AU780635B2 - Cryptate compounds and methods for diagnosis and therapy - Google Patents

Description

14. FEB. 2005 16:27 SPRUSON FERGUSON NO. 7044 P. 1 Cryptate Compounds and Methods for Diagnosis and Therapy Field of the invention The present invention relates to cryptate compounds useful as chelating agents. In particular, the present invention relates to functionalised derivatives of certain cryptate s compounds. These functionaliscd derivatives are suitable for use in radiolabelling and similar applications. The present invention also relates to a method for diagnosis or therapy of a disease utilising functionalised derivatives of cryptate compounds.

Background of the Invention Radiolabelled compounds are useful as radiopharmaceuticals, imaging agents, or to the like which are especially useful for but not limited to the diagnosis and therapy of diseases including cancer.

Known radiolabelled compounds suffer from the disadvantage that, in use, the radiolabelled nuclide can become detached from the carrier compound thereby leading to .problems and potential complications in diagnostic and therapeutic applications. Further, 15 the known radiopharmaceuticals tend to be non-specific in their biodistribution throughout a subject.

The present invention seeks to ameliorate the stated disadvantages of the prior art providing compounds which are capable of being radiolabelled more expeditiously, specifically target a localised area of tissue or an organ in a subject and which are more 20 stable than the prior art compounds and less toxic. Further, the compounds of the present invention are typically suitable for use in pharmaceutical formulations. It is a further typical object of the present invention to provide a method of diagnosis or therapy of disease in a subject.

Summary of the Invention According to a first embodiment of the present invention, there is provided a compound which is capable of chelating a metal ion or a metal ion radionuclide, of general Formula (CR4RS)n Y w w Y W )1W-X (CR4RS)n rRALBH103809.doc:NXL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:28 SPRUSON FERGUSON NO. 7044 P. 21 2 in which n represents an integer from 2 to 4, wherein each R 4 and R 5 is independently selected from H, CH 3 COOH, NO 2

CH

2 OH, H 2

PO

4

HSO

3 CN, C(-O)NH 2 and CHO; X and Y are the same or different and are selected from the group consisting of S C-R, N, P and C-Z in which R is selected from hydrogen, halogen, hydroxyl, nitro, nitroso, amino, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, cyano, -COOR', COCOOR', NH-COCH 2 Br, -NH-CO-CH=CH- COOR' in which R' is a hydrogen atom or alkyl group, wherein at least one of X and Y is C-z; W is selected from the group consisting of NH and S; Z is a functionalised group which is capable of binding said compound of Formula to a molecular recognition unit, selected from

CH

2

-NH-(CH

2 )p-Ar-NH2 and -NH-(CH 2 )p-Ar-NCS, wherein p is an integer from 1 to 4 and Ar is optionally substituted phenyl; I wherein said optional substituents are selected from the group consisting of amino, halogen, hydroxy, mercapto, nitro, cyano, thiocyano, alkyl, alkoxy, halogenoalkyl, acyl, acylamino, acyloxy, carboxyl, alkoxycarboxyl, carbamoyl, pyridoylamino, carboxyalkylcarbamoyl, N-carboxylalkylcarbamoyl, sulfo, sulfamoyl, mono- or di-alkylated or phenylated sulfamoyl optionally having one or more alkyl substituents, alkylsulfonyl, 2 0 alkoxysulfonyl, optionally hydroxy-containing phenylsulfonyl or phenoxy sulfonyl; or a pharmaceutically acceptable salt thereof.

According to a second embodiment of the invention there is provided a compound having the following structure; According to a third embodiment of the invention there is provided a pharmaceutical composition for radioimaging or diagnosis comprising a compound of Formula according to the first embodiment, a pharmaceutically acceptable salt thereof, a metal ion complex thereof, or a radiolabelled complex thereof, together with a pharmaceutically acceptable carrier, [R:\UBH]03809doc:NXL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:28 SPRUSCN FERGUSON NO. 7044 P. 22 3 According to a fourth embodiment of the invention there is provided use of a radiolabelled complex of a compound of Formula according to the first embodiment, or a pharmaceutically acceptable salt thereof for the manufacture of a .medicament for diagnosing a disease, radioimaging, or radiotherapy of a disease.

