WO1992016520A1 - Macrocyclic thioether ligands and their use as intermediates for binding ions to substrates - Google Patents

Abstract

1,4,7-Trithiacyclononane (9S3) and related compounds of formula (I), where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where A1 is -C¿2?H4- or (a), or -C3H6-, A?2¿ is -C¿2?H4- or (a), and A?3¿ is -C¿2?H3R- or (b), or except when A?1¿ is -C¿3?H6-, -C3H5R-, where R is alkyl or substituted alkyl or, if the third T contains P or As, R may also be H, may be substituted with a linker group which can bind to a substrate such as a protein and which itself retains co-ordinating functionality to chelate a radionuclide for diagnosis or therapy.

Description

MACROCYCLIC THIOETHER LIGANDS AND THEIR USE AS INTERMEDIATES

FOR BINDING IONS TO SUBSTRATES

This invention relates to certain macrocyclic thioether ligands and their use as intermediates for binding ions to substrates, especially the attachment of radionuclides to biologically active substrates such as immunoglobulins.

For a variety of uses, both diagnostic and therapeutic, for example in tracing biological processes or applying radiation to a desired part of the body, there is a need for the synthesis of bifunctional chelates which would trap metallic radionuclides and themselves be capable of reaction to covalently bond to the biologically active molecule of interest e.g. an immunoglobulin or be reactable with functions on proteins (e.g. lysine residues) or with an anti-tumour monoclonal antibody.

Not all radionuclides are equally suitable for such purposes, as will be explained below.

The potential value of radiolabelled antibodies for in vivo localisation of tumours has been recognised since the discovery of the hybridoma technique for raising monoclonal antibodies in 1976. Methods using radioiodine as the labelling nuclide (1-123 for gamma camera imaging, and 1-131 for both imaging and radioimmunotherapy) have more recently been augmented by the use of chelate-conjugated antibodies labelled with radiometals. The most widely investigated example of this method for imaging purposes employs indium-Ill chelated to a derivative of diethylenetriaminepentaacetic acid (DTPA). These procedures have been the subject of considerable clinical evaluation and pharmacokinetic analysis.

Despite the great activity and interest in this important medical field, the performance of current reagents is far from optimal. For example, radioiodine labelled antibodies are known to lose a large portion of their label (as free iodide) in vivo. This results in the need for blocking of the thyroid gland, leads to image degradation and causes dosimetric problems when therapeutic doses are being considered. A characteristic of indium-111 and yttrium-90 labelled antibodies is high non-specific uptake of radioactivity in the liver, spleen and bone marrow. This leads to excessive radiation doses to these organs if beta-emitting nuclides such as yttrium-90 are administered for radiotherapy, and restricts the diagnostic usefulness of indium-labelled antibodies in cases where liver metastates may be present.

Indium and yttrium, the radiometal ions receiving most attention at present, are "hard" Lewis acids (i.e. they are complexed most strongly by ligands containing "hard" donors such as oxygen and nitrogen). Consequently the chelates employed have been hard donors such as DTPA and EDTA. While these undoubtedly have high association constants for nuclides such as In-111 or Y-90 in vitro, human serum contains "hard" cations (Ca²⁺, Mg²⁺, Fe³⁺, Zn²⁺, K⁺ and Na⁺), in much higher concentrations than the

radiolabel and these compete with the radionuclide for the

chelate. Also present in serum are a number of iron-sequestering proteins which have high affinities for In³⁺ and Y³⁺ and are therefore able to abstract the nuclide from the antibody. In addition, at physiological pH, a kinetic destabilisation arises in that these chelates are readily protonated, further diminishing their effectiveness as radionuclide-binding chelates. The

resulting detachment of label from the antibody leads to its accumulation in the liver and other organs.

These difficulties may be circumvented by use of radionuclides of "soft" metals (i.e. those that are complexed most strongly by ligands containing "soft" donors such as sulphur and phosphorus, also known as Class B metals). Indeed, a number of such nuclides are available which have radiological properties suitable for exploitation in nuclear medicine for both diagnosis and therapy of tumours. A selection is presented in Table 1 below together with their relevant properties.

