WO2010116132A2 - Bisphosphonate compounds for chelating radionuclides - Google Patents

Abstract

Bisphosphonate compounds for chelating radionuclides are described which have separate bisphosphonate and metal chelating groups joined by a linker, so that the bisphosphonate groups are available to complex to hydroxyapatite in bone, while the metal chelating group binds to the radionuclide. This avoids problems in the prior art, where the bisphosphonate groups are used for both binding functions which compromises the bone-seeking activity of the bisphosphonate groups when they are used to chelate the radionuclide.

Description

Bisphosphonate Compounds for Chelating Radionuclides

Field of the Invention

The present invention relates to bisphosphonate compounds and to complexes formed between the compounds and chelatable radionuclides. The present invention further relates to the uses of the compounds and complexes for therapy and diagnosis.

Background of the Invention

Bisphosphonates (BPs) are a family of compounds that have been extensively used in the management of disorders of bone metabolism, see for example WO 88/00590 and US Patent No: 5,190,930. Radiopharmaceuticals based on bisphosphonates have been used in the clinic both for imaging ("scintigraphy") and palliative treatment of bone metastases. Pain due to skeletal metastases is one of the principal factors limiting the quality of life of terminal cancer patients, especially those with prostate and breast cancer. Recent evidence suggests that the use of radiolabelled bisphosphonates not only improves quality of life, but also gives a clear clinical gain in terms of life expectancy, delayed onset of new metastases, and reduction of bone metastases, especially at higher doses.

Bisphosphonates target bone by virtue of their ability to bind tightly to hydroxyapatite and the biological activity of BPs is due to their ability to accumulate in areas of high bone metabolism, such as bone metastases. The imaging properties of conventional radiolabelled BPs rely on the innate ability of bisphosphonate compounds to chelate radioisotopes. To image regions of bone metastasis in patients, nuclear medicine physicists use a combination of a BP with the gamma-emitter technetium- 99m. For example, the composition formed with methylene diphosphonate , ""¹Tc -Methylene Diphosphonate (""¹Tc-MDP) , is currently the gold standard for the detection of bone metastases . Endoradiotherapy of these metastases with beta- emitter analogues of ""¹Tc-MDP capable of producing a therapeutic effect have also been developed. For example, the rhenium compound ^186/188Re -Hydroxy Ethylidene-1, 1-I}iphosphonate (^186/188Re- HEDP) has shown promise as a palliative and therapeutic agent for bone metastases in recent clinical trials.

However, despite the proven clinical success of ""¹TcZ¹⁸⁸^⁸⁶Re-BPs, these radiopharmaceuticals are far from optimal from both a chemical and a pharmaceutical point of view. Firstly, despite years of clinical use, their exact structures and compositions remain unknown. It is generally thought that the ^99mTc-MDP preparation used in the clinic is most probably composed of a mixture of anionic polymers of different properties, and consequently is neither homogenous nor reproducible.

Secondly, although the widespread assumption that the chemistries of Tc and Re are identical has resulted in the discovery of the promising therapeutic properties of ^186/188Re-BPs, the latter show even poorer composition and in vivo properties than their ^99mτc counterparts. Particularly, BPs are not suitable for stabilising the low oxidation states (≤ 5) of Re required for coordination with other molecules. As a result, the metal in ¹⁸⁵^¹⁸⁸Re-BPs is easily oxidised in vivo to the more thermodynamically stable perrhenate anion that accumulates in the stomach as well as in the thyroid and salivary glands. This leads to problems in a clinical setting as the perrhenate anion is visualised in scans and retention in metastases is reduced.

There are examples in the prior art of complexes in which bisphosphonate groups are used for both targeting bone and chelating radionuclides.

Yan et al describe (lH-l,2,4-triazol-l-yl) -1-hydroxyethane- 1, 1- diphosphonic acid and its use for chelating ^99mTc via the phosphonic acid groups. See Yan et al Nuclear Science and Techniques, 19(3) , 165-168, 2008.

Sawicki et al discloses bisphosphonate compounds for chelating uranium (VI) via the phosphonic acid groups. See Sawicki et al, Eur. J. Med. Chem. , 43: 2768-2777, 2008.

A number of attempts have been made in the prior art to improve the properties of BPs and these have included the synthesis of bifunctional reagents in which the functional groups for targeting bone and for chelating radionuclides are distinct.

These attempts have included the synthesis of ¹⁸⁵Re- mercaptoacetylglycylglycylglycine (Rhenium-186-MAG3-BP) complex- conjugated bisphosphonate, [ [ [ [ (4 -hydroxy- 4, 4 diphosphonobutyl ) carbamoylmethyl] carbamoylmethyl] carbamoylmethyl] carbamoylmethanethiolate] oxo rhenium(V) (186Re-MAG3-HBP) . See Ogawa et al . , Bioconjug. Chem., 16, 751-757, 2005. Rhenium- 186 -monoaminemonoamidedithiol- conjugated bisphosphonate (¹⁸⁶Re-MAMA-HB) derivatives have also been made and tested for use in bone pain palliation. See Ogawa et al., Nucl . Med. Biol., 33, 513-520, 2006. The properties of these compounds are further investigated in Uehara et al . , Nuclear Medicine and Biology 34, 79-87, 2007.

The synthesis of ^99mTc-mercaptoacetylglycylglycylglycine (MAG3)- conjugated hydroxy-bisphosphonate (HBP) (^99mTc-MAG3-HBP) and a ^99mTc-6-hydrazinopyridine-3-carboxylic acid (HYNIC) -conjugated hydroxy-bisphosphonate (⁹⁹¹¹¹TC-HYNIC-HBP) for use in imaging is disclosed in Ogawa et al. (J. Nucl. Med., 47, 2042-2047, 2006) .

The synthesis of bisphosphonate conjugates with diethylenetriaminepentaacetic acid DTPA-BP 1 and 5-fluorouracil 2 and the clinically used bone imaging agent methylenediphosphonate 3 (Tc-99m-DTPA-BP and Re-188-DTPA-BP) is set out in El-Mabhouh et al., Cancer Biother. Radiopharm. , 19, 627-640, 2004.

El-Mabhouh and Mercer have prepared conjugates of diethylenetriaminepentaacetic acid and bisphosphonate (DTPA/BP) and a conjugate between 5-fluorouracil and bisphosphonate (5- FU/BP) and labelled them with ¹⁸⁸Re. See El-Mabhouh and Mercer, Appl. Radiat. Isot., 62, 541-549, 2005. However, the prior art bifunctional BP compounds suffer from one or more disadvantages. In the case of ¹⁸⁸Re-DTPA-BP, the chelator (DTPA) does not form stable complexes with Tc/Re, leading to poor in vivo properties. The production of the other bifunctional compounds mentioned above (¹⁸⁶Re-MAG3-HBP, ⁹⁹Tc -MAG3 -HBP, ¹⁸⁶Re- MAMA-BP) requires multi-step syntheses with moderate to low yields, leading to additional cost and making them unsuitable especially at large, industrial scales.

Accordingly, it remains a problem in the art to develop improved bisphosphonate reagents for the diagnosis and therapy of bone disorders .

Summary of the Invention

Broadly, the present invention is based on the realisation that the ability of prior art bisphosphonates to complex to hydroxyapatite is compromised by chelation to metal isotopes in the compositions used in the prior art, in particular arising from the observation that BPs are excellent bone- seeking agents but poor Tc/Re chelators . Accordingly, the present invention concerns the rational design of new compounds that comprise separate bisphosphonate and metal chelating moieties joined by a linker. The chemistry used to make the compounds and complexes of the present invention helps to avoid complicated multi-step syntheses used to make the prior art bifunctional bisphosphonate compounds and provides a synthesis of the compounds that can be carried out in one- step from commercially available compounds using environmentally- friendly conditions. The compounds of the present invention may form single and well-defined stable species with radionuclides such as Technetium- 99m, Rhenium-186 and/or Rhenium- 188, and Cu- 64 and may show improved in vitro and in vivo properties compared to current clinically-approved Tc/Re/Cu-BPs . In preferred embodiments, the present invention provides compounds in which the bisphosphonate moiety is capable of targeting bone without interference from the radionuclide complex and/or in which the co-ordination mode of the metal, as well as its kinetic and thermodynamic stability, is capable of being controlled according to the chelator core used.

Accordingly, in a first aspect, the present invention provides a compound for targeting bone and chelating a radionuclide represented by Formula I :

wherein:

R₁ is hydrogen or hydroxyl ; n is an integer between 1 and 6; and

R₂ is -(CH₂)_O-R₄, where o is 1, 2 or 3; and R₃ is - (CH₂) _p-R₅, where p is 1, 2, or 3; where R₄ and R₅ are independently selected from:

a) a sp2 hybridised heteroaryl group comprising a nitrogen, oxygen or sulphur heteroatom, typically in the ortho position relative to the covalent bond to the R₂ or R₃ group; or

b) a -NR₆R₇ group, wherein R₆ and R₇ are independently hydrogen or an optionally substituted C_1-4 alkyl group; or

R₂ is hydrogen or an optionally substituted C₁.₄alkyl group ; and R₃ is -C (S^~ ) =S or -C (O) - (CHSH) ₂-CO₂H ;

or

-NR₂R₃ together form a heterocyclic ring group represented by the formula:

and stereoisomers, salts, solvates, chemically protected forms, or prodrugs thereof; wherein the R₂ and R₃ substituents form a complex with a chelatable radionuclide and the bisphosphonate groups are for targeting bone .

Preferably, n is an integer selected from I₇ 2, 3, 4 or 5, and more preferably is 2, 3, 4 or 5. Alternatively or additionally, preferably o and p are 1 or 2.

In embodiments of the present invention in which one or both of R₄ and R₅ are heteroaryl groups, preferably they are 5 or 6 membered heteroaryl groups. It is further preferred that a heteroaryl group has a single heteroatom, generally nitrogen. The examples provided below illustrate preferred examples of compounds according to the present invention in which R₄ and R₅ are both heteroaryl groups, and preferably are both pyridyl . In further embodiments of the present invention, one or both of R₂ and R₃ are -CH₂-CH₂-NH₂. It is generally preferred that R₂ is hydrogen, methyl or ethyl .

In a further aspect, the compounds of the present invention may be employed to form complexes. Accordingly, the present invention provides a complex formed between a compound as described herein and a chelatable radionuclide. Suitable chelatable radionuclides are discussed further below. In some embodiments, the complex is formed between a chelated radionuclide represented by the formula [R*(CO)₃]⁺ or [*R(CO) ₂ (NO) ] ²⁺, wherein *R is a radionuclide, and a bisphosphonate compound as described herein.

In a further aspect, the present invention provides a process for producing a compound of the present invention, which comprises the steps of contacting the aminobisphosphonate and a precursor of the chelating moiety or the chelating moiety in water and maintaining the pH of the solution between pH 10.0 and 12.5 using a base, thereby to produce the compound; and optionally isolating and/or purifying the compound.

In this aspect of the present invention, a one -step reaction is provided for making the compounds of the present invention. Generally, the use of an inorganic base such as sodium hydroxide is preferred. Preferably the bisphosphonate is pamidronate, alendronate or neridronate.

