WO2014096191A1 - Bis 2h-imidazole-2-thione chelating agents as ligands for radiopharmaceutical in vivo imaging - Google Patents

Abstract

The present invention is in the field of radiopharmaceutical agents for in vivo imaging, which comprise a metal complex of a radiometal - either in free form or conjugated to a biological targeting molecule. The invention provides novel chelating agents based on thiones, useful for preparing such radiometal complexes and radiopharmaceuticals. The invention also provides radiometal complexes of the chelators, and their methods of preparation, plus radiopharmaceutical compositions, kits and methods of imaging.

Description

BIS 2H-IMIDAZOLE-2-THIONE CHELATING AGENTS AS LIGANDS FOR RADIOPHARMACEUTICAL IN VIVO IMAGING

Field of the Invention.

The present invention is in the field of radiopharmaceutical agents for in vivo imaging, which comprise a metal complex of a radiometal - either in free form or conjugated to a biological targeting molecule. The invention provides novel chelating agents based on thiones, useful for preparing such radiometal complexes and radiopharmaceuticals. The invention also provides radiometal complexes of the chelators, and their methods of preparation, plus radiopharmaceutical compositions, kits and methods of imaging.

Background to the Invention.

There is still a need for new radiopharmaceutical imaging agents. In regions where there is currently limited or no cyclotron infrastructure for ¹⁸F PET imaging agents, it may be that a SPECT agent (e.g. based on ^99mTc) is the best option. ^99mTc agents have the significant advantage that the ^99mTc radioisotope is conveniently available via a ⁹⁹Mo/^99mTc generator.

Moura et al have reported poly(mercaptoimidazolyl)borates chelators, which form Tc(I) complexes having a tricarbonyl core [Curr. Radiopharm., 5, 150-157 (2012)]:

Currently, the most important gap in technetium radiopharmaceuticals is a chelate which will rapidly form stable ^99mTc complexes at room temperature at neutral or low pH. There are examples of such chelators in the literature but most of these have issues which make them difficult to use in ^99mTc-based products. One such group of chelates are the ¾z^'s(aminethiols):

The ¾z^'s(aminethiols) form stable ^mTc complexes in high yields between pH 3-7. The thiol sulfur atoms of these ¾z^'s(aminethiols) oxidise readily in air to form disulfides (intramolecular and intermolecular). This means that the chelator thiol groups must be protected throughout the synthesis of the chelate-conjugates. The deprotection conditions for the ^-protected thiols may, however, be incompatible with the biomolecules of interest. Once deprotected, the free thiol is then unstable with respect to oxidation to the disulfide and this may lead to complications with the final ^99mTc labelled formulation.

An issue for bifunctional chelators is that non-specific binding of the ^99mTc to the vector can occur, e.g. when the kinetics of radio labelling of the chelator is less favourable than the metal coordinating groups of the bio molecule/vector. A further problem with bifunctional chelators is when the pH suitable for ^99mTc chelator radio labelling is incompatible with the functional groups of the vector.

There is therefore a need for ^99mTc radiopharmaceuticals wherein the chelator used permits labeling of small molecules, peptides and macro molecules as in vivo targeting vectors under radio labelling conditions (pH, solvent temperature etc.) which have minimal impact on the vector to be radio labelled.

The Present Invention.

The mercaptoimidazole group provides a strongly metal-coordinating sulfur atom which is stable with respect to oxidation to the disulphide. The chelators of the invention are expected to form Tc(V) complexes similar to the corresponding ^99mTc- /3z^'s(aminethiol) complexes. The stability of the mercaptoimidazole moiety should enable easier chemical synthesis and handling than the corresponding di-thiol chelates.

Tc(I) complexes having a tricarbonyl core are an alternative route to radiolabelling biomolecules, but such labeling is generally two step - the first being generation of the tricarbonyl core (which requires heating to greater than 50 °C), followed by exchange of the 3 water molecules with a tripodal chelate. In contrast, the radio labelling of the novel tetradentate chelators of the present invention will be one step, preferably at room temperature.

Detailed Description of the Invention.

In a first aspect, the present invention provides a chelating agent of Formula I:

(I)

wherein:

Y¹ and Y² are independently -NR^a-, -0-, -S- or -PR^b-,

provided that at least one of Y¹ and Y² is -NR^a- or -PR^b-^; X¹ and X² are independently -0-, -S- or -NR^b-;

R^a is independently H or an R^b group;

R^b is independently an R^c group or Z¹;

R^c is independently Ci_₄ alkyl, C₂_₄ alkoxyalkyl, Ci_₄ hydroxyalkyl or Ci_₄ fluoroalkyl;

R¹ is independently H or an R^b group, or two adjacent R¹ groups can be combined to form a 5- or 6-membered aryl, heteroaryl, alicyclic or heterocyclic ring;

Q¹ and Q² are independently -CH₂- or -CH₂CH₂-;

Q³ is a bridging group of formula -(J)_f , where f is 2, 3, 4 or 5 and each J is independently -CH₂- or -CHZ¹-;

Z¹ is -(L)_n-[BTM],

wherein n is 0 or 1 ,

BTM is a biological targeting moiety;

L is a synthetic linker group of formula -(A)_m- wherein each A is independently -CR₂- , -CR=CR- , -C≡C- , -CR₂C0₂- , -C0₂CR₂- , -NRCO- , -CONR- , -NR(C=0)NR-, -NR(C=S)NR-, -S0₂NR- , -NRSO2- , -CR2OCR2- , -CR2SCR2- , -CR2NRCR2- , a C4-8

cycloheteroalkylene group, a C₄-8 cycloalkylene group, a C_5-12 arylene group, or a C3-₁₂ heteroarylene group, an amino acid, a sugar or a monodisperse poly ethylenegly col (PEG) building block; each R is independently chosen from H, Ci_₄ alkyl, C₂_₄ alkenyl, C₂-4 alkynyl, Ci_₄ alkoxyalkyl or Ci_₄ hydroxyalkyl;

m is an integer of value 1 to 20;

wherein said chelating agent comprises 0 or 1 Z¹ group.

The term "chelator" or "chelating agent" has its conventional meaning and refers to 2 or more metal donor atoms linked via a non-coordinating backbone and arranged such that chelate rings result upon metal coordination by at least two such metal donor atoms to the same metal centre. The chelator of Formula (I) is designed to be tetradentate with the metal donor atoms derived from the Y¹ and Y² groups, and the two sulfur atoms from the C=S groups all binding to the same metal atom.