* Higher activities may be available,

nca: no carrier added

Note that the half-lives are such that the radiation dose need not be excessive, while the gamma-emission energies are within the range suitable for detection by gamma-cameras. Gamma emitters with suitable emission energies may be used for diagnostic imaging. Copper-64 and copper-62 are positron emitters of potential value in positron emission tomography. Several of the β-emitters are currently available at specific activities sufficiently high to administer therapeutic doses with reasonable amounts of antibody (e.g. lmCi/mg antibody). Moreover, some pairs of isotopes of the same element are available, e.g. gold-193 and gold-199, of which one could be used for imaging and the other for therapy using the same antibody-chelate conjugate. Thus, the target specificity of the therapeutic dose could be reliably predicted by imaging. The combination of technetium-99m and rhenium-188 or rhenium 186 (both of which metals, in their lower oxidation states, may be described as "soft") may also offer this advantage because of the close chemical similarity between the two elements. Beta emitters and those gamma emitters which also emit cell-killing secondary radiation (e.g. Auger electrons) may be used in cancer therapy.

Certain ligands containing "soft" donors such as oxygen or sulphur have been available for many years. Thus the preparation of 1 ,4,7-trithiacyclononane (9S3) is described by Sellmann e±il-. Angewandte Chemie, Int. Ed., 21, No. 10, 1984, 807-808, while a compound of similar structure in which the ethylene bridges have fused benzene rings, i.e. 2,3,7,8,12,13-hexamethoxytribenzo- [1,4,7]-trithiacyclononatriene, is described by Weiss et al., Z. Naturforsch., B. Anorg. Chem., Org. Chem., 1979, 34B (3), 448-50. The cyclically symmetrical nine-membered ring compound 9S3 is a powerful thioether ligand that is uniquely well suited to

"facial-mode" tridentate coordination through the sulphur atoms. It forms stable complexes with many transition and main group metals, especially "soft" ones, in many cases imparting unusual structural and electronic properties. The homologue 1 ,4,7-trithiacyclodecane (10S3) shows similar properties in lesser measure

(Grant et al., Inorg. Chem., 1989, 28, 4128-4132).

The 9S3 complexes have unusual and useful properties such as high stability (thioethers are typically relatively weakly

coordinating ligands), high coordination numbers, access to unusual oxidation states and high electron self-exchange rates.

The affinities of these structurally specialised chelates for the hard metal ions which occur in plasma are low, however,

suggesting that "soft"-metal-chelate combinations of this type would be well protected in vivo from cross reaction with plasma proteins and metal ions. In addition, the basicity of thioether sulphur is extremely low so that there would be no interference due to protonation at physiological pH. However, the ligands of 9S3 type described in the literature are not intrinsically suitable for complexation with biologically active substrates.

The present invention provides ligands of formula I

where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where A¹ is -C₂H₄- or or -C₃H₆-,

A² is -C₂H₄- or

and A³ is -C₂H₃R- or or except when A¹ is -C₃H₆-, -C₃H₅R-,

where R is alkyl or substituted alkyl and is preferably selected from hydroxyalkyl, alkoxyalkyl, aryloxyalkyl and arylalkoxyalkyl groups which groups may be substituted, or, if the third T contains P or As, R may also be H.

Preferably each T is S or two T's are S and the third is the group

Preferably A¹ and A² are -C₂H₄-. X may suitably be a hydrogen atom or an alkyl or phenyl group, which groups may be substituted. R is preferably a group capable of further

derivatisation and thus is preferably hydroxyalkyl, alkoxyalkyl, aryloxyalkyl or arylalkoxyalkyl optionally further substituted, for example by halo-, carboxy- or cyano-.

While not wishing to be bound by any particular theory, it is considered that the properties of 9S3 ligands may derive (at least in part) from their predisposition to facial coordination as a result of their preferred endodentate conformation. Thus,

according to one preferred aspect of the invention, the inventors have conceived the notion of further improving these ligands by substituting (9-10)S3 compounds in the thia crown in such a way as to provide (i) a phosphine donor in place of a thioether donor and/or (ii) a further co-ordinating group to satisfy the vacant co-ordination site of four-co-ordinate metal ions or a further tridentate crown to satisfy six-co-ordinate metal ions.

One aspect of the invention therefore comprises ligands of formula I above in which one T is where X is preferably a

group, such as phenyl, which may be further derivatised by the introduction of at least one substituent, preferably an ortho substituent, which is a donor group or atom or second macrocycle so as to extend the co-ordination. Thus, for example, compounds such as the 1 ,4-dithia,7-phospha(P-mono-orthosubstituted-phenyl)cyclononanes, which have the ortho substituent "pointing towards" the sulphur atoms, are postulated as being useful because a ligand substituted onto that ortho site will assist a tetrahedral complexation of a metal in co-operation with the S, S and P.