In a further aspect, the present invention provides compounds or complexes as described herein for use in therapy or diagnosis. By way of example, the compounds and complexes of the present invention may be used for the treatment of a bone disorder and for the treatment of a cancer. The treatment of cancer may involve palliative and/or therapeutic treatment. Preferred examples of cancers treatable according to the present invention include breast cancer, lung cancer, prostate cancer, myeloma, the treatment of primary bone cancer, such as osteosarcoma, melanoma, ovarian cancer, thyroid cancer, kidney cancer and head and neck cancer. The present invention is particularly applicable to the treatment of bone metastases, for example resulting from any of the types of cancer mentioned above.

In a further aspect, the present invention provides a pharmaceutical composition comprising a compound or complex as described herein in combination with the pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a kit comprising a compound as described herein and optionally a chelatable radionuclide and/or instructions for preparing a complex between the compound and the radionuclide. In a further aspect, the present invention provides the use of a compound or complex as described herein in the preparation of a medicament for the treatment or diagnosis of a bone disorders or cancer.

In a further aspect, the present invention provides a method of treating a subject having cancer or a bone disorder, the method comprising administering to the subject a therapeutically effective amount of a compound or complex of the present invention.

In a further aspect, the present invention provides a method of imaging bone, and especially bone metastases, present in a subject, the method comprising administering to the subject a therapeutically effective amount of a complex of the present invention and detecting the radiation produced by the radionuclide chelated in complex.

In a further aspect, the present invention provides a process for purifying [^18ε/188Re (CO) ₃ (H₂O) ₃] ⁺. In one embodiment, the invention provides a process for producing [^186/188Re (CO) ₃ (H₂O) ₃] ⁺ which comprises reacting ^18S/188Re0₄ ^', CO and H₃PO₄ and purifying the [^18S/188Re (CO)₃ (H₂O)₃] ⁺ using ionic chromatography to separate the compound from unreduced and/or re-oxidised ^18S/188Re0₄ ^" and colloidal ^186/188ReO₂. Preferably, the reaction is carried out between about 50 ⁰C and 70 ⁰C (e.g. at about 60 ⁰C) for about 10 to 20 (preferably about 15) minutes. This process is described in the examples for producing fac- [^186/188Re (CO) ₃ (H₂O) ₃] ⁺ that is then complexed with a compound of the present invention. However, the process has wider applicability. In a preferred embodiment, the ionic chromatography comprises a OnGuard II Ag column (Dionex) to remove chloride ions from the saline solution, followed by a strong-anion exchange (SAX) column (SAX Varian Bond Elut) to retain ^186/188ReO₄ ^" .

Embodiments of the present invention will now be further described by way of example and not limitation with reference to the accompanying figures. Brief Description of the Figures

Figure 1 shows the results of an in vitro calcium salt binding study to compare the binding of ^99mTc (CO) ₃ (DPA-alendronate) (black bars) and prior art ^99mTc-MDP (methylene diphosphonate) (grey- bars) to different calcium salts (hydroxyapatite (HA) ; beta- tricalcium phosphate (beta-triCP) ; calcium phosphate dibasic (CPdibasic) ; calcium oxalate (CO) ; calcium carbonate (CC) and calcium pyrophosphate (CPy) ) .

Figure 2 shows the results of a serum stability study in which ^99mTc (CO) 3 (DPA-alendronate) was incubated in human serum and compared with the prior art ^99mTc-MDP (methylene diphosphonate) .

Figure 3 shows the results of an in vivo imaging studies were performed in Balb/C female mice using a nanoSPECT/CT scanner after injection of 50 MBq of ^99mTc-DPA-alendronate in 200 μL. High uptake in bone is evident, especially in joints and spine.

Figure 4 shows ¹H- and ³¹P-NMR titration studies of DPA- alendronate upon increasing amounts (from top to bottom) of [Re (CO) ₃] ⁺ .

Figure 5 shows a SPECT/CT image taken 24 h post-injection showing the high uptake of 3 in bone tissue, particularly at the joints. From left to right, maximum intensity projection (M), sagittal (S), coronal (C) and transverse (T) sections.

Figure 6 shows Uptake in the left knee (decay-corrected) after injection of 3 (33 MBq, black circles, continuous line) or ¹⁸⁸Re- HEDP (29 MBq, grey squares, dashed line) obtained from ROI analysis of the imaging data. The data from 3 were scaled by a factor of 29/33 to take into account the different injected activity. Values represent the mean ± SD (n = 3 mice) . * indicates a significant difference (P < 0.05, Student's paired t- test) between the two radiotracers.

Figure 7 show a biodistribution profile of 3 (black bars) and ¹⁸⁸Re-HEDP (grey bars) at t = 48 h post-injection. Values represent the mean + SD {n = 3 mice) .

Figure 8 shows (A) TLC chromatograms of ⁶⁴Cu(OAc)₂ (top) and ^e4Cu- DTBP (bottom) ; (B) Photographs of TLC plates spotted with Cu-DTBP (a, b, c) or Cu (d) . (a) Visible light (b) UV light (254 nm) (c) Visible light (Dittmers stain) (d) Free Cu.

Figure 9 show the results of an in vitro calcium salt binding study of ⁶⁴Cu-DTBP with hydroxyapatite (HA) , /3-tricalcium phosphate (b-triCP) , calcium oxalate (CO) , calcium phosphate dibasic (CP) , and calcium pyrophosphate (CPy) in 50 mM TRIS pH 6.9

Detailed Description

Compounds

The compounds described in the present application may be represented by Formula I :

wherein:

R₁ is hydrogen or hydroxyl ; n is an integer between 1 and 6; and

R₂ is -(CH₂) _C)-R₄, where o is 1, 2 or 3; and R₃ is -(CH₂)p-R₅, where p is 1, 2 or 3; where R₄ and R₅ are independently selected from:

a) a sp2 hybridised heteroaryl group comprising a nitrogen, oxygen or sulphur heteroatom, typically present in the ortho position relative to the covalent bond to the R₂ or R₃ group ; or

b) a -NR₆R₇ group, wherein R₆ and R₇ are independently hydrogen or an optionally substituted Ci_₄ alkyl group; or

R₂ is hydrogen or an optionally substituted Ci_₄alkyl group; and R₃ is -C(S^")=S or -C(O) - (CHSH)₂-CO₂H;

or

-NR₂R₃ together form a heterocyclic ring group represented by the formula:

and stereoisomers, salts, solvates, chemically protected forms or prodrugs thereof .

Preferred examples of the compounds may be represented by general Formula Ia-Id, as set out below. Formula Ia:

Formula Ib:

Formula Ic :

Formula Id:

wherein:

R₁ is hydrogen or hydroxyl; when present, R₂ is hydrogen, methyl or ethyl; and n is an integer between 1 and 6, and preferably 2, 3, 4 or 5 ; and stereoisomers, salts, solvates, chemically protected forms, and prodrugs thereof .

The compounds of the present invention include isomers, salts, solvates, and chemically protected forms thereof, as explained in more detail below.

In the present invention, alkyl groups are generally C_x-4 alkyl groups. The term "Ci_-4 alkyl", as used herein, includes a monovalent moiety obtained by removing a hydrogen atom from a Ci_-4 hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated. In preferred embodiments, the C_1-4 alkyl group is a methyl or an ethyl group as shorter chain alkyl groups tend to make the compounds of the present invention less hydrophobic.

In the present invention, a "heteroaryl group" is generally a C_5-12 heteroaryl group, and is preferably a 5 or 6 membered heteroaryl group and as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a C_3-12 heterocyclic compound. The present invention provides example of compounds in which one or more pyridyl groups (e.g. one or more 2-pyridyl groups) are present. However, examples of heteroaryl compounds that could be employed in accordance with the present invention inc1ude :

Imidazole: a five membered aromatic ring having two nitrogen atoms and three carbon atom.

Triazole: a five membered aromatic ring having three nitrogen atoms and two carbon atoms, with two ring isomers 1, 2,3 , triazole, 1,2,4 triazole.

Tetrazole: a five membered aromatic ring having four nitrogen atoms and one carbon atom.

Pyridine: a six membered aromatic ring having one nitrogen atom and 5 carbon atoms .

Diazine: a six membered aromatic ring having two nitrogen atoms and four carbon atoms, with three ring isomers, 1,2 -diazine, 1,3- diazine and 1,4-diazine.

Triazine: a six membered aromatic ring having three nitrogen atoms and three carbon atoms, with three ring isomers, 1,2,3- triazine, 1, 2 , 4-triazine and 1, 3 , 5-triazine .

Tetrazine: a six membered aromatic ring having four nitrogen atoms and two carbon atoms, with three ring isomers 1,2,3,4- tetrazine, 1, 2 , 3 , 5-tetrazine and 1, 2,4, 5-tetrazine.

Fused ring systems such as quinoline, isoquinoline and indole.

It is generally preferred that the sp2 nitrogen containing heterocyclic group has a donor nitrogen atom in the ortho position relative to the methylene bridge of the bisphosphonate compound in order to facilitate chelation of the radionuclide by the heteroatom. A preferred heteroatom is nitrogen, i.e. providing pyridyl heteroaryl groups .

In some embodiments, the compounds of the present invention are substituted with one or more functional groups . Preferred examples of suitable functional groups include:

Halo: -F, -Cl, -Br, and -I.

Hydroxy: -OH.

Ether: -OR, wherein R is an ether substituent, for example, a Ci_₄ alkyl group (also referred to as a Ci_-4 alkoxy group) , and preferably where the ether group is methoxy or ethoxy.

Other Forms of the Substituents

Included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid ( -COOH) also includes the anionic (carboxylate) form (-C00^"), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N⁺HR¹R²) , a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0^" ), a salt or solvate thereof, as well as conventional protected forms of a hydroxyl group.

Isomers, Salts, Solvates, Protected Forms, and Prodrugs Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; C-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L- forms; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate- forms; syn- and anti-forms; synclinal- and anticlinal- forms; oi- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms") .

Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space) . For example, a reference to a methoxy group, -OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl . However, a reference to a class of structures or to a general formula includes structurally isomeric forms falling within that class or formula and, except where specifically stated or indicated, all possible conformations and configurations of the compound (s) herein are intended to be included in the general formula (e) .

The above exclusion does not pertain to tautomeric forms, for example, keto- , enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below) , imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro .

keto enol enolate

Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D) , and ³H (T) ; C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; 0 may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al. , J. Pharm. Sci. , 66, 1-19 (1977) .

For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO^") , then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺) . Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dieyelohexylamine, triethylamine , butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃J₄ ⁺.

If the compound is cationic, or has a functional group which may be cationic (e.g., -NH₂ may be -NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: acetic, propionic, succinic, glycolic, stearic, palmitic, lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric, phenylsulfonic, toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic, and gluconic. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono- hydrate, a di -hydrate, a tri -hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle the active compound in a chemically protected form. The term "chemically protected form", as used herein, includes a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that. is, are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group) . By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, 'Protective Groups in Organic Synthesis' (T. Green and P. Wuts, Wiley, 1999) . For example, a hydroxy group may be protected as an ether (-0R) or an ester (-OC(=O)R), for example, as: a t -butyl ether; a benzyl, benzhydryl (diphenylmethyl) , or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (-OCC=O)CH₃, -OAc) .