The disease may be cancer.

According to a fifth embodiment of the invention there is provided a method for diagnosing a disease, radioimaging, or radiotherapy of a disease in a mammal, comprising administering to said mammal an effective amount of a radiolabelled complex of a compound of Formula according to the first embodiment, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the third embodiment.

The disease may be cancer.

According to a sixth embodiment of the invention there is provided a conjugate I.:l compound comprising at least one compound of Formula according to the first Is embodiment, or a metal complex, or radiolabelled complex, or a pharmaceutically S' acceptable salt thereof, bonded to at least one molecular recognition unit comprising an antibody, protein, peptide, carbohydrate, oligonucleotide or oligosaccharide.

S* According to a seventh embodiment of the invention there is provided a pharmaceutical composition comprising a conjugate compound according to the sixth E' :20 embodiment, or metal complex thereof, together with a pharmaceutically acceptable carrier.

According to an eighth embodiment of the invention there is provided the use of a radiolabelled metal ion complex of a conjugate compound according to the sixth embodiment, or a pharmaccutically acceptable salt thereof, for the manufacture of a medicament for diagnosing a disease, radioimaging, or radiotherapy of a disease.

The disease may be cancer.

According to a ninth embodiment of the invention there is provided a method for diagnosing a disease, radioimaging, or radiotherapy of a disease in a mammal, comprising administering to said mammal an effective amount of a radiolabelled metal ion complex of a conjugate compound according to the sixth embodiment, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the seventh embodiment.

The disease may be cancer.

It is to be understood that throughout this specification, the term "molecular recognition unit" includes an antibody, protein, peptide, carbohydrate, nucleic acid, COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 205 16:28 SPRUSON FERGUSON NO. 7044 P. 23 4 oligonucleotide, oligosaccharide, liposome, or other molecule which can form part of a specific binding pair.

In one form of the compound of Formula the functionalised linkage group Z is selected from the group of halogen or other leaving group, nitro, nitroso, imide, dione of the formula, o vinyl group of formula Het i -C(Het 2

)-CH

2 (where Het 1 and Het 2 are the same or different and is each a nitrogen containing heterocyclic group or Het 1 is a nitrogen containing heterocyclic group and Het 2 is -C(=NH)OR 2 NCO, NCS, COR", COOR', SR2, o0 NHNR 2

R

3

NHCONHNR

2

R

3

NHCSNHNR

2

R

3

CONR

2

R

3

OR

2

NR

2

R

3 (CH2)pR 1

(CH

2 )pArR, (CH 2 0)pCH 2 R, (CH20CH20)qArRI, (CHCH)rRI, (CHCH)rArRI where

SR

2 and R 3 are the same or different and are independently selected from H, (CH2)pR' o:e1 (CH2)pArR', (CH20)pCH 2 RI, -(CH 2 OCH20)qArRI, (CHCH)rRI, (CHCH)rArRI and where R 1 is selected from SH, OH, NH 2 COOH, NCS, or -C(=NH)OCH 3

COR",

1s where R" is H, halogen, N 3 alkoxy, OAr, imidyloxy, imidazoyloxy, alkyl, or alkyl substituted with a halogen or other leaving group, where p is an integer from 1 to more typically 1 to 10, still more typically 1 to 4, even more typically 1 to 2 and yet more S* typically 1; q is an integer from 1 to 20, more typically 1 to 10, still more typically 1 to 4, even more typically 1 to 2 and yet more typically 1; r is an integer from I to 4, more 20 typically 1 or 2, still more typically 1; and Ar is optionally substituted aryl or optionally substituted aralkyl, provided that when one of X and Y is selected from C-NO 2 C-OH, C- C1, C-CH 3 or C-NH 2 then the other X or Y substituent cannot be selected from C-NO 2 C-OH, C-CI or C-NH2. In moieties of formula (CH2)pR', (CH 2 )pArRI, one or more methylene groups may also be replaced with 0, S, NH or carbonyl, for example C(O)R 1