The phrase "wherein said chelating agent comprises 0 or 1 Z¹ group" means that the chelating agent is either unconjugated such that there is no conjugated BTM, i.e. none of the R^b, R^c or Q³ groups is Z¹, or has covalently conjugated thereto, via the linker group L, one BTM such that one of the R^b, R^c or Q³ groups is Z¹ (and the others are not Z¹).

By the term "biological targeting moiety" (BTM) is meant a compound which, after administration, is taken up selectively or localises at a particular site of the mammalian body in vivo. Such sites may for example be implicated in a particular disease state or be indicative of how an organ or metabolic process is functioning.

The term "synthetic" has its conventional meaning, i.e. man-made as opposed to being isolated from natural sources e.g. from the mammalian body.

The terms "comprising" or "comprises" have their conventional meaning throughout this application and imply that the agent or composition must have the essential features or components listed, but that others may be present in addition. The term 'comprising' includes as a preferred subset "consisting essentially of which means that the composition has the components listed without other features or components^" being present.

The role of the "linker group" -(A)_m- is to distance the relatively bulky radiometal complex which results upon metal coordination, from the active site of the BTM, so that e.g. receptor binding is not impaired. This can be achieved by a combination of flexibility (e.g. simple alkyl chains), so that the bulky group has the freedom to position itself away from the active site and/or rigidity such as a cycloalkyl or aryl spacer which orientates the metal complex away from the active site. The nature of the linker group can also be used to modify the biodistribution of the resulting radiometal complex of the conjugate. Thus, e.g. the introduction of ether groups in the linker will help to minimise plasma protein binding. Preferred linker groups have a backbone chain of linked atoms which make up the -(A)_m- moiety contain 2 to 10 atoms, most preferably 2 to 5 atoms, with 2 or 3 atoms being especially preferred. A minimum linker group backbone chain of 2 atoms confers the advantage that the chelator is well-separated from the biological targeting moiety so that any interaction is minimised.

By the term "sugar" is meant a mono-, di- or tri- saccharide. Suitable sugars include: glucose, galactose, maltose, mannose, and lactose. Optionally, the sugar may be functionalised to permit facile coupling to amino acids. Thus, e.g. a glucosamine derivative of an amino acid can be conjugated to other amino acids via peptide bonds. The glucosamine derivative of asparagine (commercially available from NovaBiochem) is one example of this:

The term "poly ethylenegly col polymer" or "PEG" has its conventional meaning, as described e.g. in "The Merck Index", 14th Edition entry 7568, i.e. a liquid or solid polymer of general formula H(OCH₂CH₂)_nOH where n is an integer greater than or equal to 4. The polyethyleneglycol polymers of the present invention may be linear or branched, but are preferably linear. The polymers are also preferably non- dendrimeric. Preferred PEG-containing linker groups comprise units derived from oligomerisation of the monodisperse PEG-like structures of Formulae Biol or Bio2:

(Biol)

17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula Biol wherein p is an integer from 1 to 10. Alternatively, a PEG-like structure based on a propionic acid derivative of Formula Bio2 can be used:

(Bio2)

where p is as defined for Formula Biol and q is an integer from 3 to 15.

In Formula Bio2, p is preferably 1 or 2, and q is preferably 5 to 12.

By the term "amino acid" is meant an L- or D-amino acid, amino acid analogue (eg. naphthylalanine) which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers. Conventional 3-letter or single letter abbreviations for amino acids are used herein. Preferably the amino acids of the present invention are optically pure.

By the term "peptide" is meant a compound comprising two or more amino acids, as defined below, linked by a peptide bond (i.e. an amide bond linking the amine of one amino acid to the carboxyl of another). The term "peptide mimetic" or "mimetic" refers to biologically active compounds that mimic the biological activity of a peptide or a protein but are no longer peptidic in chemical nature, that is, they no longer contain any peptide bonds (that is, amide bonds between amino acids). Here, the term peptide mimetic is used in a broader sense to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. The term "peptide analogue" refers to peptides comprising one or more amino acid analogues, as described below. See also Synthesis of Peptides and Peptidomimetics, M. Goodman et al, Houben-Weyl Vol E22c of Methods in Organic Chemistry, Thieme (2004). Preferred embodiments.

In the chelating agent of Formula I, it is preferred that Y¹ = Y² = -NR^a- or PR^b-. More preferably, Y¹ = Y² = -NH-. When Y¹ = Y² = -PR^b-, the R^b groups are preferably R^c groups, more preferably Ci_₄ hydroxyalkyl, most preferably Y¹ = Y² = -P(CH₂OH)-. In Formula I, preferably X¹ = X² = -NR^b-. More preferably, X¹ = X² = -NCH₃-

In the chelating agent of Formula I, it is preferred that, when Y¹ = Y² = -PR^b-, Q¹ = Q² = -CH₂- and when Y¹ = Y² = -NR^a-, Q¹ = Q² = -CH₂-CH₂. In the chelating agent of Formula I, preferably each R¹ is H or an R^b group. More preferably each R¹ group is H or Ci_₄ alkyl. Most preferably, each R¹ group is H.

The chelating agent of Formula I preferably comprises a single Z¹ group, i.e. has a single BTM conjugated thereto. The Z¹ group is preferably located at Y¹, Y² or Q³, more preferably at Q³.

The chelating agent of Formula I is preferably either a bis(amhiQ mercaptoimidazole) of Formula IA, or a ¾z^'s hosphine mercaptoimidazole) of Formula IB:

IA IB

The chelating agent of Formulae IA or IB preferably comprises a single Z¹ group, i.e. has a single BTM conjugated thereto. The Z¹ group is preferably located at Q³, more preferably at the bridgehead position of Q³ (i.e. at the central carbon atom of the Q³ bridge). Preferred such chelators of the invention are of Formulae IC and ID respectivel :

IC ID

where q is 1 or 2.

In Formulae IC and ID, q is preferably 1 and each R^c is independently as defined for Formula I.

The BTM of the first aspect is preferably a single amino acid, a 3-100 mer peptide, an enzyme substrate, an enzyme antagonist an enzyme agonist, an enzyme inhibitor or a receptor-binding compound. The BTM may be of synthetic or natural origin, but is preferably synthetic. Such compounds have the advantage that their manufacture and impurity profile can be fully controlled. Monoclonal antibodies and fragments thereof of natural origin are therefore outside the scope of the term 'synthetic' as used herein.