A synthetic route to such a compound, which not only exhibits an additional donor group but also a linker group as described below, is as follows:

Such extended ligands, with or without a fourth (or three more) ligand(s) with a co-ordinating activity directed generally on the axis of symmetry of the thia crown and with such linker groups as may be expedient, may be bound to a protein or other biologically active molecule or moiety, enabling the latter to be tracked through the body, or enabling tumours to be located and/or treated, by radioimmunolocalisation.

Metal complexes of such macrocyclic thiaphosphines, which have been found to exhibit surprisingly high stability, are also included within the invention. Synthesis of such metal complexes and single crystal X-ray structure determination of the cyclic ligand shows that it is capable of facial tridentate coordination and suggests that it will ligate more strongly than 9S3, in that the phosphine donor of the non-facially coordinating ligand binds to the metal in preference to the thioethers.

It has surprisingly been found that such derivatisation of compounds of the 9S3 ring structure and variations thereon, such as the replacement of a sulphur atom by a higher valent element such as phosphorus, allows retention of the unusual and useful

co-ordination chemistry of the 9S3 ring structure.

Therefore, the invention further includes extended ligands in which at least one uni valent atom or group of a ligand of formula I has been replaced by a moiety extending the co-ordinating

functionality of the ligand of formula I and/or a linking moiety capable of facilitating binding to a substrate.

Preferred extended ligands according to the invention may be represented by replacing that atom or group by (i) a moiety derived from formula I as set forth above, or (ii) a linker group which can bind to a substrate, the linker group optionally additionally falling within the definition of said moiety and/or having

co-ordinating functionality such as carboxyl or amine or more preferably phosphine or even more preferably thiol or thioether. Examples of l i nker groups are :

such groups being linked for example to a ligand of formula I where R is hydroxyalkyl.

Alternatively, where one T is >PX or >AsX, X may suitably incorporate additionally a donor group or atom or a second macrocycle so as to extend the co-ordinating functionality of the ligand.

Compounds based on 9S3 with such a linker group (even made other than via ligands as set forth above) are within the scope of the invention.

The ligands and extended ligands according to the invention may find application as chelators for reaction with other reagents at the point of use or as intermediates in the preparation of

chelators and ready-linked chelated reagents for use in diagnosis or therapy.

Thus, the molecules set forth above can find application as chelators, which may be sold and used to bind metals such as radionuclides to substrates, e.g. in kits for labelling

antibodies. The molecules may be sold as chelates, i.e. as ready-made metal complexes, which the user may then optionally attach to a substrate. Alternatively, the molecules may be sold bound to a substrate, ready for the user then optionally to complex a metal therein. The molecule according to the invention can lastly be a metal complex bound to a substrate.

The invention therefore includes ligands and extended ligands as defined above when complexed with either a radionuclide, a biologically active substrate, such as an immunoglobulin, or both. The invention further includes a kit suitable for use in

radiodiagnosis or radiotherapy including a ligand, extended ligand or a complex thereof. Examples of reactive linking groups and substrate targets are shown in the following Table 2.

Both the anhydride and N-hydroxysuccinimide (NHS) methods have previously been used for corresponding purposes but share a number of disadvantages. They react primarily with lysine residues on the protein molecule and so there is no control over the site at which conjugation takes place.

The three other methods in the table all employ site- specific groups. Each will react only at sites distant from the antigen binding site, conferring advantages of fully-retained

immunoreactivity, improved in vivo biodistrlbution and

pharmacokinetics, and high available specific radioactivity.

The invention will now be described by way of example.

Example 1 - Preparation of ligands A to E

3-thiapentane-1,5-dithiol, HS(C₂H₄)S(C₂H₄)SH was complexed with a carbonyl Mo(CO)₃ to form a template complex

[Mo(C0)₃(SCH₂CH₂SCH₂CH₂S)]^2-. A solution of this (prepared in situ as a bistetramethylammonium salt) in acetonitrile was treated with racemic CH₂BrCHBrR to generate the corresponding trithiamacrocyclic complex. The macrocycle itself was displaced from the complex in DMSO (dimethylsulphoxide) solution by addition of

[NMe₄]₂[SCH₂CH₂SCH₂CH₂S]. In all cases a major by-product was 1,4-dithiacyclohexane, removed by silica gel chromatography.