CH

2 C(O)R1, NHCH 2 RI, NHC(O)RI, CH20R 1

CH

2 C(O)NHRI, and the like.

Typically in the compound of Fonnula the functionalised linkage group Z of the compound of Formula is selected from the group of halogen, maleimide, a vinyl N N pyidyN CH pyridyl group of formula or COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 205 16:29 SPRUSON P EGSNN.74 .2 N N 'N N

COH

2 especially or or a dione of formula 0 R4

NR

2

R

3 where R 2 and R 3 are the same or different and are independently selected Si froml H, (Cf4 2

(CH-

2 )pArR', (CH 2 Q)pCH 2

-(CH

2

OCH

2 O)qArR 2 -(CHCH)rRl, ~(CI-CH),ArRI and wvhere R1 is selected from iNm 2 COOH, NCS, NCO, or-

C(=NH)OCH

3 GOR' group where R" is halogen, alkyl, or alkyl substituted with a halogen or other leaving group, where p is an integer from I to 20, more typically 1 to still more typically I to 4, even more typically I to 2 and yet more typically 1; q is an :10 integer from I to 20, more typically I to 10, still more typically 1 to 4, even more typically 1 to 2 and yet more typically 1; r Is an integer from I to 4, more typically 1 or 2, still more typically 1; and Ar is optionally substituted aryl or optionally substituted aralkyl, provided that at least one of R 2 and R 3 is other than hydrogen, and wherein, in moieties of formula (CH 2 )pRl, (CH 2 )pArRl, one or more methylene groups may also be replaced with 0, S, NH or carbonyl, for example C(O)Rl, CH 2 C(O)RI, NHCH 2

R

1 NH4C(O)RI, OR 1 SRI, CH 2 0RI, CH 2 C(O)NHRI, and the lie.

J More typically in a compound of Formula each R 4 and R 5 is H; W is NH4; n is 2 0:6.*to 4, more typically 2 or 3, still more typically 2; Z is selected from halogen and NR 2

R

3 where R 2 and R 3 are the sarne or different and are independently selected from H,

(CH

2 )pRI, (CH 2 )PArR', provided that at least one of R 2 and R 3 is other than H; RI is selected from NI- 2 CO011, NCS, NIICOCH 2 Br and COR' where R" is halogen, typically Br; and p, is an integer from I to 4, more typically I to 2 and still more typically 1. Yet more typically, Rl is NI-1 2 Typically, R is amino, nitro, hydroxy or halogen and still more typically R is amino.

As used herein, the term "aryl' refers to single, polynuclear, conjugated and fused residues of aroma-tic hydrocarbons or aromatic heterocyclic ring systems. Examples of such groups are phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:29 SPRUSON FERGUSON NO.71044 P. 6 anthracenyl, dihydroanthracenyl, benzanthirac-enyl, dibenzanthracenyl, phenanthracenyl, fluorenyl, pyrenyl, indenyl, azulenyl, clurysenyl, pyridyl, 4-phenylpyridyl, 3phenylpyridyl, thienyl, furyl, pyrryl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrinildinyl, quinolyl, isoquinolyl, benzofurany1, benzothi enyl, purinyl, quinazolinyl, phenazinyl, acridinyl, berizoxazotyl, benzothiazolyl, heteroaryl, pyridine and liet-

CH=CH

2 and the like. Typically aryl is phenyl, pyridyl, naphthyl, anthracenyl or the like, and hieteroaryl is typically pyridine and Het-CH=CH 2 Still more typically, aryl is pheny].

As used herein, the term "aralkyl" refers to alkyl groups substitu~ted with one or more aryl groups as previously defined. Examples of such groups are benzyl, 2phenylcthyl and I -phenylethyl.

As used herein, the term "optionally substituted" means that the moiety described as optionally substituted may carry one or more substituents selected firom amino, halogen, hydroxy, n-ercapto, nitro, cyano, thiocyano, alkyl, alkoxy, halogenoalkyl, acyl, acylamino, acyloxy, carboxyl, alkoxycarboxyl, carbamoyl, pyridoylamino, carboxyalkylcarbamoyl, N-carboxyalkylcarbanxoyl, suipho, sulpharnoyl, mono- or dialkylated or phenylated sulphamoyl which can carry one or more alkyl substituents, alkylsulphonyl, alkoxysuiphonyl, optionally hydroxy-containing phenylsulphonyl or phenoxy sulphonyl.