The molecular weight of the BTM is preferably up to 15,000 Daltons. More preferably, the molecular weight is in the range 200 to 12,000 Daltons, most preferably 300 to 10,000 Daltons, with 400 to 9,000 Daltons being especially preferred. When the BTM is a non-peptide, the molecular weight of the BTM is preferably up to 3,000 Daltons, more preferably 200 to 2,500 Daltons, most preferably 300 to 2,000 Daltons, with 400 to 1,500 Daltons being especially preferred.

When the BTM is an enzyme substrate, enzyme antagonist, enzyme agonist, enzyme inhibitor or receptor-binding compound it is preferably a non-peptide, and more preferably is synthetic. By the term "non-peptide" is meant a compound which does not comprise any peptide bonds, i.e. an amide bond between two amino acid residues Suitable enzyme substrates, antagonists, agonists or inhibitors include glucose and glucose analogues such as fluorodeoxyglucose; fatty acids, or elastase, Angiotensin II or metalloproteinase inhibitors. A preferred non-peptide Angiotensin II antagonist is Losartan. Suitable synthetic receptor-binding compounds include estradiol, estrogen, progestin, progesterone and other steroid hormones; ligands for the dopamine D-1 or D-2 receptor, or dopamine transporter such as tropanes; and ligands for the serotonin receptor.

The BTM is most preferably a 3-100 mer peptide or peptide analogue. When the BTM is a peptide, it is preferably a 4-30 mer peptide, and most preferably a 5 to 28- mer peptide. When the BTM is a peptide, preferred such peptides include:

somatostatin, octreotide and analogues,

- peptides which bind to the ST receptor, where ST refers to the heat-stable toxin produced by E.coli and other micro-organisms;

- bombesin;

- vasoactive intestinal peptide;

- neurotensin;

- laminin fragments eg. YIGSR, PDSGR, IKVAV, LRE and

KCQAGTFALRGDPQG,

N-formyl chemotactic peptides for targeting sites of leucocyte accumulation, Platelet factor 4 (PF4) and fragments thereof,

RGD (Arg-Gly-Asp)-containing peptides, which may eg. target angiogenesis

[R.Pasqualini et al, Nat Biotechnol. 1997 Jun;15(6):542-6]; [E. Ruoslahti,

Kidney Int. 1997 May;51(5): 1413-7].

peptide fragments of a₂-antiplasmin, fibronectin or beta-casein, fibrinogen or thrombospondin. The amino acid sequences of a₂-antiplasmin, fibronectin, beta-casein, fibrinogen and thrombospondin can be found in the following references: a₂-antiplasmin precursor [M.Tone et al., J.Biochem, 102, 1033,

(1987)]; beta-casein [L.Hansson et al, Gene, 139, 193, (1994)]; fibronectin

[A.Gutman et al, FEBS Lett., 207, 145, (1996)]; thrombospondin- 1 precursor

[V.Dixit et al, Proc. Natl. Acad. Sci., USA, 83, 5449, (1986)]; R.F.Doolittle,

Ann. Rev. Biochem., 53, 195, (1984);

- peptides which are substrates or inhibitors of angiotensin, such as: angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (E. C. Jorgensen et al, J. Med. Chem., 1979, Vol 22, 9, 1038-1044)

[Sar, He] Angiotensin II: Sar-Arg-Val-Tyr-Ile-His-Pro-Ile (R.K. Turker et al, Science, 1972, 177, 1203).

- Angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu;

c-Met targeting peptides.

When the BTM is a peptide, one or both termini of the peptide, preferably both, have conjugated thereto a metabolism inhibiting group (M^IG). Having both peptide termini protected in this way is important for in vivo imaging applications, since otherwise rapid metabolism would be expected with consequent loss of selective binding affinity for the BTM peptide. By the term "metabolism inhibiting group" (M^IG) is meant a biocompatible group which inhibits or suppresses enzyme, especially peptidase such as carboxypeptidase, metabolism of the BTM peptide at either the amino terminus or carboxy terminus. Such groups are particularly important for in vivo applications, and are well known to those skilled in the art and are suitably chosen from, for the peptide amine terminus:

N-acylated groups -NH(C=0)R^G where the acyl group -(C=0)R^G has R^G chosen from: Ci_₆ alkyl, C_3-10 aryl groups or comprises a poly ethylenegly col (PEG) building block. Suitable PEG groups are described for the linker group (L¹), above. Preferred such PEG groups are the biomodifiers of Formulae Biol or Bio2 (above). Preferred such amino terminus M^IG groups are acetyl, benzyloxycarbonyl or trifluoroacetyl, most preferably acetyl. Suitable metabolism inhibiting groups for the peptide carboxyl terminus include: carboxamide, tert-butyl ester, benzyl ester, cyclohexyl ester, amino alcohol or a poly ethylenegly col (PEG) building block. A suitable M^IG group for the carboxy terminal amino acid residue of the BTM peptide is where the terminal amine of the amino acid residue is N-alkylated with a Ci_₄ alkyl group, preferably a methyl group. Preferred such M^IG groups are carboxamide or PEG, most preferred such groups are carboxamide.

Preferred BTM peptides are RGD peptides or c-Met targeting peptides. A more preferred such RGD peptide comprises the fragment:

A most preferred such RGD peptide is when the BTM is a peptide of Formula BTM1):

BTM1

wherei ⁷ is either -NH₂ or PEGl, wherein PEGl is:

PEGl

wherein a is an integer of from 1 to 10.

In Formula BTM1, X⁷ is preferably PEGl with 'a' preferably equal to 1.

A preferred functionalised biological targeting molecule is of Formula BTM2:

(BTM2)

The c-Met targeting peptide is preferably an 18 to 30-mer cyclic peptide of Formula BTM3:

Ζ^!-[ΰΜΒΡ]-Ζ² (BTM3)

where:

cMBP is of Formula (II):

-(A)_x-Q-(A')_y (II)

where Q is the amino acid sequence (SEQ-1):

-Cys^X^Cys^-Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶- wherein X¹ is Asn, His or Tyr;

X² is Gly, Ser, Thr or Asn;

X³ is Thr or Arg;

X⁴ is Ala, Asp, Glu, Gly or Ser;

X⁵ is Ser or Thr;

X⁶ is Asp or Glu;

and Cys^a_d are each cysteine residues such that residues a and b as well as c and d are cyclised to form two separate disulfide bonds;

A and A' are independently any amino acid other than Cys, with the proviso that at least one of A and A' is present and is Lys;

x and y are independently integers of value 0 to 13, and are chosen such that [x + y] = 1 to 13;

Z¹ is attached to the N-terminus of cMBP, and is H or M^IG;

Z² is attached to the C-terminus of cMBP and is OH, OB^c, or M^IG,

where B^c is a biocompatible cation;

each M^IG is independently a metabolism inhibiting group which is a biocompatible group which inhibits or suppresses in vivo metabolism of the cMBP peptide;

wherein cMBP is conjugated at the Lys residue of the A or A^' groups with the chelating agent of the invention.