For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (>C=0) is converted to a diether (>C(OR)₂) , by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amide or a urethane, for example, as: a methyl amide (-NHCO-CH₃) ; a benzyloxy amide (-NHCO-OCH₂C_SH_S, -NH-Cbz) ; as a t-butoxy amide

(-NHCO-OC(CH₃) ₃, -NH-Boc) ; a 2-biphenyl-2-propoxy amide (-NHC0- OC (CH₃) ₂C₆H₄C₆H₅, -NH-Bpoc) , as a 9-fluorenylmethoxy amide (-NH- Fmoc) , as a 6-nitroveratryloxy amide (-NH-Nvoc) , as a 2- trimethylsilylethyloxy amide (-NH-Teoc) , as a 2,2,2- trichloroethyloxy amide (-NH-Troc) , as an allyloxy amide

(-NH-Alloc) , as a 2 (-phenylsulphonyl) ethyloxy amide (-NH-Psec) ; or, in sμitable cases, as an N-oxide (>N0$) .

For example, a carboxylic acid group may be protected as an ester for example, as: an Ci_-7 alkyl ester (e.g. a methyl ester,- a t- butyl ester) ; a C_1-7 haloalkyl ester (e.g., a Ci_-7 trihaloalkyl ester) ; a triCa.._? alkylsilyl-Cχ_₇ alkyl ester; or a C_5-20 aryl-C_1-7 alkyl ester (e.g. a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide.

It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term "prodrug" , as used herein, includes a compound which, when metabolised (e.g. in vivo) , yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.

For example, some prodrugs are esters of the active compound

(e.g. a physiologically acceptable metabolically labile ester) . During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Examples of such metabolically labile esters include those wherein R is C_1-7 alkyl

(e.g. -Me, -Et) ; C_1-7 aminoalkyl (e.g. aminoethyl; 2- (N, N- diethylamino) ethyl; 2- (4-morpholino) ethyl) ; and acyloxy- C_1-7 alkyl

(e.g. acyloxymethyl ; acyloxyethyl ; e.g. pivaloyloxymethyl ; acetoxymethyl ; 1-acetoxyethyl; 1- (1-methoxy-l -methyl) ethyl - carbonxyloxyethyl ; 1- (benzoyloxy) ethyl; isopropoxy- carbonyloxymethyl ; 1-isopropoxy-carbonyloxyethyl; cyclohexyl- carbonyloxymethyl ; 1 - cyclohexyl - carbonyloxyethyl ; cyclohexyloxy- carbonyloxymethyl ; 1-cyclohexyloxy-carbonyloxyethyl; (4- tetrahydropyranyloxy) carbonyloxymethyl ; 1- (4- tetrahydropyranyloxy) carbonyloxyethyl ;

(4-tetrahydropyranyl ) carbonyloxymethyl ; and 1- (4-tetrahydropyranyl) carbonyloxyethyl) .

Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Complexes of the Compounds and Their Uses

The compounds of the present invention may be used for therapy, in particular the treatment of bone disorders or cancer in an analogous manner to the prior art compounds described in the introduction above. In addition, the compounds of the present invention may be used to chelate radionuclides, for example to enable them to be employed in imaging studies or for therapeutic purposes. Examples of radionuclides that are chelatable by the compounds of the present invention include technetium, rhenium and copper isotopes such as ^99mTc, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁶⁰Cu, ⁶¹Cu₇ ⁶²Cu, ⁶⁷Cu. The present invention may employ the radionuclides alone or in combinations. For example, one commonly used combination is ^18e/188Re. In general, technetium isotopes are employed for imaging purposes, rhenium isotopes for therapeutic purposes and copper isotopes for both imaging and therapy.

The results presented in the example demonstrate that complexes of the present invention have superior binding to hydroxyapatite than the prior art compound ""¹Tc-MDP and have significantly improved serum stability, and additionally can be made without recourse to the complex multi-step syntheses used in the prior art.

The present invention provides active compounds for use in a method of treatment of the human or animal body. Such a method may comprise administering to such a subject a therapeutically- effective amount of an active compound, preferably in the form of a pharmaceutical composition.

The term "treatment", as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, relief of pain, and cure of the condition. Treatment as a preventative measure, i.e. prophylaxis, is also included. By way of example, the compounds and complexes of the present invention may be used for the treatment of bone disorders and for the treatment of cancer. The treatment of cancer may involve palliative and/or therapeutic treatment . Preferred examples of cancers treatable according to the present invention include breast cancer, lung cancer, prostate cancer, myeloma, melanoma, ovarian cancer, thyroid cancer, kidney cancer, head and neck cancer, and the treatment of primary bone cancer, such as osteosarcoma. The present invention is particularly applicable to the treatment of bone metastases, for example resulting from any of the types of cancer mentioned above . In one embodiment , the treatment of breast cancer using the compounds and complexes of the present invention may be based on the fact that hydroxyapatite and calcium oxalate calcifications are often present in malignant and benign breast tumours, respectively. In view of the results of the calcium salt binding studies disclosed herein, it is clear that ""¹TC-MDP is unable to distinguish between the two calcification, whereas compounds of the present invention are capable of selectively detecting HA-containing malignant breast tumours with higher sensitivity. This is turn provides specificity in the application of the compounds of the present invention for the treatment of malignant breast tumours .

The term "therapeutically-effective amount" as used herein, includes that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.

Formulations and Dosage

While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) comprising at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein. The term "pharmaceutically acceptable" as used herein includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, 'Remington's Pharmaceutical Sciences', 18th edition, Mack Publishing Company, Easton, Pa., 1990.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution or suspension which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient . The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects .

Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

Examples

Materials and Methods

General. Reagents were obtained from commercial sources and used as received unless otherwise noted. Pamidronate, alendronate and neridronate were synthesized following a reported procedure (see

US7411087) . NMR spectra were obtained using a Bruker Avance 400 at 20⁰C in D₂O (Cambridge Isotope Laboratories) unless otherwise noted. ¹H chemical shifts are referenced with respect to the residual solvent peak (δ_H 4.79 ppm) . ³¹P resonances were referenced to an external solution of 85% H₃PO₄ (δ_P 0 ppm) . 13/- chemical shifts were left unreferenced. High-resolution mass spectra were obtained at the EPSRC National Mass Spectrometry Service Centre at Swansea University using a Thermo Scientific LTQ Orbitrap XL spectrometer coupled to an Advion TriVersa NanoMate using nanoelectrospray ionization. Elemental analyses were carried out at the Elemental Analysis Service at London Metropolitan University. Reverse phase (RP) and size exclusion (SE) HPLC analyses were carried out using an Agilent 1200 series system equipped with a quadruple pump, a UV detector set at 254 nm (RP) or 280 nm (SE) and a radiodetector (Lablogic) optimized for the detection of y-photons . For RP studies, Agilent Zorbax Eclipse XDB-C18 columns (5 μm, 4.6 x 150 mm (Analytical) ; 5 μm, 21.2 x 150 mm (Preparative)) were used, whereas a TOSOH TSK-GEL G2500PW_XL was used for SE chromatography. [""¹TcO₄]Na and "¹¹¹Tc-MDP were obtained from the Radiopharmacy at Guy's and St Thomas' Hospital NHS Trust, London.

RP-HPLC Methods:

The following methods were employed using the stated conditions.

Method A: Solvent A: H₂O 0.1% TFA; Solvent B: CH₃CN 0.1% TFA.

Time Flow rate % A % B

1 mL/min (analytical)

0 100 0 5 mL/min (preparative)

1 mL/min (analytical)

3 min 100 0 5 mL/min (preparative)

1 mL/min (analytical)

16 min 70 30 5 mL/min (preparative)

1 mL/min (analytical)

18 min 100 0 5 mL/min (preparative)

1 mL/min (analytical)

20 min 100 0 5 mL/min (preparative)

Method B: Solvent A: 0.05 M TEAP (Triethylammonium phosphate pH=2.25) ; Solvent B: MeOH.

Method C: Solvent A: H₂O 0.1% TFA; Solvent B: CH₃CN 0.1% TFA.

Time Flow rate % A % B

0 2 mL/min 100 0

20 min 2 mL/min 50 50

24 mm 2 mL/min 50 50

25 min 2 mL/min 100 0

30 min 2 mL/min 100 0

General Synthesis

DPA-Pamidrona te

To a suspension of pamidronate (400 mg, 1.43 mmol) in 12 mL of distilled water was added NaOH (228 mg, 5.72 mmol) and the mixture stirred at room temperature until all pamidronate had dissolved. At this point, 2-picolyl chloride-HCl (472 mg, 2.86 mmol) was added, which resulted in the solution turning slightly orange. The pH of the reaction mixture was 12.2. After 16 h stirring at room temperature the pH was 10.6 and was increased back to 12.2 by adding more NaOH (56 mg, 1.43 mmol) . The mixture was left stirring at room temperature for a further 24 h, after which the pH had lowered to 11.25. At this point, the reaction mixture was washed with dichloromethane (3 x 12 mL) and the organic washings discarded. The water layer was evaporated to dryness to yield the crude product as a salmon pink solid (827 mg) . DPA-Pamidronate -Na₂ was purified as needed as the disodium salt by dissolving in 0.1 % TFA in water and adjusting its pH to 8 and purifying using preparative RP-HPLC using method A (Rt = 06:59 mm: ss ) . ¹H NMR (D₂O, 100 mM Sodium carbonate-bicarbonate buffer pH 9, 400.1 MHz, 298 K) : δ_H (ppm) ; 8.36 (d, J = 4.9 Hz, 2H, Py-H) , 7.70 (td, J = 7.6, 1.5 Hz, 2H, Py-H) , 7.37 (d, J = 7.7 Hz, 2H, Py-H), 7.27 (t, J = 6.2 Hz, 2H, Py-H), 3.95 (s, 4H, Py-CH₂-N) , 3.19 (br, 2H, -N-CH₂-CH₂-) , 2.34 (b, 2H, -N-CH₂-CH₂-) ; ³¹P(¹Hj NMR (D₂O, 100 mM Sodium carbonate -bicarbonate buffer pH 9, 161.9 MHz, 298 K) : <5p (ppm) ; 18.77. ESI-MS (- ion) : found: 207.6 (100%) , calc. 207.5 (100%) for [DPA-Pamidronate]^2". Found: C, 38.89; H, 4.10; N, 9.01. C₁₅H₁₉N₃Na₂O₇P₂ requires : C, 39.06; H, 4.15; N, 9.11.

