein samples were resolved by reducing SDS-PAGE (10% gel).

    [0336] The best levels of conjugation were achieved at pH=7.5. In all cases, ortho-(1,2- and 3,4-) substituted linker compounds are more selective (less LC-LC and inter-HC-HC) than the meta-(1,3- and 3,5-) modified linker compounds.

    Example 6

    Reaction of excess bis-(2-iodoacetamide) linkers with Trastuzumab

    [0337] Linker compounds 3, 6, 10 and 13 were reacted in excess with Trastuzumab and the reaction products analysed using SDS-PAGE.

    ##STR00041##

    [0338] The reactions used fully reduced Tmab (0.033 mM) in Tris.HCI buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 7.5) with bis-iodoacetamide linkers. Tmab (0.033 mM) was reduced with 5 equiv. of TCEP for 2 hs at room temperature.

    [0339] The analysis and attributed products are shown in FIG. 7.

    [0340] L: protein ladder,

    [0341] lane 1: Tmab control (Non-reducing dye),

    [0342] lane 2: Tmab control (reducing dye),

    [0343] lane 3: Tmab incubated with 5 equiv. of methyl 3,4-bis(2-iodooacetamido)benzoate (6),

    [0344] Lane 4: Tmab incubated with 5 equiv. of methyl 3,5-bis(2-iodoacetamido)benzoate (3),

    [0345] Lane 5: Tmab incubated with 5 equiv. of N,N′-(1,2-phenylene)bis(2-iodoacetamide) (13),

    [0346] Lane 6: Tmab incubated with 5 equiv. of N,N′-(1,3-phenylene)bis(2-bromoacetamide) (10).

    [0347] HC: heavy chain, LC: light chain, LC-LC: light chain homodimers, HC-HC: heavy chain homodimers. Protein samples were resolved by reducing SDS-PAGE (10% gel).

    [0348] HC: heavy chain, LC: light chain, LC-LC: light chain homodimers, HC-HC: heavy chain homodimers. Protein samples were resolved by reducing SDS-PAGE (10% gel).

    [0349] Similar selectively was observed as for the analogous bromo linker compounds.

    Example 7

    Selectivity of Reaction of Amide Substituted Linkers with Trastuzumab

    [0350] The reaction of amide functionalised linker compounds 14, 15, 16 and 17 with Trastuzumab was investigated.

    ##STR00042##

    [0351] SDS-PAGE analysis of cross-linking of reduced Tmab (0.013 mM) in Tris.HCl buffer (0.5 M, pH 7.5, 5 mM EDTA) with 5 equiv. of TCEP at room temperature overnight is shown in FIG. 8.

    [0352] L: ladder,

    [0353] lane 1: Tmab incubated with 5 equiv. of 16,

    [0354] lane 2: Tmab incubated with 5 equiv. of 14,

    [0355] lane 3: Tmab incubated with 5 equiv. of 17,

    [0356] lane 4: Tmab incubated with 5 equiv. of 15.

    [0357] Deconvoluted spectrum protein MS of rebridged Tmab with Compound 17 linker showing a major peak at 75,059.82 Da confirmed the attachment of two linkers to the half-antibody product.

    Example 8

    Selectivity with Other Monoclonal Antibodies

    [0358] SDS-PAGE analysis of cross-linking of fully reduced Rituximab (0.035 mM) in Tris.HCl buffer with bis-bromoacetamide linkers is shown in FIG. 9. Rituximab (0.035 mM) was reduced with 5 equiv. of TCEP for 2 hrs at room temperature.

    [0359] L: protein ladder,

    [0360] lane 1: Rmab incubated with 5 equiv. of methyl 3,4-bis(2-bromoacetamido)benzoate (6),

    [0361] lane 2: Rmab incubated with 5 equiv. of methyl 3,5-bis(2-bromoacetamido)benzoate (3),

    [0362] lane 3: Rmab incubated with 5 equiv. of N,N′-(1,2-phenylene)bis(2-bromoacetamide) (12),

    [0363] lane 4: Rmab incubated with 5 equiv. of N,N′-(1,3-phenylene)bis(2-bromoacetamide) (9).