More preferably, the cMBP peptide is of Formula IIA:

-(A)_x-Q-(A^')_Z-Lys- (IIA)

wherein:

z is an integer of value 0 to 12, and [x + z] = 0 to 12,

and cMBP comprises only one Lys residue.

In Formulae II and IIA, Q preferably comprises the amino acid sequence of either SEQ-2 or SEQ-3:

Ser-Cys^X^Cys^-Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X⁶

(SEQ-2);

Ala-Gly-Ser-Cys^X^Cys^-Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr- X⁴-X⁵-X⁶-Gly-Thr (SEQ-3).

In Formulae II and IIA, X³ is preferably Arg.

The cMBP peptide most preferably has the amino acid sequence (SEQ-7):

Ala-Gly-Ser-Cys^a-Tyr-Cys^c-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys^d-Trp-Cys^b-Tyr-Glu-

Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys.

When Q¹ and Q² are CH₂CH₂, the chelators of the invention can be prepared as outlined in Scheme 1 : Sche

Compound 5

The synthesis of Compound 5 is provided in the Examples. It is envisaged that this synthesis can be adapted to the situation where Q¹ and Q² are C¾, and different bridging groups Q³.

Phosphine chelators of the invention can be prepared according to the Examples. An alternative route is shown in Scheme 2: Scheme 2.

The phosphine starting materials can be obtained as follows: when R = Me, Chem. Ber., 122, 1465-1472 (1989) and when R = Ph, Synth. Inorg. Metal-org. Chem., 6, p. 179, 181, 186, 188 (1976):

In a second aspect, the present invention provides a radiometal complex of the chelator of the first aspect.

Preferred aspects of the chelator in the second aspect are as described in the first aspect (above). The radiometal complex of the second aspect is preferably useful as an in vivo imaging agent. By the term "imaging agent" is meant a compound suitable for imaging the mammalian body. Preferably, the mammal is an intact mammalian body in vivo, and is more preferably a human subject. The imaging agent is typically administered in a non-pharmaco logic amount, i.e. at a dosage designed to have a minimal biological effect on the mammalian subject. Preferably, the imaging agent can be administered to the mammalian body in a minimally invasive manner, i.e. without a substantial health risk to the mammalian subject when carried out under professional medical expertise. Such minimally invasive administration is preferably intravenous administration into a peripheral vein of said subject, without the need for local or general anaesthetic. The term "m vivo imaging" as used herein refers to those techniques that noninvasive ly produce images of all or part of an internal aspect of a mammalian subject. Preferred such imaging techniques are SPECT (Single Photon Emission Tomography) and PET (Positron Emission Tomography).

In the radiometal complex of the second aspect, the radiometal is preferably chosen r- _m 99mrr 9 mrr 18_6γ) 188^ 62^ 64^ 67^ 67^ 68 -ι 45

from: Tc, Tc, Re, Re, Cu, Cu, Cu, Ga, Ga or Ti, and is more preferably ^99mTc or ^94mTc, most preferably ^99mTc.

The radiometal complexes of the second aspect can be prepared as described third aspect (below).

In a third aspect, the present invention provides a method of preparation of the radiometal complex of the second aspect, which comprises reaction of the chelator of the first aspect with a supply of the radiometal in a suitable solvent.

Preferred aspects of the chelator and metal complex in the third aspect are as described in the first and second aspects respectively (above).

The suitable solvent is typically aqueous in nature, and is preferably a biocompatible carrier solvent as defined in the fourth aspect (below).

The radiometal complexes of the thirds aspect may be prepared by reaction of a solution of the radiometal in the appropriate oxidation state with the chelator at the appropriate pH. The solution may optionally contain a ligand which complexes weakly to the radiometal (such as gluconate or citrate for ^99mTc) i.e. using ligand exchange or transchelation. Such conditions are often useful to suppress undesirable side reactions such as hydrolysis of the radiometal ion. When the radioisotope is ^99mTc, the usual starting material is sodium pertechnetate from a ⁹⁹Mo/^99mTc generator. Technetium is present in ^99mTc-pertechnetate in the Tc(VII) oxidation state, which is relatively unreactive. The preparation of technetium complexes of lower oxidation state Tc(I) to Tc(V) therefore usually requires the addition of a suitable pharmaceutically acceptable reducing agent such as sodium dithionite, sodium bisulphite, ascorbic acid, formamidine sulphinic acid, stannous ion, Fe(II) or Cu(I), to facilitate complexation. The pharmaceutically acceptable reducing agent is preferably a stannous salt, most preferably stannous chloride, stannous fluoride or stannous tartrate.

Further details on the preparation of non-technetium radiometal complexes, including chelate-BTM conjugates are provided by Wangler et al [Mini Rev.Med.Chem., 11(11), 968-983 (2011)] and Zeglis et al [Dalton Trans. 40(23) 6168-6195 (2011)].

In a fourth aspect, the present invention provides a radiopharmaceutical composition which comprises the radiometal complex of the second aspect, together with a biocompatible carrier, in a form suitable for mammalian administration.

Preferred aspects of the chelator and metal complex in the fourth aspect are as described in the first and second aspects respectively (above).

By the phrase "in a form suitable for mammalian administration" is meant a composition which is sterile, pyrogen- free, lacks compounds which produce toxic or adverse effects, and is formulated at a biocompatible pH (approximately pH 4.0 to 10.5). Such compositions lack particulates which could risk causing emboli in vivo, and are formulated so that precipitation does not occur on contact with biological fluids (e.g. blood). Such compositions also contain only biologically compatible excipients, and are preferably isotonic.

The "biocompatible carrier" is a fluid, especially a liquid, in which the imaging agent can be suspended or preferably dissolved, such that the composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is isotonic); an aqueous buffer solution comprising a biocompatible buffering agent (e.g. phosphate buffer); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. poly ethylenegly cols, propylene glycols and the like). Preferably the biocompatible carrier is pyrogen-free water for injection, isotonic saline or phosphate buffer.