R was as follows:

Ligand A: -CH₃ (yield 21%)

Ligand B: -CH₂OH (yield 24%)

Ligand C: -CH₂OCH₂-phenyl (yield 6%)

Ligand D: -CH₂OCH₂-parabromophenyl (yield 20%)

Ligand E: -CH₂OCH₂-parabenzoate (yield 8%)

The reaction scheme may be depicted as

The coordination chemistry of Ligand A with Fe(II), Ni(II), Cu(II), Ag(I) and Hg(II), and of Ligand C with Fe(II), has surprisingly been found to be closely analogous to the parent (unsubstituted) 9S3 complexes, as regards stoichiometry, e l ectroni c spectra , stabi l i ty , co-ordi nation number and el ectrochemical behaviour.

Representative Ligands were synthesised in detail as follows:- Preparation of Lloand B. viz 9S3-CH₂OH

Molybdenum hexacarbonyl, Mo(CO)₆, 0.02 mol, and acetonitrile (60 mL) were heated together to reflux under a di nitrogen

atmosphere with stirring for 2 hours. The resulting solution of Mo(CO)₃(MeCN)₃ was transferred anaerobically to a flask containing the bis(tetramethylammonium) salt of 3-thiapentane-1,5-dithiol ([NMe₄]₂[SCH₂CH₂SCH₂CH₂S], 0.02 mol). The resulting suspension was stirred for 24h at room temperature, after which a solution of racemic 1,2-dibromopropan-3-ol (0.02 mol) in acetonitrile (10 ml) was added. After stirring for 2h the solvent was removed in vacuo and the residue treated with [NMe₄]₂CSCH₂CH₂SCH₂CH₂S], (0.02 mol) in dimethyl sulphoxide (50 ml). After stirring at room temperature under di nitrogen for 24h the mixture was extracted with di ethyl ether (4 × 25ml) and the combined extracts evaporated to dryness. The residue was dissolved in chloroform and chromatographed on silica gel using chloroform as eluent. Pure 9S3-CH₂OH (as a viscous oil which crystallised on prolonged vacuum drying,

24% yield) was obtained by evaporation of the relevant fractions. Preparation of Liαand D. 9S3-CH₂OCH₂C₆H₄-Br

This compound was prepared by a method analogous to that for B (9S3-CH₂OH), substituting 1,2-dibromo-3-(4'-bromobenzyloxy) propane for 2,3-dibromopropane. It was purified by silica gel chromatography as described for C (later). Yield 20%.

Preparation of Liαand E. 9S3-CH₂OCH₂C₆H₄-4-COOH

This compound was prepared by a method analogous to that for B (9S3-CH₂OH), substituting 1,2-dibromo-3-(4'-carboxybenzyloxy)propane for 2,3-dibromopropane. It was purified as follows. The DMSO reaction solution was diluted with water (200 ml) and the mixture extracted with chloroform. The chloroform extracts were washed with water, dried over MgSO₄ and evaporated to dryness. The residue was extracted into 10% sodium hydroxide solution, the extract washed with chloroform and acidified with concentrated hydrochloric acid, resulting in precipitation of a solid. This was dissolved in ethanol, the solution filtered and treated with an equal volume of iced water, and the resulting precipitate collected and dissolved in chloroform. This solution was dried over MgSO₄ and evaporated to dryness leaving the product as a white powder. Yield 8%.

Example 2 - Preparation of Lioands C. F and G

The above synthesis can be developed, building on Ligand B. This Ligand may be reacted with the bromide RW according to the reaction scheme

R¹ may be as follows:

Ligand C (= Ligand C above): -CH₂-phenyl (yield 87%)

Ligand F: -CH₂-paracyanophenyl (yield 43%)

Ligand G: -CH₂-meta-monobromo-phenyl (yield 57%)

These ligands were synthesised in detail as follows:Preparation of Lioand C. 9S3-CH₂OCH₂Ph

Ligand B (1 mmol) was reacted with sodium hydride (1 mmol) in dry dimethylformamide (5 ml) followed by addition of benzyl

chloride (1 mmol) and stirring at room temperature for 3 days.