DPA-Alendrona te

DPA-Alendronate was synthesized following the same procedure as for DPA-Pamidronate -Na₂ to yield 811 mg (based on a 1.43 mM scale) of a salmon pink solid. The pure product was purified as needed as the TFA salt by using preparative RP-HPLC using method A (Rt = 06:28 mm:ss) . ¹H NMR (D₂O, 400.1 MHz, 298 K) : δ_H (ppm) ; 8.69 (d, J = 5.2 Hz, 2H, Py-H) , 8.47 (t, J = 7.9 Hz, 2H, Py-H), 7.99 (d, J = 8.0 Hz, 2H, Py-H), 7.90 (t, J = 6.5 Hz, 2H, Py-H), 4.24 (s, 4H, py-CH₂-N) , 2.65 (br, 2H, -N-CH₂-CH₂-) , 1.82 (br, 4H, -CH₂-CH₂-CH₂- ); ¹³C NMR (D₂O, 100.6 MHz, 298 K) : δ_c (ppm) ; 152.5 [2C₁ Py), 147.06 (2CH, Py) , 141.45 (2CH, Py) , 127.23 (2CH, Py) , 126.21 (2CH, Py) , 116.23 (q, ¹Jc_-1. = 291 Hz, CF₃-COOH) , 72.97 (t, ¹Jc_-P = 140 Hz, CH₂-C(PO₃) ₂ (OH) ) , 55.51 (C, PyCH₂-) , 55.07 (C, PyCH₂) , 38.67 (-CH₂-) , 30.92 (-CH₂-) , 20.64 ( -CH₂-C (PO₃) ₂ (OH) ) ; ³¹P(¹H) NMR (D₂O, 161.9 MHz, 298 K) : δ_P (ppm); 19.09. HR-ESI-MS (- ion) : found: 430.0931 (100 %) , calc. 430.0938 for [DPA-alendronate] ^", and matches theoretical isotope distribution. Found: C, 36.42; H, 3.52; N, 6.30. C₂₀H₂₃F₆N₃O₁₁P₂ requires : C, 36.54; H, 3.53; N, 6.39. DPA~Neridrona te

DPA-Alendronate was synthesized following the same procedure as for DPA-Pamidronate and DPA-Alendronate to yield 1.040 g (based on a 1.67 mM scale) of a salmon pink solid. The pure product was purified as needed as the TFA salt by using preparative RP-HPLC using method A (Rt = 08:31 mm:ss) . ¹H NMR (D₂O, 400.1 MHz, 298 K) : δ_H (ppm) ; 8.63 (d, J = 4.5 Hz, 2H, Py-H) , 8.38 (t, J = 7.8 Hz, 2H, Py-H) , 7.91 (d, J = 8.0 Hz , 2H, Py-H) , 7.82 (t, J = 6.5 Hz , 2H, Py-H) , 4.19 (s, 4H, Py-Ci_-2-N), 2.57 (t, J = 7.3 Hz, 2H, -N-CH₂- CH₂-) , 1.78 (br, 2H, -N-CH₂-CH₂-CH₂-) , 1.39 (br, 4H, -N- (CH₂) ₂-CH₂- CH₂-) , 1.12 (t, 2H, -N-(CH₂)₄-CH₂-) ; ¹³C NMR (D₂O, 100.6 MHz, 298 K) : δ_c (ppm) ; 162.8 (q, ²Jc-_F= 35 Hz, CF₃-COOH) , 152.5 (2C, Py), 146.8 (2CH, Py) , 141.6 (2CH, Py) , 127.0 (2CH, Py) , 126.2 (2CH, Py), 116.25 (q, ¹Jc_-F = 291 Hz, CF₃-COOH) , 73.37 (t, ¹J₀-_P = 143 Hz, - CH₂-C(POs)₂(OH)) , 55.48 (C, PyCH₂), 54.55 (C, N-CH₂-), 33.28(-CH₂- ) , 26.80 (-CH₂-) , 24.79 (-CH₂-), 22.77 ( -CH₂-C (PO₃) ₂ (OH) ); ³¹P(¹H) NMR (D₂O, 161.9 MHz, 298 K) : δ_P (ppm) ; 19.48.

Re (CO) 3-dipycolylamine-alendronate

Re (CO) ₃-Dpa-Alendronate was synthesized in an NMR tube by mixing equimolar amounts of a 2.54 mM solution of DPA-alendronate in 0.5 mL of 100 mM carbonate buffer pH 9 in D₂O with [Re(CO)₃(H₂O)₃I ⁺ from a stock solution in D₂O and heating for 30 minutes at 90 ⁰C. To confirm the formation of Re (CO) ₃-Dpa-Alendronate, the crude reaction was monitored by RP-HPLC to show a single peak with Rt 12:58 mm: ss (method B)

¹H NMR (D₂O, 400.1 MHz, 298 K) : δ_H (ppm) ; 8.84 (d, J^" = 5.2 Hz, 2H, Py-H) , 7.84 (t, J = 7.8 Hz, 2H, Py-H), 7.46 (d, J = 8.0 Hz, 2H, Py-H), 7.46 (d, J = 7.9 Hz, 2H, Py-H) , 7.26 (t, J = 6.5 Hz , 2H, Py-H), 4.24 (s, 4H, py-CH₂-N) , 2.65 (br, 2H, -N-CH₂-CH₂-), 1.82 (br, 4H, -CH₂-CH₂-CH₂-) ; ³¹P(¹H) NMR (D₂O, 161.9 MHz, 298 K) : δ_P (ppm) ; 18.20; HR-ESI-MS (- ion) : found: 701.0325 (100 %) , calc. 701.0310 for Ci₉H₂₂N₃Oi₀P₂Re and matches theoretical isotope distribution.

Dithiocarbamate-pamidronate

Pamidronate (0.400 g) was dissolved in 25 mL of H₂O. The pH of this solution was adjusted to 12 by adding NaOH while stirring at 15⁰C. At this point, 2.5 mL of CS₂ were added. The reaction solution was left stirring vigorously at room temperature for two hours before adding more NaOH to bring back the pH back to 12. This last step was repeated after 4, 36 and 6O h. At this point, the remaining CS₂ layer was removed using a syringe (~ 0.5 mL) and any traces of dissolved CS₂ were evaporated using a rotary evaporator. To the yellow aqueous solution was added EtOH until a white precipitate appeared. This mixture was left at 4 "C for 48 h, when a transparent oil had precipitated. The oil separated from the aqueous layer and evaporated to yield 300 mg of the product as a semi-crystalline powder. ¹H NMR (D₂O, 400.1 MHz, 298 K) : δ_H (ppm) ; 3.73 (t, J = 7.7 Hz , 2H, NH-CB₂-) , 2.13 (m, , 2H, - CH₂-CH₂-), ¹³C NMR (D₂O, 100.6 MHz, 298 K) : δ_c (ppm) ; 209.1 (1C, S₂C-CH₂-) , 74.48 (t, ¹Jc_-P = 140 Hz, -CH₂-C(PO₃) ₂ (OH) ) , 44.96 (CH₂), 32.30 (CH₂); ³¹P(¹Hj NMR (D₂O, 161.9 MHz, 298 K) : δ_P (ppm) ; 18.13. ESI-MS (- ion) : found: 309.9 (100 %) , calc. 309.9 for [Dithiocarbamate-Pamidronate] ^"

Re-Dimercaptosuccinic acid (DMSA) -Pamidronate

100 mg (0.1 mmol) of [ReO (dimercaptosuccinic acid anhydride) ₂] NBu₄ were dissolved in 2 mL of acetonitrile. To this solution was added a mixture of pamidronate (146 mg, 0.4 mmol) and triethylamine (400 microL) in 2 mL of water. After 3 h stirring at room temperature the solvents were evaporated to yield the crude product as an orange crystalline solid. RP-HPLC analysis shows two products that elute at 05:30 mm:ss (64 %) and 07:02 mm: ss (28 %) as well as the hydrolysed starting material at 08:44 mm: ss (3 %) . The products were separated from starting materials using semi -preparative RP-HPLC using method C and analysed by IR spectroscopy. IR (KBr, cm^"1) : 960 (Re=O) , 1065-1286 (P=O_stretc_h) / 1535 (amide) , 1673 (sec. amide), 1693 (carboxylic acid), 3000- 3700 (caboxylic acid and sec. amide) .

Example 1

Radiolabelling of DPA-alendronate with ^99mTc

[Tc^99ra (CO) ₃ (H₂O)₃J⁺ was synthesized from an Isolink™ kit (Mallinckrodt Medical B. V.) as described in the manufacturer's instructions. Briefly, 1 mL of [Tc⁹⁹¹¹O₄]^" (100 MBq) was added to the kit and the vial heated at 100 degrees for 20 minutes. After cooling to room temperature, the contents were neutralized with 120 μL of IM HCl and analysed by HPLC to confirm the purity of the newly formed [Tc^99m (CO) ₃ (H₂O) ₃] ⁺ . To establish the minimum concentration at which DPA-alendronate could be efficiently labelled with [Tc^99m (CO) ₃ (H₂O) ₃] ⁺, five different solutions of different concentrations of DPA-alendronate were prepared (1.7x10^{" 3} M, 1.7xlO^"5 M, 1.7xlO^"7 M and 1.7xlO^~9 M) in 100 mM carbonate buffer at pH 9. An aliquot (100 μL) of each solution was mixed with 100 μL of [Tc^99m (CO) ₃ (H₂O) ₃] ⁺ (15 MBq) , before being heated for 30 minutes at 100⁰C. After cooling the solutions in a water bath, each sample was analyzed by analytical RP-HPLC. Very efficient labelling (radiochemical yield > 98 %) was found when concentrations higher than 1.7xlO^"5 M were used and no labelling when the concentration was lower than 1.7xlO^"7M. Using HPLC method B, Tc^99m(CO) ₃ (DPA-alendronate) elutes at 13:22 mm:ss, whereas [Tc^99m (CO) ₃ (H₂O) ₃] ⁺ and [Tc⁹⁹¹¹¹O₄]^" elute at 02.55 mm:ss and 08:00 mm: ss, respectively.

Jn vitro calcium salt binding studies

In order to assess the binding of Tc^99m (CO) ₃ (DPA-alendronate) and Tc⁹⁹¹¹¹MDP to different calcium salts, 1 mg/mL suspensions of hydroxyapatite, beta-tricalcium phosphate, calcium phosphate dibasic, calcium oxalate, calcium carbonate and calcium pyrophosphate in 50 mM TRIS pH 6.9 were prepared. Immediately after, 10 μL (0.8 MBq) of either Tc^99m (CO) ₃ (DPA-alendronate) or Tc^99τnMDP were added to each suspension and vortexed for 1 h at room temperature. The solutions were then centrifuged (5 min, 10.000 rpm) and a 100 μL aliquot of the supernatant of each sample analyzed using a gamma counter. A control sample lacking calcium salt was also prepared and counted as a standard for 0 % binding. The results are expressed as % binding using the following equation:

where CPM₃ are the counts per minute of each sample and CPM_C are the counts per minute of the control . The results are shown in Figure 1.

Serum Stability and Binding Studies

Human serum samples (1 mL) were incubated with 200 μL (14 MBq) of ""¹TC(CO)₃ (DPA-alendronate) in a 5% C0₂/95% air atmosphere at 37 ⁰C for 24 h. Aliquots (20 μL) were taken at 0 , 3, 6 and 18 hours after mixing and analysed by SE-HPLC using a flow of 1 mL/min of PBS buffer as eluent. Serum proteins eluted at 5 minutes, whereas ^99mTc (CO) ₃ (DPA-alendronate) eluted at 10 minutes. The percentages were obtained by measuring the area under the peaks for both species in the gamma chromatogram. In the case of ^99mτc- MDP, human serum samples (1 mL) were incubated with 200 μL (14 MBq) ""¹Tc-MDP in a 5% CO₂/95% air atmosphere at 37⁰C for 24 h. Aliquots (50 μL) were taken at 0, 3, 6 and 18 hours after mixing and the proteins of each timepoint were precipitated by addition of EtOH (70 μL) . The samples were centrifuged and the supernatant separated. The precipitated proteins were washed with another 70 μL of EtOH. Supernatant and precipitated proteins were counted using a gamma counter. The results are shown schematically in Figure 2.