The radiopharmaceutical composition is supplied in a suitable vial or vessel which comprises a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (e.g. nitrogen or argon), whilst permitting addition and withdrawal of solutions by syringe or cannula. A preferred such container is a septum-sealed vial, wherein the gas-tight closure is crimped on with an overseal (typically of aluminium). The closure is suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on septum seal closure) whilst maintaining sterile integrity. Such containers have the additional advantage that the closure can withstand vacuum if desired (e.g. to change the headspace gas or degas solutions), and withstand pressure changes such as reductions in pressure without permitting ingress of external atmospheric gases, such as oxygen or water vapour.

Preferred multiple dose containers comprise a single bulk vial which contains multiple patient doses, whereby single patient doses can thus be withdrawn into clinical grade syringes at various time intervals during the viable lifetime of the preparation to suit the clinical situation. Pre-filled syringes are designed to contain a single human dose, or "unit dose" and are therefore preferably a disposable or other syringe suitable for clinical use. The pharmaceutical compositions of the present invention preferably have a dosage suitable for a single patient and are provided in a suitable syringe or container, as described above.

The pharmaceutical composition may contain additional optional excipients such as: an antimicrobial preservative, pH-adjusting agent, filler, radioprotectant, solubiliser or osmolality adjusting agent. By the term "radioprotectant" is meant a compound which inhibits degradation reactions, such as redox processes, by trapping highly- reactive free radicals, such as oxygen-containing free radicals arising from the radio lysis of water. The radioprotectants of the present invention are suitably chosen from: ascorbic acid, /?ara-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e. 2,5-dihydroxybenzoic acid) and salts thereof with a biocompatible cation. By the term "biocompatible cation" (B^c) is meant a positively charged counterion which forms a salt with an ionised, negatively charged group, where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body. Examples of suitable biocompatible cations include: the alkali metals sodium or potassium; the alkaline earth metals calcium and magnesium; and the ammonium ion. Preferred biocompatible cations are sodium and potassium, most preferably sodium.

By the term "solubiliser" is meant an additive present in the composition which increases solubility in the solvent. A preferred such solvent is aqueous media, and hence the solubiliser preferably improves solubility in water. Suitable such

solubilisers include: Ci_₄ alcohols; glycerine; polyethylene glycol (PEG); propylene glycol; polyoxy ethylene sorbitan monooleate; sorbitan monooloeate; polysorbates; poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers

(Pluronics™); cyclodextrins (e.g. alpha, beta or gamma cyclodextrin, hydroxypropyl- β-cyclodextrin or hydro xypropyl-y-cyclodextrin) and lecithin.

By the term "antimicrobial preservative" is meant an agent which inhibits the growth of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The antimicrobial preservative may also exhibit some bactericidal properties, depending on the dosage employed. The main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such micro-organism in the pharmaceutical composition. The antimicrobial preservative may, however, also optionally be used to inhibit the growth of potentially harmful micro-organisms in one or more components of kits used to prepare said composition prior to administration. Suitable antimicrobial preservative(s) include: the parabens, i.e. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial preservative(s) are the parabens.

The term "pH-adjusting agent" means a compound or mixture of compounds useful to ensure that the pH of the composition is within acceptable limits (approximately pH 4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as tricine, phosphate, citrate or TRIS [i.e. tm(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition is employed in kit form, the pH adjusting agent may optionally be provided in a separate vial or container, so that the user of the kit can adjust the pH as part of a multi-step procedure.

By the term "filler" is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophilisation. Suitable fillers include inorganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose.

The radiopharmaceutical compositions may be prepared under aseptic manufacture (i.e. clean room) conditions to give the desired sterile, non-pyrogenic product. It is preferred that the key components, especially the associated reagents plus those parts of the apparatus which come into contact with the imaging agent (e.g. vials) are sterile. The components and reagents can be sterilised by methods known in the art, including: sterile filtration, terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). It is preferred to sterilise some components in advance, so that the minimum number of

manipulations needs to be carried out. As a precaution, however, it is preferred to include at least a sterile filtration step as the final step in the preparation of the pharmaceutical composition.

In a fifth aspect, the present invention provides a kit for the preparation of the radiopharmaceutical composition of the fourth aspect, which comprises the chelator of the first aspect in sterile, solid form such that upon reconstitution with a sterile supply of the radiometal in a biocompatible carrier, dissolution occurs to give the desired radiopharmaceutical composition. Preferred aspects of the chelator and metal complex in the fifth aspect are as described in the first and second aspects respectively (above).

The sterile, solid form is preferably a lyophilised solid. In a sixth aspect, the present invention provides a method of imaging the human or animal body which comprises generating an image of at least a part of said body to which the radiometal complex of the second aspect, or the composition of the fourth aspect has distributed using PET or SPECT, wherein said imaging agent or composition has been previously administered to said body.

Preferred aspects of the chelator, metal complex and radiopharmaceutical composition in the sixth aspect are as described in the first, second and fourth aspects respectively (above).

The method of the sixth aspect is preferably carried out repeatedly to monitor the effect of treatment of a human or animal body with a drug, said imaging being effected before and after treatment with said drug, and optionally also during treatment with said drug.

In a seventh aspect, the present invention provides the use of the radiometal complex of the second aspect, the composition of the fourth aspect, or the kit of the fifth aspect in a method of diagnosis of the human or animal body.

Preferred aspects of the chelator, metal complex, radiopharmaceutical composition and kit in the seventh aspect are as described in the first, second, fourth and fifth aspects respectively (above).

In an eighth aspect, the present invention provides a method of diagnosis of the human or animal body which comprises the method of imaging of the seventh aspect.

Preferred aspects of the chelator, metal complex, radiopharmaceutical composition and kit in the seventh aspect are as described in the first, second, fourth and fifth aspects respectively (above). The invention is illustrated by the following Examples. Example 1 provides the synthesis of a mercaptoimidazole precursor. Example 2 provides the synthesis of a l,3-£z^'s(phosphonium salt) precursor to Compound 3. Example 3 provides the synthesis P2S2 tetradentate chelator ("Compound 3").

Examples 4 and 5 provide the syntheses of palladium and rhenium complexes of Compound 3 respectively. These show that the chelator acts a tetradentate, with binding of the 2 phosphine donor atoms, and the two mercaptoimidazole sulfur atoms. Example 6 provides the synthesis of an N₂S₂ tetradentate chelator ("Compound 5") having -CH₂CH₂- linkages.

Examples 7 and 8 show that Compound 5 labels ^99mTc cleanly to form a ^99mTc metal complex. Example 9 provides the purification of ^99mTc-Compound 5.