25 ml of water was then added and the resulting mixture extracted with diethyl ether and purified by silica gel chromatography using toluene followed by 20% dichloromethane in toluene, then

dichloromethane, as eluant. Yield 87% Preparation of Liαand F. 9S3-CH₂OCH₂C₆H₄-4-CN

This compound was prepared by a method analogous to that for C, substituting 4-cyanobenzyl chloride for benzyl chloride. The product was purified by silica gel chromatography also as described for C. Yield 43%.

Preparation of Liαand G. 9S3-CH₂OCH₂C₆H₄-3-Br

This compound was prepared by a method analogous to that for C, substituting 3-bromobenzyl chloride for benzyl chloride. The product was purified by silica gel chromatography as described for C. Yield 57%

Example 3 - Preparation of 1-phenyl-1-phospha-4,7-dithiacyclononane(9PhPS2)

Caesium carbonate (0.013 mol) was stirred in dry

dimethylformamide (250 ml) at 70ºC under a dinltrogen atmosphere. 1,2-dichloroethane (0.01 mol) in DMF (50 ml) and 3-phenyl-3-phosphapentane-1,5-dithiol (prepared by a literature method,

0.01 mol) in DMF (50 ml) were added dropwise, simultaneously and at the same rate (a peristaltic pump was used to maintain the equal and constant rate of addition at 4.5 mlh^-1). After completion of the addition the mixture was stirred for a further 11h at 70ºC, then cooled to room temperature. The solvent was removed in vacuo and the residue extracted with diethyl ether (30 ml). The extract was washed with water (2 × 30 ml) and dried over MgSO₄. The solvent was removed and the residue chromatographed on silica gel, eluting first with dichloromethane/chloroform 1:1, then toluene. The product was obtained by removal of solvent from the relevant fractions in 37% yield as a colourless, viscous oil which

crystallised below room temperature. Example 4 - Preparation of Metal Complexes of 9PhPS2 Mercury (II) perchlorate complex with 9PhPS2

1-phenyl-1-phospha-4,7-dithiacyclononane (L) (60mg, 0.23 mmole) was dissolved in acetonitrile (3ml), and stirred at room

temperature while mercury (II) perchlorate (50mg, 0.11 mmole) was added. The solution was stirred for 1 hour, then ether was slowly added until the solution turned cloudy (about 10ml). The product came out of solution as white crystals on refrigeration.

67mg of (9PhPS2)₂Hg(ClO₄)₂ (61%) were obtained.

Microanalysis Found C 31.53%, H 3.33%

C₂₄H₃₄Cl₂HgO₈P₂S₄ requires C 31.60%, H 3.76%

IR (mull, cm^-1) 1095(s), 1080(s), 930(s), 875, 830, 820, 810, 765, 745, 725, 700, 695, 635.

Nickel (II) tetraf1 uoroborate complex with 9PhPS2

Nickel (II) tetrafluoroborate hexahydrate (0.0357g,

0.0105 mmole) in ethanol (2ml) was added to a stirred solution of L (0.055g, 0.215 mmole) in ethanol (12ml). A pea-green colour formed at once. The solution was left at 0°C for 1 hour, and the resulting green complex was filtered off, washed with ether and dried under vacuum. 0.059g (67%) of (9PhPS2)₂Ni(BF₄)₂.H₂O was obtained.

Microanalysis Found C 38.14%, H 4.39%

C₂₄H₃₅B₂F₈NiO_1/2P₂S₄ requires C 38.22%, H 4.68%

Copper (II) tetrafluoroborate complex with 9PhPS2

L (64.5mg, 0.25 mmole) was stirred in ether (1ml), and ethanol

(10ml) added. Copper (II) tetrafluoroborate (28.5mg, 0.12 mmole) in ethanol (2ml) was added, and the red product was stirred for 10 min. All attempts to remove the fine solid by filtration failed.

The mixture was centrifuged, and the supernatant decanted off. The red solid was shaken with ether (12ml) and again centrifuged. The solvent was poured off and the product dried under high vacuum.

45mg of (9PhPS2)₂Cu(BF₄)₂ (51%) were obtained.