    [0364] HC: heavy chain, LC: light chain, LC-LC: light chain homodimers, HC-HC: heavy chain homodimers. Protein samples were resolved by reducing SDS-PAGE (10% gel).

    [0365] Similar selectivity and reaction products observed to reaction with Trastuzumab. 1,2-compounds show less LC-LC and HC-HC products with more effective formation of HC-LC product at 75kDa. Results observed using Trastuzumab can therefore be considered a reasonable exemplar for antibodies.

    Example 9

    Selective Functionalisation of Antibodies

    [0366] The inventors have demonstrated that selectivity of linkers can be exploited to selectively mono functionalise and hetero-bi-functionalise antibodies such as Trastuzumab.

    [0367] The site selective functionalisation of Trastuzumab was investigated. FIG. 10 shows SDS-PAGE analysis of cross-linking of partially reduced Tmab in Tris.HCl buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 6, 7.5, or 8) with bis-bromoacetamide linkers. Tmab was reduced with 1.1 equiv. of TCEP for 2 hs at 4° C. and incubated with 1.1 equiv. each linker at room temperature overnight.

    [0368] L: protein ladder,

    [0369] Lanes 1, 9 and 17: 1 equiv. of methyl 3,4-bis(2-bromoacetamido)benzoate (5),

    [0370] Lanes 2, 10 and 18: 1.1 equiv. of methyl 3,5-bis(2-bromoacetamido)benzoate (2),

    [0371] Lanes 3, 11 and 19: 1.1 equiv. of N,N′-(1,2-phenylene)bis(2-bromoacetamide) (12),

    [0372] Lanes 4, 12 and 20: 1.1 equiv. of N,N′-(1,3-phenylene)bis(2-bromoacetamide) (9).

    [0373] Lane 5-8, 13-16, and 21-24 represent the same reactions as above, in the same order described, but run under non-reducing conditions (non-reducing dye).

    [0374] HC: heavy chain, LC: light chain, LC-LC: light chain homodimers, HC-HC: heavy chain homodimers. Protein samples were resolved by reducing SDS-PAGE (10% gel).

    [0375] Equivalent intensity of bands at 75kDa and 50kDa indicate selective functionalisation of a single disulphide bridge to produce one HC-LC conjugate per mAb. In other words, monofunctionalisation. Unfunctionalized linkers 12 and 9 appear to perform better, as fewer lower molecular weight bands are seen under non-reducing conditions, indicating reaction has proceeded to a greater extent.

    [0376] Through sequential partial reduction, incubation, further reduction and incubation the selective hetero-bi-functionalisation of Trastuzumab was effected. FIG. 11a shows SDS-PAGE of bifunctional cross-linking of Tmab (0.033 mM) in Tris.HCl buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 7.5) using sequential method. Tmab (0.033 mM) was reduced with 2.2 equiv. of TCEP for 2 hs and incubated with 2.2 equiv. of methyl 3,4-bis(2-bromoacetamido)benzoate (5) at room temperature overnight (lane 1). Functionalised Tmab (0.033 mM) was then further reduced with 2.2 equiv. of TCEP for 2 hs and incubated with 4 equiv. of N,N′-(5-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-iodoacetamide) (15) at room temperature overnight (lane 2).

    [0377] Protein samples were resolved by reducing SDS-Page (10% gel).

    [0378] The deconvoluted spectrum protein MS shows a major peak at 74,711.99 Da corresponding to reaction with one molecule of linker 5 and one molecule of linker 15.

    [0379] This is a demonstration of the ability to form hetero-bi-functionalised half-antibodies through sequential addition of 2 different linkers.

    [0380] The structure is stylistically represented in FIG. 11b. Linker 5 shows rebridging of the disulphide that links the light and heavy chains of the antibody. Linker 15 is referred to herein as ‘intra-chain’ rebridging, linking the two thiols that made up the hinge region of the antibody.