Abbreviations.

DCM: dichloromethane;

Et₂0: diethylether.

HPLC: high performance liquid chromatography.

MDP: methylenediphosphonic acid.

MeCN: Acetonitrile.

MeOH: methanol;

NEt₃ : triethy lamine ;

Ph: phenyl.

Compounds of the Invention.

where Ph = phenyl.

Example 1: Synthesis of l-Methyl-2H-imidazole-2-thione (Compound 1).

Compound 1 was prepared by the method of Guziec et al [J.Org.Chem., 59, 4691-

4692 (1994)]. Yield 5.5 g, 64 %.

1H NMR (400 MHz, CDC1₃, ppm):

δ 12.01 (bs, 1H, NH), 6.71 (d, 1Η, CH), 6.68 (d, 1Η, CH), 3.58 (s, 3Η, CH₃). ¹³C { 1H} NMR (100 MHz, CDC1₃, ppm): δ 160.42 (OS), 1 19.13 (CH), 1 14.26 (CH), 34.29 (CH₃). ESI MS (+): 1 15 m/z [M]+. Example 2: Synthesis of l-,3-6 s[6 sfhvdroxymethyl)phenylphosphoniumlpropane Dichloride (Compound 2).

This method is adapted from the method of Miller et al [J.Organomet.Chem., 695, 1138 (2010)]. To a stirred sample of l,3-£z^'s(phenylphosphino)propane (Sigma- Aldrich; 1.2 g, 4.6 mmol), degassed formaldehyde (2 ml, 23 mmol, 35 %) and cone. HC1 (0.8 mL, 9.2 mmol, 36 %) were added and the mixture was stirred for 2 hours. Et₂0 was added to afford colourless crystals which were isolated by vacuum filtration. Yield 1.97 g, 94 %.

1H NMR (400 MHz, D2O, ppm): δ 7.70 (m, 6H, Ph), 7.58 (m, 4H, Ph), 4.70 (s, 8H, CH2OH), 2.73 (m, 4H, PCH2), 1.87 (quintet, 2Η, PCH2CH2CH2P).

3¹P NMR (162 MHz, D2O, ppm): δ 22.52 (s, P).

¹³C NMR (100 MHz, D2O, ppm): δ 135.42 (s, Ph), 132.32 (s, Ph), 130.21 (s, Ph), 113.33 (s, Ph P-C) 112.58 (s, Ph P-Q, 52.40 (s, PCH2OH), 51.82 (s, PCH2OH), 17.03 (d, iJpc = 16.59 Hz, PCH2), 16.41 (d, iJpc = 16.59 Hz, PCH2), 14.55 (s,

CI MS (+):205 m/z [Ci9H28P₂04Na]2+.

Example 3: Synthesis of P2S2 Chelator (Compound 3).

To a solution of Compound 2 (Example 2; 0.1 g, 0.22 mmol) in dry MeOH, NEt3 (0.15 mL, 1.1 mmol) and Compound 1 (Example 1; 0.44 mmol) were added, the reaction was heated to reflux for 2 hours. Upon cooling, the solvent was removed in vacuo and the product isolated by extraction with dry Et20 (10 mL), washed with degassed K2CO3 (5 mL, 1M), dried over MgSOt and filtered via cannula. The solvent was removed in vacuo to yield a colourless viscous oil. Yield 0.094g, 83 %.

1H NMR (400 MHz, CDCb, ppm): δ 7.48 (m, 4H, ph), 7.34 (m, 6H, ph), 6.83 (d, 0.4H, MMI), 6.66 (s, 4H, CH), 4.06 (m, 4H,PCH2N), 3.58 (s, 6Η, CHs), 2.08 (m, 2Η, PCH2), 2.01 (m, 2Η, PCH2), 1.73 (m, 2Η PCH2CH2CH2P).

³¹P NMR (162 MHz, CDCb, ppm): δ -20.96 (Compound 3), -21.44 (Compound 3), - 53.48 (l,3-bis(phenylphosphino)propane).

TOF ES MS (+): 513 [M]+ m/z. Example 4: Synthesis of Palladium Complex of P2S2 Chelator (Compound 4A).

The P2S2 tetradentate ligand (Compound 3, Example 3) was dissolved in dry MeCN (5 mL), then PdCb and NaPF₆ were added and the reaction mixture heated overnight. The solution was filtered through celite and the solvent removed in vacuo. The Compound 4A product was isolated as a dark red solid. Yield 0.087 g, 66 %.

1H NMR (400 MHz, MeCN-d4, ppm): δ 7.90 (m, 4H, Ph), 7.49 (m, 6H, Ph), 7.18 (bs, 2H, CH), 7.08 (bs, 2Η, CH), 4.71 (m, 4Η, PCH2N), 3.86 (s, 6Η, CHs), 2.51 (m, 2Η, PCH2), 1.64 (m, 2Η, PCH2), 1.26 (m, 2Η PCH2CH2CH2P).

3¹P{1H} NMR (162 MHz, MeCN-d₄, ppm): δ 9.88, 8.11, -144.20 (PF₆)₂.

TOF ES MS (+): 311 m/z [M]₂+.

Example 5: Synthesis of Rhenium Complex of P2S2 Chelator (Compound 4B).

The P2S2 tetradentate ligand (Compound 3, Example 3) was dissolved in dry toluene (5 mL) and ReOCb(PPh3)2 (Sigma- Aldrich) was added, the reaction mixture heated overnight at 80°C. The dark green precipitate formed was isolated by vacuum filtration and washed with toluene and diethyl ether.

Crude 1H NMR (400 MHz, MeCN-d4, ppm): δ 7.85 (m), 7.53 (m), 7.36 (m), 6.92 (m), 4.72 (m), 4.33 (bs), 3.67 (bm), 3.50(bm), 3.20 (bm), 2.14 (bs), 1.80 (m).

3¹P{1H} NMR (162 MHz, MeCNd4, ppm): δ 36.67 (Compound 3 dioxide), 36.57 (Compound 3 dioxide), 25.57 (OPPhs), 20.91 (Compound 4B), 20.44 (Compound 4B), 17.57.

TOF ES MS (+): 732 m/z [M] +.

Example 6: Synthesis of N2S2 Chelator (Compound 5).

Reference is made to Scheme 1 (above).

Step (a): Synthesis of Intermediate II.