Microanalysis Found C 38.23%, H 4.27%

C₂₄H₃₄B₂CuF₈P₂S₄ requires C 38.44%, H 4.57%

IR (mull, cm^-1) 1325, 1095(s), 1060(s), 1045(s), 1000, 930, 885,

820, 810, 760, 725, 700, 525, 495. Copper (I) hexafluorophosphate complex with 9PhPS2

L (120mg, 0.468 mmole) was stirred in acetonitrile (4ml). Cu(CH₃CN)₄PF₆ (82mg, 0.22 mmole) was added to the solution which became very faint pink in colour as the copper complex dissolved. Ether (20ml) was added, the solution lost Its colour and became cloudy. The solution was left overnight at 4ºC to give off-white crystals. These were filtered off and dried under vacuum to yield 134mg (80%) of (9PhPS2)₂CuPF₆.CH₃CN.

Microanalysis Found C 40.74%, H 4.58%, N 1.81%

C₂₆H₃₇CuFNP₃S₄ requires C 40.96%, H 4.89%, N 1.84%

IR (mull, cm^-1) 1410, 840(s), 745(s), 695, 550, 480.

The ligand structure was determined by single crystal X-ray to confirm the structure Cu(9PhPS2)₂ ⁺ as having tetrahedral

coordination with the copper bound by one facially tridentate ligand and a single phosphine donor from the second ligand. The structures is shown in Figure 1.

Example 5 - Preparation of extended 9S3 ligands

1. N-hydroxysul phosucci ni mi de ester, sodium salt

Ligand E (see Example 1) (51mg, 0.15 mmol), dicyclohexylcarbodiimide (31mg, 0.16 mmol), and N-hydroxysulphosuccinimide, sodium salt (32.5mg, 0.15 mmol) were stirred in dry dimethylformamide (1ml) under dry dinitrogen at room temperature for 48h. A white solid was precipitated (dicyclohexylurea) and was removed by filtration. The filtrate was diluted with 15ml diethylether, resulting in formation of a gummy solid which was collected and freed of solvent under high vacuum. Infra red absorption (cm^-1): 3500 (br), 2900, 2840, 1760, 1730, 1655, 1615, 1600, 1405, 1355, 1225 (br), 1095, 1070, 1035, 985. The product was dissolved in dimethyl sulphoxide (DMSO) to a concentration of lOmg/ml for use in protein labelling, and stored below 0°C. (Hereafter the product is identified as "bifunctional chelator solution").

2. N-hydroxysuccinimlde ester

Ligand E (0.103g, 0.3 mmol), dicyclohexylcarbodiimide (0.062g, 0.3 mmol), and N-hydroxysuccimide (0.062g, 0.3 mmol) were stirred at 4°C in dry tetrahydrofuran (3ml) under dry dinitrogen for 3h. The white precipitate (dicyclohexyl urea) that formed was removed after overnight refrigeration at 4ºC, by filtration, and the filtrate dried under vacuum. The IR spectrum of this material showed the presence of impurities (dicyclohexylcarbodiimlde) which were partially removed by reprecipitation from chloroform (2ml) by addition of cyclohexane (20ml) followed by decantation of the supernatant and drying the oily residue under vacuum. IR: 2920, 2845, 1795, 1765, 1735, 1620, 1605, 1570, 1530, 1445, 1410, 1359. Example 6 - Preparation of labelled chelate - protein conjugate

Demonstration of the preparation and stability of the conjugate is described as follows with reference to Figures 2 to 7 wherein:

Figure 2 gives the results for gel filtration of rabbit IgG incubated with ¹⁹⁷Hg pre-chelated with the bifunctional chelator (9S3-activated ester): radioactivity and absorbance elution profi 1e;

Figure 3 shows gel filtration of rabbit IgG incubated with 1⁹⁷Hg pre-chelated with intermediate E (9S3-carboxylate):

radioactivity and absorbance elution profile;

Figures 4 and 5 show gel filtration of ¹⁹⁷Hg-labelled rabbit IgG incubated with whole human serum for 24h: radioactivity and absorbance elution profiles respectively; and

Figures 6 and 7 show gel filtration of ¹⁹⁷Hg-labelled rabbit IgG in PBS: radioactivity and absorbance profiles respectively.