    Example 10

    Formation of Stable Thio-Bridged Fab Derivatives and Selective Preparation of a mAb-Protein Conjugate

    [0381] The linker compounds of the invention are able to form stable thio-bridged Fab derivatives. These can be subsequently conjugated to reduced or partially reduced antibodies or other proteins. First, IFab was buffer exchanged to conjugation Tris.HCl buffer (pH 7.5) and diluted to (5 mg/mL, 0.1 μmol) using Amicon® Ultra-0.5 mL (3 KDa) centrifugal filters. Stock solution of TCEP (4.0 mg/mL, 0.042 mmol, 14 mM) was prepared in the same conjugation buffer. IFab was reduced by incubation with TCEP (2 equiv. relative to IFab) for 1 h at room temperature. Then a TCEP quenching step using penta-PEG azide for 1 h was followed.

    [0382] The reduced IFab was incubated with bis-o-diiodoacetamide (PEG).sub.7 linker 18 (10 μL of stock solution (7 mg/mL), 5 equiv.) for 3 hs at room temperature.

    [0383] Excess reagent was removed by using quick step of purification using protein A column (1 mL HisTrap, FF) to remove excess linkers from functionalized IFab solution prior to conjugation with partially reduced Tmab. The binding buffer Tris.HCI (20 mM, 500 mM NaCl, pH 7.4) was used to collect Fab fractions, then the collected faction were concentrated using were concentrated using Amicon® Ultra-15 mL (3 KDa) centrifugal filters and finally buffer exchanged into conjugation buffer.

    [0384] A deconvoluted protein MS spectrum of IfAb control (non-reduced) showed major peaks at 47,636.32 Da, while the deconvoluted protein MS spectrum of functionalised IfAb with bis-o-diiodoacetamide (PEG)7 linker (18) showed major peaks at 48,690.90 Da, confirming the functionalised had occurred. As schematic structure is shown in FIG. 12a.

    [0385] Tmab was buffer exchanged to Tris.HCl (100 mM, 150 mM NaCl, 5 mM EDTA, pH 7.5) and diluted to (5.0 mg/mL, 0.03 μmol) using Amicon® Ultra-0.5 mL (10 KDa) centrifugal filters. Tmab was reduced by incubation with TCEP (1.1 equiv. relative to Tmab) for 2 hs at 40. Functionalised IFab (4 equiv. relative to Tmab) was then added to the reduced Tmab and left at room temperature overnight. SEC was then performed using superdex column (HiLoad 16/600, Superdex 200 pg, GE Healthcare) for purification of reaction products. Prior to loading onto the column, samples were centrifuged at 20,000 g for 10 minutes. The collected fractions containing the conjugate were buffer exchanged into conjugation buffer and sterilised using 0.45 μm membrane filters.

    [0386] See FIG. 12b.

    [0387] L: protein ladder

    [0388] Lane 1: Tmab incubated with 1.1 equiv. of methyl 3,4-bis(2-bromoacetamido)benzoate (5) as control,

    [0389] Lane 2: IfAb conjugate,

    [0390] Lane 3: Tmab conjugation with IfAb at room temperature overnight showing the correct estimated mass of the conjugate around 125 KDa with significant reduction of the 75 KDa band (half-antibody) before performing size exclusion chromatography,

    [0391] Lane 4: size exclusion column purification collected fractions (F6-G2),

    [0392] Lane 5: size exclusion column purification collected fractions (G4-G13),

    [0393] Lane 6: size exclusion column purification collected fractions (H1-H7).

    [0394] Lanes 4 and 5 contain the desired mAb-fAb conjugate (some HC-HC impurity in Lane 5). A schematic representation of the antibody conjugate is shown in FIG. 12c. It may also be termed a mAb-protein conjugate or tri-functional monoclonal antibody. The inventors have demonstrated its selective preparation and stability to AKTA purification on ProteinA column.