2,2'-(ethane-l,2-diyl¾ 5(azanediyl))diethanol I (Sigma- Aldrich; 1.0 g, 6.8 mmol) and N-ethyl-N-isopropylpropan-2-amine (Sigma- Aldrich; 1.7 g, 13.5 mmol) were dissolved in DCM (10 mL) under a nitrogen atmosphere and cooled to 0°C. Benzyl chloro formate (2.1 mL, 14.8 mmol) in DCM (10 mL) was added dropwise over 5 min and the resulting suspension was stirred at 0°C for 30 min followed by 1 hour at room temperature. To the reaction mixture was added water (50 mL), then DCM (50 mL) and stirred for 5 minutes before the phases were separated. The aqueous phase was extracted twice with DCM (50ml) before the combined organic phases were dried with MgS0₄, filtered and concentrated to give crude dibenzyl ethane- l,2-diyl£z^'s((2- hydroxy ethyl)carbamate) II (2.6 g, 6.24 mmol, 93%) as a yellow oil.

m/z [M+H expected 417.20 found 417.13

The material was used in the next step without any further purification.

Step (b): Synthesis of Intermediate III.

Intermediate II [Step (a); 1.1 g, 2.6 mmol] was dissolved in DCM (10 mL), and triethylamine (1.1 mL, 7.9 mmol) added under a nitrogen atmosphere. The clear solution was cooled to 0°C, then methanesulfonyl chloride (0.5 mL, 5.8 mmol) was added dropwise over 2 min. The suspension was left stirring at 0°C for 30 min followed by 30 min at room temperature before water (50 mL) and DCM (50 mL) were added. The resulting biphasic system was stirred for 5 minutes before the phases were separated. The aqueous phase was extracted twice with DCM (50 mL) and the combined organic phases dried with MgS0₄, filtered and concentrated to dryness to give crude (3 ,8-dioxo- 1 , 10-diphenyl-2,9-dioxa-4,7-diazadecane-4,7-diyl)¾z5(ethane- 2,1-diyl) dimethanesulfonate III (1.3g, 2.3mmol, 88%) as a white wax.

m/z [M+H⁺]¹ expected 573.16 found 573.16

The material was used in the next step without any further purification. Step (c): Synthesis of Intermediate IV.

Intermediate III [Step (b); 620 mg, 1.1 mrnol] was dissolved in N-methyl imidazole 83.5 mL) in a vial. The vial was capped and heated at 50 °C for 16 hours then allowed to cool to room temperature before being slowly poured out into diethylether (100 mL). The ether phase was decanted off, and the remaining oil was dissolved in water (0.1% TFA) and purified with prep. HPLC³ to give 3,3^*-((3,8-dioxo-l,10- diphenyl-2,9-dioxa-4,7-diazadecane-4,7-diyl)bis(ethane-2,l-diyl))bis(l -methyl- 1H- imidazol-3-ium) 2,2,2-trifluoroacetate IV (287 mg, 0.4 mrnol, 34%) as a clear oil after freeze-drying.

m/z [M²⁺/2f expected 273.15 found 273.28.

Step (d): Synthesis of Intermediate V.

Intermediate IV [Step (c); (200mg, 0.3 mrnol] was dissolved in MeOH (4 ml) in a vial and added sulfur (166 mg, 5.2 mrnol) and sodium methoxide (700 μΐ, 2.6 mrnol). The vial was capped and heated to 50 °C for 1 hour after which the solids were filtered off and the solution was added acetic acid (148 μΐ, 2.6 mrnol) before concentrating to dryness. The crude was dissolved in water (0. P/oTFA) and purified via preparative HPLC³ to give dibenzyl ethane-l,2-diyl¾z5((2-(3-methyl-2-thioxo-2,3-dihydro-lH- imidazol-l-yl)ethyl)carbamate) V (152 mg, 0.3 mrnol, 96%) as an off-white solid. m/z [M+FT expected 609.23 found 609.26

Step (e): Synthesis of Compound 5.

Intermediate V [Step (d); 152 mg, 0.3 mrnol] was dissolved in DCM (15 ml), and BBr₃ (1M solution in DCM, 0.75 ml) added dropwise and left stirring 30 min at room temperature under an nitrogen atmosphere. The excess BBr₃ was quenched using MeOH (2 mL), then the mixture was concentrated to dryness. The crude was dissolved in water (0.1%> TFA) and purified using preparative HPLC⁴ to give 3,3'- ((ethane- 1 ,2-diyl3zs(azanediyl))3zs(ethane-2, 1 -diyl))bis( 1 -methyl- 1 H-imidazole- 2(3H)-thione) [Compound 5; 35 mg, 0.10 mrnol, 41 % yield].

m/z [M+H⁺]² expected 341.16 found 341.11

1H NMR (400 MHz, CD₃OD): δ_Η 3.42 (3H, s, NCH₃), 3.55-3.59 (11H, m, NCH₃ and CH₂NCH₂CH₂NCH₂), 4.46 (4H, t, J= 6.4 Hz, CH₂NAr), 7.07 (2H, d, J = 1.2 Hz, NCH=CHNCH₃) and 7.13 (2H, d, J = 1.2 Hz, NCH=CHNCH₃). Analytical HPLC

Thermo Finnigan Surveyor System with Surveyor MSQ Plus detector.

'Column Phenomenex Luna 3μ C18 (2) 20 x 2 mm, detection: 214 and 254nm, solvent A: H₂O/0.1 % TFA, solvent B: CH₃CN flow: 0.6 mL/min, gradient: 5-95 % B over 5 min

²Column Phenomenex Luna 3μ C18 (2) 20 x 2 mm, detection: 214 and 254nm, solvent A: H₂O/0.1 % TFA, solvent B: CH₃CN flow: 0.6 mL/min, gradient: 0-20 % B over 5 min Preparative HPLC

Waters Delta Prep 4000 w. Waters 2487 dual λ absorbance detector and Foxy200 fraction collector.

³Column: Phenomenex Luna 5μ C18 (2) 250 x 50 mm, detection: 214 and 254nm, solvent A: H₂O/0.1 % TFA, solvent B: CH₃CN, flow: 50 mL/min, gradient: 5-95 % B over 60 min

⁴Column: Phenomenex Luna 5μ C18 (2) 250 x 21.20 mm, detection: 214 and 254 nm solvent A: H₂O/0.1 % TFA, solvent B: CH₃CN, flow: 10 mL/min, gradient: 0-20 % B over 40 min

Example 7: ^99mTc Labelling of N?S? Chelator Compound 5 at pH 9.

Step (Ϊ).