Bifunctional chelator solution (prepared as described in

Example 5-1) (3.62μl) was diluted to 46μl with DMSO and the solution added to 2μl aqueous carrier-free ¹⁹⁷HgCl₂ (37 MBq/ml , Medgenix pic). After 2 minutes this solution was added to

rabbit IgG (Sigma, 2mg in 1ml phosphate buffered saline, PBS). After incubation at room temperature for lh, the sample was chromatographed on a sephadex G25M gel filtration column

(Pharmacia PD10) eluting with PBS. 30 fractions of 1.4ml were collected. The bulk of the radioactivity (52-93%) eluted with the protein fractions (4ml). The results are shown in Figure 2. As a control, the ligand E (which contains a carboxylate group instead of the activated ester), was employed in place of the bifunctional chelator. This was expected to bind mercury ions but to be incapable of reacting with the protein. In this case, less than 5% of the radioactivity eluted as an identifiable protein peak. The results are shown in Figure 3. In a second control, the

radioactive mercuric chloride was incubated with protein in the absence of any chelator; in this case 32% of the activity was eluted with protein, indicating significant non-specific binding. Thus, the chelating moiety binds mercury sufficiently strongly to suppress non-specific binding, while the activated ester

functionality causes efficient binding to protein.

The above results indicate that the chelated mercury is stable towards transfer of mercury ions to non-specific protein binding sites on rabbit IgG. The types of co-ordinating functional groups available under these circumstances are broadly typical of the biological milieu. To further define the stability under

biological conditions, the conjugate was incubated with human serum, and with bovine serum albumin which is known to be capable of binding class b metals (e.g. copper).

Stability to bovine serum albumin

Rabbit IgG labelled with ²⁰³Hg by the procedure described above, taken from the most radioactive IgG fraction obtained from the gel filtration step, was incubated with an excess of bovine serum albumin (1ml of lmg/ml) for lh at 37°C. The sample was then loaded onto a protein-A-derivatised affinity column (Pharmacia HiTrap 1ml) and eluted with pH 8.9 NaCI/glycine buffer (6ml) followed by pH4 citrate buffer (7ml). 13 × 1ml fractions were collected and assayed for protein, by measuring absorbance at 280nm, and for radioactivity. 90% of the radioactivity eluted with the IgG fraction (compared to 94% in the control in which labelled IgG was incubated alone). Thus transfer of radioactivity to albumin was slight (of the order of 4%) after the first hour. However, the loss may become more significant after longer periods.

Stability to human serum

Rabbit IgG labelled with ¹⁹⁷Hg by the procedure described above (O.Tml) was added to human serum (2ml) obtained by clotting whole blood, and incubated for 24h at 37°C under sterile conditions. A sample of the serum was then loaded onto a 300 x 8mm gel filtration column (LKB GlasPak, TSK G3000SW) and eluted with phosphate buffered saline. Absorbance at 280nm was monitored and 0.5ml fractions were collected and counted. Results are shown in

Figure 5. Although the A₂₈₀ trace showed several peaks, the radioactivity was confined to a single well defined peak as shown in Figure 4. A control in which labelled IgG was incubated alone showed an identical eluate radioactivity profile as shown in

Figures 6 and 7. These results thus fail to demonstrate any transfer of radioactivity to serum components that are separable from IgG and so do not reveal any major instability over the time period studied.

Claims

1 . A l igand of formula I

where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where A¹ is -C₂H₄- or or -C₃H₆-,

A² is -C₂H₄- or

and A³ is -C₂H₃R- or or except when A¹ is -C₃H₆-, -C₃H₅R-,

where R is alkyl or substituted alkyl or, if the third T contains P or As, R may also be H.

2. A ligand according to claim 1 wherein R is selected from hydroxyalkyl, alkoxyalkyl, aryloxyalkyl and arylalkoxyalkyl groups which may be further substituted.

3. A ligand according to claim 1 or 2 wherein A¹ and A² are -C₂H₄-.

4. A ligand according to any one of claims 1 to 3 wherein each T is S.

5. A ligand according to any one of claims 1 to 3 wherein two T's and S and the third is the group

6. A ligand according to claim 5 wherein X is a phenyl group which may be substituted.

7. A ligand according to claim 6 wherein X is ortho-substituted.

8. An extended ligand comprising a ligand in accordance with any one of the preceding claims in which at least one univalent atom or group thereof has been replaced by a moiety extending the

co-ordinating functionality of the ligand and/or a linking moiety capable of facilitating binding to a substrate.

9. A metal complex of a ligand or extended ligand according to any one of the preceding claims.

10. A metal complex according to claim 9 wherein the metal is a radionuclide.

11. A ligand extended ligand or metal complex in accordance with any one of the preceding claims when complexed with a biologically active substrate.

12. A kit suitable for use in radiodiagnosis or radiotherapy including a ligand, extended ligand or complex in accordance with any one of the preceding claims.