In order to assess the serum stability of the binding of "^mTc (CO) ₃ (DPA-alendronate) to the calcium salt, an identical experiment was set in the presence of 10 mg of HA. In this case, at each time point, 50 μL were withdrawn after centrifugation of the solution and analyzed using a gamma counter. After the aliquot was taken, the HA pellet was re- suspended by gently pipetting up and down. ⁹⁹¹¹¹Tc (CO) ₃ (DPA-alendronate) remained bound to HA in serum for at least 18 h.

To assess the chemical stability of ^99mTc (CO) ₃ (DPA-alendronate) , the serum samples were centrifuged after 18 h using a Vivaspin filter with a molecular cut-off of 5000. The entire radioactivity transferred to the filtrate and was analysed by analytical RP- HPLC (Method B) to confirm that ^99mTc (CO) ₃ (DPA-alendronate) had not decomposed.

Discussion

The design of the compounds of the present invention was based on two concepts. Firstly, the BP part of the molecule was separated from the chelator by a spacer, in order to help to avoid BP-metal interactions . Secondly, the need for the radionuclide to selectively coordinate into the chelating group and remain stable and chemically inert under in vivo conditions was addressed. The organometallic precursor fac- [M (CO) ₃ (H₂O) ₃] ⁺ (M = Tc, Re) , pioneered by Jaouen et al . and developed by Alberto et al . facilitates the latter requirement. In particular, when the three labile water molecules are displaced by an appropriate ligand system, the d^s low-spin octahedral Tc (I) /Re (I) centre formed is fully protected from oxidation and ligand substitution. Furthermore, imaging probes containing a coordinatively saturated fa c- [M(CO) ₃] ⁺ core have shown high in vivo stability and negligible binding to human serum proteins, both of which are highly desirable properties of any targeted agent. Particularly favourable ligands for this metal core are N3-tridentate chelators containing two sp² N-heterocycles, hence our choice of dipicolylamine (DPA) as our chelating group. As the targeting vector we selected alendronate, a clinically-approved BP that binds very avidly to hydroxyapatite (HA) , the main component of bone mineral . In addition, alendronate provides an amino group separated from the BP group by a spacer, allowing the facile conjugation in a single step of two picolyl units to form a DPA unit for coordination of fac- [M (CO) ₃] ⁺ .

Two major obstacles were encountered during the development of DPA-ale. First was the extreme insolubility of alendronate in organic solvents which complicates any conjugation reaction. Second is the high basicity of its amino group (pKa 12.7), which inhibits nucleophilic attack of alendronate using standard organic bases. Taking all these problems into account we developed a set of conditions that allow the reaction to occur. Important factors are pH and the concentration of base. Using strong organic bases such as triethylamine was unsuccessful . High concentrations of inorganic bases and high temperatures, however, had to be avoided as hydrolysis of 2-picolyl chloride and rearrangements of the bisphosphonate may occur. We found that using water as solvent and maintaining the pH of the solution at 12.0 with the minimum amount of NaOH was sufficient to drive the reaction to completion after 36 hours at room temperature. Under these conditions, no hydrolysis nor bisphosphonate rearrangements were detected. The yield of the reaction was found to be higher than 90% as assessed by RP-HPLC analysis.

The complexation of DPA-alendronate with fac- [Re (CO) ₃ (H₂O) ₃] ⁺ and its solution properties were examined using HPLC and NMR and MS spectroscopy. The aim was to evaluate if the organometallic core selectively coordinated the chelating DPA group. NMR/HPLC titration studies revealed that fac- [Re (CO) ₃ (H₂O) ₃] ⁺ stoichiometrically binds DPA-alendronate in the designed facial conformation when less than 1.5 equivalents were used. The presence of a single species in solution was also confirmed by ¹HZ³¹P-NMR and HPLC, see Figure 4. HR-ESI-MS also demonstrates the formation of the desired product. When higher amounts of metal were added, however, new ³¹P-NMR signals as well as the general upfield shift of the aromatic protons of the ¹H-NMR spectrum strongly suggest coordination of more metal centres to the BP group. These putative multinuclear compounds, however, are extremely unlikely to form during radiosynthesis as the concentration of ligand always exceeds that of the radionuclide by several orders of magnitude.

DPA-alendronate in concentrations as low as 10^"5 M can be efficiently labelled (> 98% radiochemical yield) with fac- [⁹⁹¹¹¹Tc (CO)₃] ⁺ in water. RP-HPLC analysis show that fac- [""¹Tc(CO)₃] -DPA-alendronate and fac- [Re (CO) ₃] -DPA-alendronate coeluted, consistent with their analogous structure.

One of the factors that makes alendronate one of the most potent BPs is its high skeletal uptake and retention, which is directly related to its affinity towards HA (Nancollas et al . , Bone, 38, 617-627, 2006). The affinity and selectivity of fac- [^99mTc (CO) ₃] -DPA-alendronate and ""¹Tc-MDP towards several calcium salts were evaluated using an in vitro assay. Fac- [^99mTc (CO) ₃] - DPA-alendronate is selective and has a very high affinity (> 80 %) towards HA, see Figure 1. ^99mTc-MDP, on the other hand, is not selective and binds HA and calcium oxalate (CO) with comparable affinity (~ 40 %) . Remarkably, the compound of the present invention shows higher affinity for HA despite having a concentration of free (non-labelled) BP 10 times higher than that of free MDP in ⁹⁹¹¹¹Tc-MDP.

The fate of a targeted imaging probe or radiopharmaceutical once in the blood stream is one of the most important factors to consider. It is well stablished that strong binding to serum proteins such as albumins results in slow blood clearance and hence low target-to-background ratios. Interestingly, the exemplified compound of the present invention showed negligible binding to serum proteins after incubation with human plasma for at least 18 h. ^99mTc-MDP, on the other hand, remains mostly bound to serum proteins over 16 h.

In vivo imaging studies were performed in Balb/C female mice using a nanoSPECT/CT scanner. After injection of fac-

[^99mTc (CO) ₃] -DPA-alendronate, scans were taken at 1, 2 and 6 h. The results show almost quantitative and rapid uptake of the tracer in bone tissue. In order to compare with the current gold standard, control imaging studies were also performed with ^99mTc-MDP. Thus, fac- [⁹⁹¹¹¹Tc (CO)₃] -DPA-alendronate shows identical bone uptake compared to ^99mTc-MDP, and highlights its usefulness as a bone-seeking tracer. Biodistribution studies were also performed post-mortem to assess the uptake of the two tracers in different internal organs. Bone uptake of both compounds was almost identical (-33% ID/g for the femurs) . Soft tissue uptake, while still very low, was slightly higher with fac-

[""¹Tc(CO)₃] -DPA-alendronate (-6% ID/g), than with ""¹Tc-MDP (-2% ID/g) . This is fully consistent with the more lipophilic nature of DPA-ale and the tricarbonyl core. The negligible uptake in the stomach provides further evidence of the in vivo chemical stability of fac- [^99mTc (CO) ₃] -DPA-alendronate . These results are shown in Figure 3. Thus, these results demonstrate the high potential of DPA-alendronate as an improved agent for the treatment of bone metastases.

Example 2

Radiolabelling of DPA-alendronate with ¹⁸⁸Re

Synthesis of ¹⁸⁸Re-hydroxyethylidene-l,l-diphosphonate (¹⁸⁸Re-HEDP)

¹⁸⁸Re-HEDP was synthesised and characterised according to the reported method (Lewington, J. Nucl. Med., 46: 38s-47s, 2005) .. Labelling kits containing a mixture of the lyophilised reagents

(8.3 mg of 1-hydroxy ethylidene-1, 1-diphosphonic acid (HEDP), 3.0 mg of gentisic acid and 3.9 mg of SnCl₂-H₂O) were prepared in- house. To these kits were added a 1 mL solution containing ¹⁸⁸ReO₄ ^"

(200 MBq) and 1 μmol of HReO₄, followed by heating at 100 °C for 15 min. After cooling, a 1 mL buffer solution containing 39 mg of sodium acetate and 10 μL of a 32% NaOH solution was added to increase the pH to 6. This solution was diluted at least twofold with saline and filtered with a sterile 0.22 μm filter for animal studies. Quality control was achieved using ITLC-SG strips developed with acetone and saline. In acetone, ¹⁸⁸Re-HEDP and ¹⁸⁸ReO₂ colloids have a R_F = 0 whereas ¹⁸⁸ReO₄ ^" has a R_F = 1. In saline, ¹⁸⁸Re-HEDP and ¹⁸⁸ReO₄ ^" have a R_F = 1 whereas ¹⁸⁸ReO₂ colloids have a R_F = 0. Radiochemical yields were >97%.

Synthesis and purification of [¹⁸⁸Re(CO)₃ (H₂O)₃]⁴ (1)

1 was synthesised and characterised according to the published method (Lewington, Eur. J. Cancer 27, 954-958, 1991) . A rubber- sealed 10 mL serum vial containing 3 mg of BH₃-NH₃ was slowly flushed with CO for 10 min inside a fumehood. 1 mL of ¹⁸⁸ReO₄ ^" (up to 4 GBq) in saline was then mixed with 6 μL of 85% H₃PO₄ using a 20 mL syringe. This mixture was then injected into the sealed vial . The syringe and needle were left inserted into the vial in order to allow the volume expansion due to the generation of approximately 10 mL of H₂. The vial (with the syringe in place) was then heated at 60 ⁰C for 15 tnin. and cooled to room temperature afterwards. For analysis, 2 μL of the crude solution were spotted into a glass-backed silica gel TLC plate, dried under a gentle stream of N₂ and developed with 99% MeOH/1% HCl_COnc- Using this system, "¹⁸⁸ReO₂" colloids appear at R_F = 0.05, 1 appears as two broad peaks with R_F = 0.20-0.50 and ¹⁸⁸ReO₄ ^" appears at R_F = 0.90) . Radiochemical yield of 1 varied between 80-85%.

In order to separate 1 from the by-products, the crude solution was passed at a flow of 5 mL/min through a system composed, in this order, of a cation exchange column in the silver form (OnGuard II Ag Dionex, 1 cm³) followed by a strong-anion exchange (SAX) column (SAX Varian Bond Elut, 100 mg) and a hydrophilic 13 mm PTFE 0.22 μm filter (Millipore Millex IC) . Both columns had been previously conditioned according to the manufacturers' instructions followed by drying by passing air. Furthermore, as a preventive measure, the OnGuard II Ag column was shielded from light with aluminium foil in order to prevent oxidation of 1 that might occur due to photoreduction of the precipitated AgCl salt . After the 1 mL solution was passed through the system, the two columns and the filter were washed with 100 μL of water to obtain a 1.1 mL eluate containing 1 in 65% radiochemical yield

(calculated from initial ¹⁸⁸ReO₄ ^" activity) and > 99% radiochemical purity.