Compound 5 (146 nmoles) in 50μ1 H₂0 was added to a lyophilised kit containing the following formulation:

Step (ii)

^99mTc-pertechnetate eluate from a Drytec™ generator (GE Healthcare; 1 mL, 631 MBq) was then added to the vial, and the solution allowed to stand at room temperature for 80 min before HPLC analysis. The RCP was 100%.

Example 8: ^wmTc Labelling of N?S? Chelator Compound 5 at pH 7.4.

^99mTc-pertechnetate eluate from a Drytec™ generator (GE Healthcare; 1 mL, 725 MBq) was added to a vial containing Compound 5 (26 nmoles) in phosphate buffer (0.5 mL, 50 mM, pH 7.4). After the addition of SnCl₂/MDP solution (0.1 mL, 0.07 μιηοΐεβ SnCl₂, 0.15 μιηοΐεβ MDP(H4), the solution was allowed to stand for 35 min before HPLC analysis (see Example 9). The RCP was 100%.

Example 9: Purification of ^wmTc Labelled N?S? Chelator Compound 5.

A 100 HPLC injection was used for the purification. ^99mTc labelled Compound 5 was HPLC purified in 92% yield from excess Compound 5 directly into phosphate buffer (0.5 mL, 50 mM, pH 7.4). The residual acetonitrile was removed on a vacuum line and ^99mTc labelled compound 5 was formulated as a 29 MBq/mL solution. The formulation was analysed after 75 min by HPLC. (RCP = 100%).

Analytical and preparative HPLC

Gilson 322 pump with a UV/ViS 156 detector, g-detector (Bioscan Flow-count, PMT probe, Thermo MS3083; LabLogic Systems Limited).

Column Phemonenex Luna 5μ C18 (2) 150 x 4.6 mm, UV detection 253nm, radioactivity detection. Solvent A: H₂O/0.1% TFA, solvent B: CH₃CN, flow:

lmL/min, gradient: 0-3 min 4% B, 3-15 min 4-60% B, 15-16 min 60-90%, 16-20 min 90% B, 20-21 min 90-4% B, 21-25 min 4% B.

Claims

CLAIMS. 1. A chelating agent of Formula I:

(I)

wherein:

Y¹ and Y² are independently -NR^a-, -0-, -S- or -PR^b-,

provided that at least one of Y¹ and Y² is -NR^a- or -PR^b-^; X¹ and X² are independently -0-, -S- or -NR^b-;

R^a is independently H or an R^b group;

R^b is independently an R^c group or Z¹;

R^c is independently Ci_₄ alkyl, C₂_₄ alkoxyalkyl, Ci_₄ hydroxyalkyl or Ci_₄ fluoroalkyl;

R¹ is independently H or an R^b group, or two adjacent R¹ groups can be combined to form a 5- or 6-membered aryl, heteroaryl, alicyclic or heterocyclic ring;

Q¹ and Q² are independently -CH₂- or -CH₂CH₂-;

Q³ is a bridging group of formula -(J)_f , where f is 2, 3, 4 or 5 and each J is independently -CH₂- or -CHZ¹-;

Z¹ is -(L)_n-[BTM],

wherein n is 0 or 1 ,

BTM is a biological targeting moiety;

L is a synthetic linker group of formula -(A)_m- wherein each A is independently -CR₂- , -CR=CR- , -C≡C- , -CR₂C0₂- , -C0₂CR₂- , -NRCO- , -CONR- , -NR(C=0)NR-, -NR(C=S)NR-, -S0₂NR- , -NRS0₂- , -CR₂OCR₂- , -CR₂SCR₂- , -CR₂NRCR₂- , a C₄_₈ cycloheteroalkylene group, a C₄_s cycloalkylene group, a C_5-12 arylene group, or a C3-12 heteroarylene group, an amino acid, a sugar or a monodisperse poly ethylenegly col (PEG) building block; each R is independently chosen from H, Ci_₄ alkyl, C₂_₄ alkenyl, C₂_₄ alkynyl, Ci_₄ alkoxyalkyl or Ci_₄ hydroxyalkyl;

m is an integer of value 1 to 20;

wherein said chelating agent comprises 0 or 1 Z¹ group.

2. The chelating agent of claim 1, where Y¹ = Y² = -NH-.

3. The chelating agent of claim 1 or claim 2, where Y¹ = Y² = -PR^b-.

4. The chelating agent of any one of claims 1 to 3, where BTM is a single amino acid, a 3-100 mer peptide, an enzyme substrate, an enzyme antagonist an enzyme agonist, an enzyme inhibitor or a receptor-binding compound.

5. The chelating agent of any one of claims 1 to 4, where X¹ = X² = -NR^b-.

6. The chelating agent of any one of claims 1 to 5, where Q¹ = Q² = -CH₂-.

7. The chelating agent of any one of claims 1 to 6, which comprises a single Z¹ group.

8. The chelating agent of claim 7, where the Z¹ group is located at Q³.

9. A radiometal complex of the chelator of any one of claims 1 to 8.

10. The radiometal complex of claim 9, where the radiometal is chosen from:

Ti.

11. The radiometal complex of claim 10, where the radiometal is ^99mTc.

12. A method of preparation of the radiometal complex of any one of claims 9 to

11 , which comprises reaction of the chelator of any one of claims 1 to 8 with a supply of the radiometal in a suitable solvent.

13. A radiopharmaceutical composition which comprises the radiometal complex of any one of claims 9 to 11, together with a biocompatible carrier, in a form suitable for mammalian administration.

14. A kit for the preparation of the radiopharmaceutical composition of claim 13, which comprises the chelator of any one of claims 1 to 8 in sterile, solid form such that upon reconstitution with a sterile supply of the radiometal in a biocompatible carrier, dissolution occurs to give the desired radiopharmaceutical composition.

15. The kit of claim 14, where the sterile, solid form is a lyophilised solid.

16. A method of imaging the human or animal body which comprises generating an image of at least a part of said body to which the radiometal complex of any one of claims 9 to 11, or the composition of claim 13 has distributed using PET or SPECT, wherein said imaging agent or composition has been previously administered to said body.

17. The method of claim 16, which is carried out repeatedly to monitor the effect of treatment of a human or animal body with a drug, said imaging being effected before and after treatment with said drug, and optionally also during treatment with said drug.

18. The use of the radiometal complex of any one of claims 9 to 11, the composition of claim 13, or the kit of claim 14 or claim 15 in a method of diagnosis of the human or animal body.

19. A method of diagnosis of the human or animal body which comprises the method of imaging of claim 16 or claim 17.