In another foxrn of the compound of Formula the Z group of said compound of Formula isselected from the group of NR 2

R

3 where R 2 and R 3 together with the nitrogen atom to which they are attached form a substituted saturated or unsaturated 3 to 8 membered ring optionally containing one or more additional heteroatoms 0, S or N and wherein there is at least one substituent capable of binding said compound of Formula (1) with a molecular recognition unit.

Also disclosed herein is a compound of Formula as described in the first embodiment of the present invention which is complexed with a metal ion.

The metal ion is typically selected from 64 CU, 6 7 CU, 99 mTe, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Mo, Ni, V, Pb, k, Pt, Zn, Cd, Mn, Rxi, Pd, 1-1g, Ti, TI, Sn, Zr, and the lanthanide group of elements in thc Periodic Table such as Sm, Ho, Gd, Tb, Sc, Y, and the actinides.

The metal ion is further typically a radionuclide selected from the group of 64Cu 67 Cu, 99 m1Tc, and radionuclides of In(III), Ga (III), Fe (III), Cu Ti (IV) and other radionuclides from the Lanthanides, Re, Sm, Ho, and Y.

Detailed Description Of The Invention The compounds of Formula typically comprise those compounds where n represents an integer from 2 to 4, more typically 2 or 3, still more typically 2. Typically, COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:29 SPRUSON FERGUSON NO. 7044 P. 26 6a one of X and Y is a C-Z group where Z is typically NR 2

R

3 where R 2 and R 3 are the same or different and are selected from H, (CH 2 )pRI and (CH 2 )pArRI, where R 1 is as previously defined, and p is I to 4, more typically 1 to 2 and still more typically 1, provided that at least one of R 2 and R 3 is other than hydrogen. Usually, one of X is a C-Z group and the other is a group C-R, where R is as previously defined, typically amino, lower alky), nitro, hydroxy or halogen. Generally, R 1 is NH 2 and p is 1.

An example of a compound of Formula is: NH NH

NH

2 NH NH NH-CH 2 NH 2 NH NH_ The R I group in a compound of Formula may provide a point of attachment of a compound of Formula to a molecular recognition unit.

The molecular recognition unit is typically an antibody, protein, peptide, oligonucleotide, oligosaccharide. In particular, the molecular recognition unit is typically an antibody and more typically a monoclonal antibody.

4 444* COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. EB. 2005 16:30 S PRUSON FERGUSON NO. 1044 P. 27 7 Thus, compounds of Formula provide a method of attachment of radionuclide metal ions such as In(IJI), Ga(III), Fe(III), Tc([V) or Tc(V), Re(VII), Cu(I1), Ti(IV), other radionuclides from the Lanthanides, Rhenium, Samar-ium, Holmium, Yttrium and the like to molecular recognition units such as monoclonal antibodies, receptor specific proteins, peptides or oligonucleotides for in vivo imaging and therapy.

The compounds of general Formnula are typically prepared by attachment of a functional linking group to a suitable cryptate. Methods of synthesis of cryptates useful as precursors to compounds of Formula in which W is NH- are described in United States Patent No. 4,497,737 in the name of Sargeson et a, the disclosure of which is incorporated herein by reference. Other cryptates where W is S mnay be prepared by analogous methods. Sargeson et a/ describe synthesis of mctal cage "cryptate" compounds by the condensation of a tris-{dian-tine) metal ion complex as described at column 3 lines 30 to with fornialdehyde and an appropriate nucleophile. In order to obtain the compounds of Formula an, appropriate nucleophile is selected so as to obtain the desired functionalised 115 linkage Lgroup Z as defined in Formula In particular, reference is made to column 4 lines 7 to 27 of United States Patent No. 4,497,737. Alternatively, a fanctionalised linkage group Z may be attached to a functional group of a cryptate prepared by the methods taught by Sargeson et a[ (for example see Example 9 at column 9 line 65 to column 12 lind 10 of Sargeson el al) by standard synthetic techniques. If necessary, a protecting group may be 20 introduced into the cryptate structure to protect latent functionality for the desired finctionalised linkage group Z as defined in Formula during synthesis of the desired cryptate precursor. Suitable protecting groups are described, for example in Greene, T.W., Protective Groups in Organic Synthesis (John Wiley Sons, New York, 1981) and 25McOrnie, Protective Groups In Organic Chemislyy (Plenumn Press, London, 1973).