Synthesis and characterisation of ¹⁸⁸Re (CO) ₃-dipicolylamine- alendronate (3)

To establish the minimum concentration at which 2 could be efficiently labelled, four solutions of different concentrations of 2 in PBS buffer (1 mg/mL, 1.7xlO^"3 M; 0.1 mg/mL, 1.7X10^"4 M; 0.01 mg/mL, 1.7xlO^"5 M and IxIO^"4 mg/mL, 1.7xlO^"7 M) were tested. 3 was synthesised by mixing a 100 μL solution of freshly-made 1 (150 MBq) and 100 μL of the solution of 2 into a N₂-purged rubber- sealed vial that contained 2 mg of a powdered Ix PBS tablet followed by heating at 75 ⁰C for 30 min. After this time the vial was cooled to room temperature. Very efficient labelling (up to 18.8 GBq/mg, radiochemical yield ≥ 95%) was achieved when the concentration of 2 was 1.7xlO^"4 M or higher. Lower concentrations resulted in lower radiochemical yields of ≤ 94% (1.7XlO^"5 M) or < 60% (1.7xlO^~7 M) . Using RP-HPLC, 3 elutes with a retention time (t_R) of 13:12 mm:ss (γ//T detection) . Characterisation was achieved by comparison with the t_R of 1^85/187Re (CO) ₃-dipicolylamine-alendronate using the same RP-HPLC method (13:14 mm:ss, IJV detection) . For TLC analysis, 2 μL of the solution were spotted into a glass-backed silica gel TLC plate, dried under a gentle stream of N₂ and developed with 99% MeOH/1% HCl_conc. Using this system, 3 appears at R_F = 0.1. For in vivo studies, the solution of 3 was diluted with saline as needed and sterilised prior to i.v. injection with a 0.2 μm sterile syringe filter.

Stability and serum protein binding in vitro studies

Studies in PBS. To assess the in vitro stability (stability towards oxidation to ¹⁸⁸ReO₄ ^") of 3 and ¹⁸⁸Re-HEDP, 10 μL solutions of these compounds (5 MBq of 3; 3.5 MBq of ¹⁸⁸Re-HEDP) were incubated in PBS (500 μL) in a 5% CO₂/95% air atmosphere at 37 ⁰C and constant shaking for 48 h. Aliquots (2 μL, ¹⁸⁸Re-HEDP; 20 μL, 3) were taken at 1, 24 and 48 h after mixing and analysed by ITLC (¹⁸⁸Re-HEDP) using acetone as the mobile phase or RP-HPLC (3) .

Studies in human serum. Human serum samples (500 μL) were incubated with 10 μL (5 MBq) of 3 in a 5% CO₂/95% air atmosphere and constant shaking at 37 ⁰C for 48 h. Aliquots (20 μL) were taken at 1, 24 and 48 h after mixing and analysed by SE-HPLC using a flow of 1 mL/min of PBS as eluent . Serum proteins eluted at 5 minutes, whereas 3 eluted at 10 minutes. The proportions in each component were obtained by measuring the area under the peaks for both species in the gamma chromatogram. ¹⁸⁸Re-HEDP eluted with similar t_R as serum proteins even in the absence of serum (consistent with its polymeric nature) ruling out the use of SE-HPLC as a method of analysis. An established alternative method for measuring serum binding was used. Thus, human serum samples (500 μL) were incubated with 10 μL (3.5 MBq) of ¹⁸⁸Re-HEDP in a 5% CO₂/95% air atmosphere and constant shaking at 37 ⁰C for 48 h. Aliquots (50 μL) were taken at 1, 24 and 48 hours and the proteins precipitated by addition of EtOH (70 μL) . The samples were centrifuged and the supernatant separated. The precipitated proteins were washed twice with 70 μL of EtOH. The radioactivity of supernatant and precipitated proteins was counted using a gamma counter .

To assess the fate of 3 and ¹⁸⁸Re-HEDP after incubation in serum, the samples were centrifuged after 48 h using a Vivaspin filter with a molecular cut-off of 5000 Da. For 3, the entire radioactivity eluted from the filter, confirming it does not bind to serum proteins. Furthermore, RP-HPLC of the filtrate confirmed that 3 had not oxidised to ¹⁸⁸ReO₄ ^" . In the case of ¹⁸⁸Re-HEDP, 20% of the activity (protein-bound ¹⁸⁸Re-HEDP and/or intact ¹⁸⁸Re-HEDP) remained in the filter. ITLC analysis of the filtrate (remaining 80% of the radioactivity) demonstrated the activity was solely due to ¹⁸⁸ReO₄ ^", consistent with the results from the PBS studies .

Imaging studies

Adult female BALB/c mice were injected i.v. in the tail vein with 33 MBq of 3 in 200 μL [n = 3) or 29 MBq of ¹⁸⁸Re-HEDP in 200 μL (n = 3) . At 1, 5 and 24 and 48 hour time -points, the animals were anaesthetised using isoflurane and imaged using a NanoSPECT/CT animal scanner (Bioscan Inc.) . Whole-body SPECT images were obtained in 20 projections over 45 min using a 4-head scanner with 4 x 9 (2 mm) pinhole collimators in helical scanning mode and CT images with a 45 kVP X-ray source, 1000 ms exposure time in 180 projections over 7.5 min. Images were reconstructed in a 256 x 256 matrix using HiSPECT (Scivis GmbH) , a reconstruction software package, and images were fused using proprietary Bioscan InVivoScope (IVS) software. Quantification of the images to obtain the uptake at the knee was performed by locating the left knee as the region of interest (ROI) using the quantification tool in the Bioscan InVivoScope (IVS) software. The instrument had been previously pre-calibrated using a syringe of known ¹⁸⁸Re activity. Significant differences (P < 0.05, Student's paired t- test) between the two radiotracers were found for t = 24 h (P = 0.020} and t = 48 h (P = 0.033) .

Biodistribution studies

Biodistribution studies were carried out in accordance with British Home Office regulations governing animal experimentation. The mice from the imaging studies were used for the biodistribution studies. After 48 hours, the mice were culled by cervical dislocation and the following organs were dissected: femur, pancreas, kidneys, heart, stomach, spleen, intestine, liver, lung, muscle, tail, thyroid and a sample of blood. Each sample was weighed and counted with a gamma counter (LKB compugamma) , together with standards prepared from a sample of the injected material. The percent of injected dose per gram of tissue was calculated for each tissue type.

Results

The synthesis, characterisation and preclinical in vivo studies of its ¹⁸⁸Re complex, ¹⁸⁸Re (CO) ₃-DPA- alendronate (3, Scheme 1), in comparison with ¹⁸⁸Re-HEDP to evaluate its potential as a new improved radiopharmaceutical for the therapy of bone metastases.

3 was made from generator-eluted ReO₄ ^" in two steps (Scheme 1) . The precursor fac- [¹⁸⁸Re (CO) ₃ (H₂O) ₃] ⁺ (1, Scheme 1) was synthesised following the method of Schibli et al (supra) . The radiochemical yields of 1 ranged between 80-85%, in agreement with the published method, with the remaining by-products being unreduced and/or re-oxidised ¹⁸⁸ReO₄ ^" and colloidal "¹⁸⁸ReO₂". A new purification method was required since none was described in the original report . The inventors deduced that ionic chromatography could be used to separate the two by-products based on their ionic and colloidal character. Thus, the crude solution was passed through a system composed of two solid-phase extraction columns connected in series, an OnGuard II Ag column (Dionex) to remove chloride ions from the saline solution followed by a strong-anion exchange (SAX) column (SAX Varian Bond Elut 100 mg) to retain ¹⁸⁸ReO₄ ^". Using this system, 1 is obtained in the eluate in good radiochemical yields (65%, based on initial ¹⁸⁸ReO₄ ^" activity) and excellent purities (> 99%) . The OnGuard II Ag column was used to remove the chloride ions from the saline solution that otherwise compete with ¹⁸⁸ReO₄ ^" in the SAX column, at the expense of 1 being retained to some extent in the OnGuard II Ag column (10%) .

3 was synthesised by mixing a solution of freshly-made 1 (100 μL, 150 MBq) with 2 (0.01 mg/mL in PBS, 100 μL) in a N₂-purged vial followed by heating at 75 ⁰C for 30 min. RP-HPLC analysis of the reaction solution revealed the formation of 3 with a specific activity of 18.8 GBq/mg and ≥ 96% radiochemical yield with the remainder of the activity being ¹⁸⁸ReO₄ ^". In contrast to [⁹⁹¹¹¹Tc(CO)₃ (OHa)₃] ⁺, re-oxidation of 1 to ¹⁸⁸ReO₄ ^" during labelling conditions has been observed previously with other ligands and can be rationalised to be the result of the lower redox potential of Re compared to that of Tc. Longer reaction times (up to 60 min) and lower reaction temperatures (60 ⁰C) led to lower yields of 3. A comparison with the chromatogram of the well- characterised non-radioactive Re (CO) ₃-DPA-alendronate complex, and its ^99mTc analogue, demonstrates the formation of the desired compound as a single species.

In order to achieve the maximum therapeutic efficiency, ¹⁸⁸Re compounds must remain stable and bound to the target during at least one to three half -lives of ¹⁸⁸Re (16.9 h) . One of the most important drawbacks of ¹⁸⁸Re-HEDP is its lack of stability both in vivo and in vitro. In order to assess the in vitro stability of 3 in comparison with ¹⁸⁸Re-HEDP, both compounds were incubated in PBS for 48 h at 37 ⁰C. RP-HPLC and TLC analyses demonstrated that 3 did not degrade over this time, whereas most of ¹⁸⁸Re-HEDP oxidised to ¹⁸⁸ReO₄ ^" (up to 75%) . Incubation of both compounds in human serum show most of the radioactivity from the ¹⁸⁸Re-HEDP sample remains bound to serum proteins during the first 24 h. After this time, ~70% of the radioactivity was free in solution. ITLC analyses demonstrated, however, that the non-protein bound radioactivity was ¹⁸⁸ReO₄ ^", the decomposition product of ¹⁸⁸Re-HEDP. 3, on the other hand, remained non-protein bound and unmodified throughout the 48 h incubation period.

In vivo imaging studies with 3 and ¹⁸⁸Re-HEDP were carried out at 1, 5, 24 and 48 h post-injection with adult BALB/c female mice using a nanoSPECT/CT scanner (Figure 5) . These studies confirmed the ability of 3 to accumulate in areas of metabolically-active bone such as the joints, whilst soft- tissue organ uptake was very low throughout the experiment. Quantification of the images provided an interesting comparison of the pharmacokinetics of each compound (Figure 6) . Thus, both compounds show an increase in uptake in the knee for the first 5 hours. After this time, however, the uptake of 3 increased during the next 24 h, whereas that of ¹⁸⁸Re-HEDP diminished until the end of the 48 h experiment . We propose the increased uptake in bone of 3 during the first 24 h is the result of recycling of the unmetabolised, chemically intact complex from soft tissues, coupled with its excellent retention and slow release from bone compared to ¹⁸⁸Re- HEDP. This is in agreement with the biodistribution profiles at 48 h (vide infra) as well as with previous in vitro experiments with its ^99mτc analogue, demonstrating the superior capabilities of Re/Tc-DPA-alendronate for binding, and remaining bound, to the main component of bone mineral (hydroxyapatite) .