    Example 11

    Selectivity of Thio-Bridging Compounds is Dependent on Their Regio-Chemistry: Ortho Substituted Versus Meta Substituted

    [0395] Selectivity of compounds 15 and 17 was evaluated where fully reduced Tmab (4 equiv.) was incubated with only 2 equivalents of each bis-iodoacetamide linker 15 and 17. The reaction products were resolved using SDS-PAGE analysis (shown in FIG. 13a) and further characterised by protein MS (shown in FIG. 13b and c).

    [0396] First, Tmab was buffer exchanged into Tris.HCl buffer (pH 7.5) and diluted to (5 mg/mL, 34 μM, 1 ml) using Amicon® Ultra-0.5 mL (10 kDa) centrifugal filters. Tmab was reduced by incubation with TCEP (4 equiv. relative to Tmab) for 2 h at 4° C. The reduced protein was aliquoted into 100 μL samples for each reaction. A stock solution of N,N-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide) (17) was prepared in DMF at final concentration (1.0 mg/mL, 1.4 μmol, 1.4 mM). Working solution of 17 (0.68 mg/mL) was prepared by serial dilution with DMF. 2 equiv. of working solution of 17 (7 μL, 2 equiv. relative to Tmab aliquot) was added to the reduced protein and held at room temperature overnight. An analogous procedure was followed for the reaction of compound 15 with Tmab.

    [0397] The azide modified bis-iodoacetamide compounds 15 and 17 displayed an interesting manner of selectivity. SDS-PAGE analysis of the reaction with the meta-substituted compound 15 showed the HC (half-antibody bridged intra) of Tmab being the major protein product present (FIG. 13a, lane 2). Characterisation with protein MS confirmed that this HC protein product has undergone conjugation with one molecule of 15 through rebridging of the intra-chain heavy-heavy disulphide (FIG. 13b).

    [0398] In contrast, the ortho-substituted compound 17 displayed greater preference to cross-link heavy-light disulfide bonds with a significant amount of a higher molecular weight product observed by SDS-PAGE analysis (FIG. 13a, Lane 1). Characterisation with protein MS confirmed that this protein product has undergone HC-LC disulphide rebridging with one molecule of 17 (FIG. 13c). Importantly, MS analysis also demonstrated that the remaining HC protein was native, and had not undergone any appreciable conjugation with compound 17.

    [0399] FIG. 13a shows an SDS-PAGE gel (10%) evaluating selectivity of bis-iodoacetamide linkers in cross-linking Tmab (5 mg/mL, 34 μM) in Tris.HCI buffer (100 mM, pH 7.5) containing 150 mM NaCl, and 5 mM EDTA, reduced with 4 equiv. of TCEP (136 μM) and incubated with (6.8 μM, 2 equiv.) of each bis-iodooacetamide linker at room temperature overnight.

    [0400] L: protein ladder,

    [0401] Lane 1: Tmab incubated with 2 equiv. of N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide) (17),

    [0402] Lane 2: Tmab incubated with 2 equiv. of N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-carbamoyl)-1,3-phenylene)bis(2-iodoacetamide) (15).

    [0403] Deconvoluted protein MS spectra of Tmab cross-linked with 2 equiv. of N,N-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-iodoacetamide) (15) (FIG. 13b) and N,N′-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide) (17) (FIG. 13c).

    REFERENCES

    [0404] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

    [0405] EP333573

    [0406] S M Andrew and J A Titus Current Protocols in Cell Biology (2000) 16.4.1-16.4.10.

    [0407] E J Smith, L Visai, S W Kerrigan, P Speziale, and T J Foster* Infect Immun. 2011, 79(9): 3801-3809.

    [0408] T Kantner, B Alkhawaja, A G Watts (2017) ACS Omega, 2, 5785-5791.

    [0409] For discussion of protecting groups and their synthesis and use, see Peter G. M. Wuts and Theodora W. Greene, Greene's Protective Groups in Organic Synthesis 4.sup.th Ed 2006, Wiley-Blackwell.

    [0410] For standard molecular biology techniques, see Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.