Ex-vivo biodistribution studies at 48 h demonstrate that 3 exhibits higher uptake in bone tissue than ¹⁸⁸Re-HEDP (percentage of injected dose per gram of tissue (% ID/g) in one whole femur: 21.2 + 6.6% for 3; c.f. 13.4 ± 0.2% for ¹⁸⁸Re-HEDP) , consistent with its higher stability and/or better targeting properties (Figure 7) . As shown in the above-mentioned imaging studies, soft -tissue uptake was very low for both compounds with most organs having an uptake of less than 0.6% ID/g. 3 consistently shows higher uptake than ¹⁸⁸Re-HEDP in these organs, especially in the liver (0.96 ± 0.2% ID/g) . This may be explained by the lipophilic nature of the tricarbonyl core. An interesting exception, however, is the lower uptake of 3 in the thyroid. We attribute this to the tendency of ¹⁸⁸Re-HEDP, but not 3, to decompose into ¹⁸⁸ReO₄ ^", which is known to be taken up by sodium- iodide symporter (NIS) -expressing organs such as the thyroid.

Discussion

This example describes a simple and convenient method to purify 1 that will facilitate the labelling of other small molecules and biomolecules such as His-tagged peptides/proteins with ¹⁸⁸Re in high radiochemical yields and purities. The synthesis of 3 as a new radiopharmaceutical for the radionuclide therapy of bone metastases is also described. 3 can be easily synthesised with high specific activities in two steps using kit-based methodology and, in contrast with the clinicalIy-approved ^18S/188Re-HEDP, it forms an inert, single species that has been well characterised. The strategy of using a designed chelating agent for rhenium rather than relying on the chelating properties of the bisphosphonate group is vindicated in that 3 displays superior stability, bone targeting and retention properties. 3 is therefore an attractive candidate for further clinical studies.

Example 3

Synthesis and in vitro evaluation of ⁶⁴Cu-DTBP as a PET agent for imaging calcified tissues

Synthesis of ⁶⁴Cu-DTBP

Synthesis

Methyl 3- (benzyl (methyl) amino) propanoate

Methyl 3 -bromopropanoate (5.00 g, 29.9 mmol) and N-methyl-1- benzylamine (3.63 g, 29.9 mmol) were dissolved in acetonitrile (200 mli) . After 2 min. stirring at room temperature, Na₂CO₃ (31 g, 299 mmol) was added and the temperature increased to 70 ⁰C for 70 h. After cooling, the reaction solution was filtered, and to the filtrate were added 100 mL of 2M NaOH. The product was extracted with 3 x 100 mL of CH₂Cl₂ and the extracts dried over Na₂SO₄. After filtration, the solvents were evaporated under vacuum to yield the product as a colourless oil (5.89 g, 95 %) . ¹H-NMR (CDCl₃, 400.3 MHz, 298 K) <5_H (ppm) 7.34 (m, 5H, C₆H₅ -CH₂ -N) , 3.70 (s, 3H, -COOCH₃) , 3.54 (s, 2H, C₆H₅-CH₂-N) , 2.77 (t, J = 7.6 Hz, 2H, -N-CH₂-CH₂-) , 2.55 (t, J = 7.6 Hz, 2H, -CH₂-COOCH₃) , 2.23 (s, 3H, -N-CH₃) ; ¹³C-NMR (CDCl₃, 100.7 MHz, 298 K) δ_c (ppm) 173.03 (-COOCH₃) , 138.88, 128.91, 128.20 and 127.03 (C₆H₅-CH₂-) , 62.11 (C₆H₅-CH₂-) , 52.74 (-N-CH₂-CH₂-) , 51.56 (-COOCH₃) , 41.91 (-N-CH₃) , 32.75 (-N-CH₂-CH₂-) . Found: C, 69.57; H, 8.18; N, 6.63. Ci₂H₁₇NO₂ requires: C, 69.54; H, 8.27; N, 6.76. MS (ESI) 208.1339 (M+H⁺, found), 208.1338 (M+H⁺, calculated)

3- (Methylamino) propanoic acid hydrochloride

A Schlenk tube was charged with methyl 3-

(benzyl (methyl) amino) propanoate (2 g, 9.64 mmol) and dissolved in EtOH (60 mL) . To this solution were added 500 mg of 10% Pd/C in small portions . The atmosphere of the tube was purged first with oxygen-free N₂ followed by H₂. A double-layer latex balloon filled with H₂ ( ~ 1 L) was then attached to the Schlenk tube and the reaction was left stirring at room temperature for 48 h, during which the balloon was refilled as needed with H₂. At this point, the flask was opened and Celite^® added to the reaction mixture followed by filtration with Celite . The filtrate was evaporated to dryness to yield a yellowish oil. ¹H- and ¹³C-NMR confirmed complete amine deprotection. However, it was also noted that transesterification had occurred to some extent:

The crude product was used for the next step without modification. Thus, the mixture of methyl and ethyl esters was dissolved in 40 mL of 5 M HCl and refluxed for 16 h. The solvent was then evaporated under vacuum to yield the product as a hygroscopic white solid (0.969 g, 72 %) . ¹H-NMR (D₂O, 400.3 MHz, 298 K) δ_H (ppm) 3.33 (t, J = 6.4 Hz, 2H, -N-CIi₂-CH₂-), 2.85 (t, J = 6.4 Hz, 2H, -CH₂-COOH) , 2.76 (s, 3H, -N-CH₃) ; ¹³C-NMR (D₂O, 100.7 MHZ, 298 K) S_c (ppm) 174.32 (- COOH) , 44.56 (-NH-CH₂-) , 33.08 (- CH₂-CH₂-), 30.05 (-NH-CH₃) . Found: C, 34.37; H, 7.35; N, 9.88. C₄H₁₀ClNO₂ requires: C, 34.42; H, 7.22; N, 10.03.

l-Hydroxy-3- (methylamino) propane-1 , 1-diphosphonic acid hydrochloride

3- (Methylamino) propanoic acid hydrochloride (0.969 g, 6.9 mmol) and phosphorous acid (0.849 g, 10.4 mmol) were suspended in 3.5 mL of sulfolane and heated to 75 °C for 30 min, resulting in the complete dissolution of both reagents. The mixture was then cooled to 35 ⁰C and PCl₃ (3.22 g, 23.4 mmol), added dropwise over 5 min. followed by heating at 67 °C for 3 h. At this point, the reaction solution was cooled to 0 °C and 10 mL of H₂O were added dropwise over 5 mins . Charcoal was added to the flask and the mixture was then refluxed for 1 h. , filtered through Celite⁸ and cooled to 0 ⁰C. To this solution was added EtOH until a white precipitate appeared. After 12 h standing at 4 ⁰C, an oily- residue had separated. The residue was washed with ethanol and recrystallised from EtOH/H₂O at 4 ⁰C to yield the pure product as a white solid (781 mg, 39%) . ¹H-NMR (D₂O, 400.3 MHz, 298 K) δ_H (ppm) 3.39 (t, J = 6.6 Hz, 2H, -N-CiJ₂-CH₂-), 2.73 (s, 3H, -N-CH₃), 2.85 (m, 2H, -CH₂-C (OH) (PO₃H₂) ₂) ; ¹³C-NMR (D₂O, 100.7 MHz, 298 K) δ_c (ppm) 72.16 (t, ¹Jc_-P = 140 Hz, -C(OH) (PO₃H₂) ₂) , 45.59 (-NH-CH₂-) , 32.93 (-NH-CH₃) , 29.40 (-CH₂-CH₂-); ³¹P(¹HJ NMR (D₂O, 162.1 MHz, 298 K) : δ_P (ppm) ; 17.47. Found: C, 16.91; H, 4.95; N, 4.82. C₄H₁₄ClNO₇P₂ requires: C, 16.82; H, 4.94; N, 4.91. HR-ESI-MS: 248.0103 ([M-H] ^", found) , 248.0094 ([M-H] ^", calculated) .

Pentasodium mono (3-hydroxy-3, 3- diphosphonatopropyl (methyl) dithiocarbamate) (DTBP) l-Hydroxy-3- (methylamino) propane-1, 1-diphosphonic acid hydrochloride (70 mg, 0.25 mmol) was suspended in 10 mL THF. To this suspension was added NaOH (69 mg, 1.75 mmol, 7 eq) dissolved in 0.3 mL H₂O, followed by 0.7 mL of H₂O. The clear solution had a pH of 12. The solution was cooled to 0 ⁰C and CS₂ (373 mg, 4.9 mmol, 19.6 eq) , dissolved in 1 mL of THF, was slowly added over 5 min. The reaction solution became cloudy. After 24 h stirring at room temperature the pH had dropped to 10 and the colour had changed from colourless to faint yellow. At this point, the excess CS₂ and THF were evaporated using a rotary evaporator and 20 mL of acetone were added to the mixture, resulting in the separation of an oil after storage at 4 ⁰C overnight. The oil was isolated by decantation of the supernatant, washed with 5 mL of acetone, dissolved in H₂O and lyophilised to yield the product as a hygroscopic white crystalline powder (120 mg, 77 %) . ¹H-NMR (D₂O, 400.3 MHz, 298 K) δ_H (ppm) 4.34 (t, J = 7.7 Hz, 2H, -CH₂-N- CS₂) , 3.49 (s, 3H, -N-CH₃) , 2.24 (m, 2H, -CH₂-C (OH) (PO₃) ₂) ; ¹³C-NMR (D₂O, 100.7 MHz, 298 K) δ_c (ppm) 206.2 (-CS₂) , 74.85 (t, ¹Jc-_P = 140 Hz, -C(OH) (PO₃Ha)₂), 53.93 (-N-CH₂-), 42.98 (-N-CH₃) , 31.92 (-CH₂- CH₂-) ; ³¹P(¹Hj NMR (D₂O, 162.1 MHz, 298 K) : δ_P (ppm) ; 18.03.

Found: C, 9.63; H, 3.20; N, 1.96. (C₅H₈NNa₅O₇P₂S₂) • (NaCl) ^₅ (H₂O) ₁₀ requires: C, 9.66; H, 3.08; N, 2.25. MS (ESI) ; 161.2 ( [M-2H]^2", found) , 161.5 ( [M-2H]^2", calculated) ; 172.5 ( [M-3H+Na] ^2", found) , 172.5 ( [M-3H+Na]^2", calculated) ; 323.9 ( [M-H]^", found) , 324.0 ( [M- H]^", calculated) ; 346.0 ( [M-2H+Na] ^" found) , 345.9 ( ( [M-2H+Na] ^", calculated) ; 368.0 ( [M-3H+Na] ^" found) , 367.9 ( ( [M-3H+Na] ^", calculated) .

Radiosynthesis

To establish the minimum concentration at which DTBP could be efficiently labelled with ⁶⁴Cu, four solutions of different concentrations of DTBP were prepared (1.5xlO^~3 M, 1.5xlO^"4 M, 1.7xlO^"s M and 1.7xlO^"8 M) in 100 mM carbonate buffer at pH 9. An aliquot (100 μL) of each solution was mixed with 100 μL of a ⁶⁴Cu(OAc)₂ solution at pH > 7 (15-100 MBq) and left stirring for 5 minutes at room temperature. Each sample was then analysed by silica gel TLC using 15 mM EDTA in 10% ammonium acetate :MeOH (50:50) as the mobile phase. Using this system, free ⁶⁴Cu has an R_F = 0.6, whereas ⁶⁴Cu-DTBP has a R_F = 0 (See Fig 8A) . Very efficient labelling (10 GBq/mg, radiochemical yield >99 %) was found when concentrations equal or higher to 1.5xlO^"4 M were used. In order to prove the chemical identity of ⁶⁴Cu-DTBP, the nonradioactive version was synthesised and analysed using the same TLC method. Thus, Cu-DTBP stays at the baseline of the TLC plate, in agreement with its radioactive version (Fig 8B(A, B and C) . The spot can be seen by visible light due to its absorbance at 450 nm, characteristic of copper-dithiocarbamates and it is UV active at 254 nm, (Fig 8B(B)) . Furthermore, the spot becomes light green after staining the TLC plate with Dittmers reagent, as expected if the spot contain phosphorus atoms (Fig 8B(C)) . Free copper, on the other hand, moves to R_F = 0.6, as seen for ⁶⁴Cu.

To confirm that the ⁶⁴Cu does not bind to the DTBP ligand via the bisphosphonate groups, UV spectroscopy was used. The UV spectrum showed a characteristic peak for -CS₂-Cu-CS₂- complexes (absorbance at 450 nm) which increased with increasing amounts of Cu to the DTBP ligand. This continued until a 2:1 (DTBP : Cu) ratio was reached, proving that the Cu binds through the CS2 chelator and not the bisphosphonate.

Serum binding and stability studies

Human serum samples (0.5 mL, Sigma-Aldrich) were incubated with 50 μL (10 MBq) of ⁵⁴Cu-DTBP in a 5% CO₂/95% air atmosphere at 37 "C for 24 h. Aliquots (20 μL) were taken at 1, 17 and 24 hours after mixing and the proteins of each time point were precipitated by addition of EtOH (70 μL) . The samples were centrifuged and the supernatant separated. The precipitated proteins were washed (2 times) with another 70 μL of EtOH. The radioactivity of supernatant and precipitated proteins was counted using a gamma counter. ^β4Cu-DTBP appears completely bound to serum proteins during 24 h. The binding, however, seems to be weak and the association of ⁶⁴Cu with the DTBP is retained, as addition of just 0.5 mg of hydroxyapatite is enough to displace the complete radioactivity from the proteins in solution to the insoluble calcium salt. Stability studies were also performed in PBS. Thus, 50 μL (10 MBq) of ⁶⁴Cu-DTBP was added to a 1 mL solution and the mixture was shaked for 24 h at 37 ⁰C. At 1, 17 h and 24 h, samples were taken and analysed by silica gel TLC using 15 mM EDTA in 10% Ammonium acetate: MeOH (50:50) as the mobile phase. ⁶⁴Cu-DTBP remained stable for the first 17 h, after which new radioactive species are observed.

In vitro calcium salt binding studies

In order to assess the binding of ⁶⁴Cu-DTBP to different calcium salts, 0.5 mg/mL suspensions of hydroxyapatite (HA) , /3-tricalcium phosphate, calcium phosphate dibasic, calcium oxalate and calcium pyrophosphate in 50 mM TRIS pH 6.9 were prepared. Immediately after, 10 μL (1 MBq) of ⁶⁴Cu-DTBP were added to each suspension and vortexed for 1 h at room temperature. The solutions were then centrifuged (5 min, 10,000 rpm) and a 100 μL aliquot of the supernatant of each sample analyzed using a gamma counter. A control sample lacking calcium salt was also prepared and counted as a control. The results are expressed as % binding using the following equation: CPM.

% binding = 1- UlOO

CPM, where CPM₃ are the counts per minute of each sample and CPM_C are the counts per minute of the control .

The results are shown in Figure 9. ⁶⁴Cu-DTBP binds avidly to biologically-relevant calcium salts like hydroxyapatite, calcium oxalate and calcium phosphate.

***

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail may be made. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

References

Ogawa et al . , Bioconjug. Chem. , 16, 751-757, 2005.

Ogawa et al . , Nucl. Med. Biol., 33, 513-520, 2006;

Ogawa et al . , J. Nucl. Med., 47, 2042-2047, 2006.

El-Mabhouh et al . , Cancer Biother. Radiopharm. , 19, 627-640, 2004.

El-Mabhouh and Mercer, Appl . Radiat . Isot., 62, 541-549, 2005.

Uehara et al . , Nuclear Medicine and Biology 34, 79-87, 2007.

US Patent No: 7,411,087.

Nancollas et al . , Bone, 38, 617-627, 2006.

WO 88/00590.

US Patent No: 5,190,930

Yan et al, Nuclear Science and Techniques, 19(3) , 165-168, 2008.

Sawicki et al, Eur. J. Med. Chem., 43: 2768-2777, 2008.

Schibli et al . , Bioconjugate Chem. 13, 750-756, 2002.

Claims

Claims :

1. A compound for targeting bone and chelating a radionuclide represented by Formula I :

wherein:

R₁ is hydrogen or hydroxyl; n is an integer between 1 and 6; and

R₂ is -(CH₂)_O-R4/ where o is 1, 2 or 3; and R₃ is - (CH₂) p-R₅, where p is 1, 2, or 3; where R₄ and R₅ are independently selected from:

a) a sp2 hybridised heteroaryl group comprising a nitrogen, oxygen or sulphur heteroatom in the ortho position relative to the covalent bond to the R₂ or R₃ group

b) -NR₆R₇ group, wherein R₆ and R₇ are independently hydrogen or an optionally substituted Ci_-7 alkyl group; or or

R₂ is hydrogen or an optionally substituted C_halky1 group; and

R₃ is -C(S^")=S or -C(O)-(CHSH)₂-CO₂H;

or

-NR₂R₃ together form a heterocyclic ring group represented by the formula:

and stereoisomers, salts, solvates, chemically protected forms, or prodrugs thereof ; wherein the R₂ and R₃ substituents form a complex with a chelatable radionuclide and the bisphosphonate groups are for targeting bone.

2. The compound of claim 1 wherein n is 2, 3, 4 or 5.

3. The compound of claim 1 or claim 2 , wherein o and p are 1 or 2.

4. The compound of any one of the preceding claims, wherein the heteroaryl group is 5 or 6 membered.

5. The compound of any one of the preceding claims, wherein the heteroaryl group has a single heteroatom.

6. The compound of any one of the preceding claims, wherein the heteroatom of the heteroaryl group is nitrogen

7. The compound of any one of the preceding claims , wherein R₄ and R₅ are both heteroaryl groups .

8. The compound of any one of the preceding claims, wherein R₄ and R₅ are both pyridyl .

9. The compound of any one of claims 1 to 3 , wherein R₂ and R₃ are both -CH₂-CH₂-NH₂.

10. The compound of any one of the preceding claims, wherein R₂ is hydrogen, methyl or ethyl.

11. The compound of any one of the preceding claims which is represented by the Formula Ia:

or the compound which is represented by the Formula Ib:

or the compound which is represented by the Formula Ic;

or the compound which is represented by Formula Id:

wherein:

R₁ is hydrogen or hydroxy1 ; when present, R₂ is hydrogen, methyl or ethyl; and n is 2, 3, 4 or 5;

and stereoisomers, salts, solvates, chemically protected forms, or prodrugs thereof .

12. A complex formed between one or more of the compounds of any one of claims 1 to 11 and a chelatable radionuclide, wherein the complex is formed between the R₂ and R₃ substituents and the chelatable radionuclide.

13. The complex of claim 12, wherein the radionuclide is an isotope of technetium, rhenium or copper.

14. The complex of claim 12 or claim 13 , wherein the radionuclide is ^99mTc, ¹⁸⁶Re, ¹⁸⁸Re, ^S4Cu, ⁶⁰Cu, ⁵¹Cu, ⁶²Cu or ⁶⁷Cu, and combinations thereof.

15. The complex of any one of claims 12 to 14, wherein the complex is formed between a chelated radionuclide represented by the formula [R*(CO)₃]⁺ or [*R (CO) ₂ (NO) ] ²⁺, wherein *R is a radionuclide, and a compound of any one of claims 1 to 11.

16. A process for producing a compound according to any one of claims 1 to 11, wherein the process comprises contacting the aminobisphosphonate and a precursor of the chelating moiety or the chelating moiety in water and maintaining the pH of the solution between pH 10.0 and 12.5 using a base, thereby to produce the compound; and optionally isolating and/or purifying the compound.

17. The process of claim 16, wherein the base is an inorganic base such as sodium hydroxide .

18. The process of claim 17 or claim 18, wherein the bisphosphonate is pamidronate, alendronate or neridronate .

19. A compound or a complex of any one of claims 1 to 15 for use in therapy or diagnosis.

20. The compound or a complex for use in therapy or diagnosis according to claim 19, for the therapy or diagnosis of a bone disorder or a cancer.

21. The compound or a complex for use in therapy or diagnosis according to claim 20, for the therapy or diagnosis of (a) breast cancer, lung cancer, prostate cancer, myeloma, melanoma, ovarian cancer, thyroid cancer, kidney cancer or head and neck cancer, (b) primary bone cancer, such as osteosarcoma, or (c) bone metastases.

22. The compound or a complex for use in therapy or diagnosis according to claim 19, for use in imaging studies.

23. A pharmaceutical composition comprising a compound or complex of any one of claims 1 to 15 in combination with the pharmaceutically acceptable carrier.

24. A kit comprising a compound of any one of claims 1 to 11 and optionally a chelatable radionuclide.

25. Use of a compound or complex of any one of claims 1 to 15 in the preparation of a medicament for the treatment or diagnosis of a bone disorder or a cancer.

26. A process for producing [^18S/188Re (CO) ₃ (H₂O) ₃] ⁺ comprising reacting ^18S/188Re0₄ ^", CO and H₃PO₄ and purifying the

[^18S/188Re (CO) ₃ (H₂O) ₃] ⁺ using ionic chromatography to separate the compound from unreduced and/or re-oxidised ¹⁸⁶^¹⁸⁸ReO₄ ^" and colloidal ^18S/188Re0₂.

27. The process of claim 26, wherein the reaction is carried out at about 60 ⁰C for about 15 minutes.

28. The process of claim 26 or claim 27 for producing fac- [^186/188Re (CO) 3 (H₂O)₃I⁺.

29. The process of any one of claims 26 to 28, wherein the ionic chromatography comprises a OnGuard II Ag column (Dionex) to remove chloride ions from the saline solution, followed by a strong- anion exchange (SAX) column (SAX Varian Bond Elut) to retain i8_S/i88_Reθ4- _