For example, compounds of Formula where said Z group comprises a mono- or C..'.di-substituted amino group and where the substituent is optionally substituted alkyl, are readily prepared by treating the amino compound with the appropriate halo -substituted alkyl. Typically, the compound of Formula where Z is -NH4-CH 2 -CH2-NH 2 can be prepared by treating a compound of Formula where R is NH 2 with BrCH 2

CH

2

NH

2 in the :3o. presen~ce of NaHCQ 3 or the like with suitable protection.

Compounds of formula where R 1 is -NCS may be prepared by reacting the amino compound with thiophosgene (see W087/1 263 Kozak et al., Cancer Res. 49, 2639 (1989). Substituted acid halide compounds are produced by reacting a compound of formula where R is 142 with BrCH 2 CO&r at 4'C according to the procedure of C J Mathias el al., Bioconjugate Chem., 1, 204 (1990). Compounds with an electrophilic moiety can also be prepared by methods known in the art, such as in ACC Chenm. Res. 17 202-209 (1984).

Compounds with active esters (CH)p-C(O)-X may be formed by the procedures of Bodanszky M, T'he Peplida. Analysis Synthesiy Biology, Ed. E. Gross and J Meienhofer, Vol pp 105-196. Academic Press, Inc., Orlando, FL. (1979) and Bodanszky M, Principles of Peptide Synthesis, pp 9-58, Springerverlag, New Y ork, (1984). Other compounds of COMS ID No: 58M1-01 119416 Received by IP Australia: Time 16:37 Date 2005-02-14 WO 00/40585 PCT/AU00/00003 8 formula may be prepared from such compounds by standard procedures such as described in Modem Synthetic Reactions, H 0 House, 2 nd Edition, Benjamin, Inc.

Philippines, 1972.

In a typical embodiment, compounds of formula in which X or Y is C-Z where the group Z is a group NH(CH 2 )pRI or NH(CH 2 )pArR I may be prepared by a Schiff Base condensation reaction of a compound of formula (or a metal complex thereof) in which X or Y is NH, with an aldehyde of formula HC(O)(CH 2 )p.RI or HC(0)(CH 2 )p-ArR

I

(Still typically. the Schiff Base condensation reaction is most appropriately conducted between a copper complex of compounds of Formula in which X or Y is NH 2 with an aldehyde of formula HC(0)(CH 2 )p.-RI or HC(0)(CH 2 In one particular form of this embodiment, a compound of formula is obtained by the reaction of nitrobenzaldehyde with the copper complex of an aminocryptate such as described by Sargeson et al. The reaction is typically performed in an inert-gas atmosphere and in the presence of solvent and diluents typically inert to the reactants. Suitable solvents comprise aliphatic, aromatic, or halogenated hydrocarbons such as benzene, toluene, xylenes; chlorobenzene, chloroform, methylene chloride, ethylene chloride; ethers and ethereal compounds such as dialkyl ether, ethylene glycol mono or -dialkyl ether, THF, dioxane; alkanols such as methanol, ethanol, n-propanol, isopropanol; ketones such as acetone, methyl ethyl ketone; nitriles such as acetonitrile or 2-methoxypropionitrile; N,N-dialkylated amides such as dimethylformamide; dimethylsulphoxide, tetramethylurea; as well as mixtures of these solvents with each other.

If the amine or a salt thereof is soluble in water, then the reaction medium may be water at low temperature. The compounds of formula may be converted to pharmaceutically .acceptable salts by way of recognised procedures.

Typically, for medical use salts of the compounds of the present invention will be pharmaceutically acceptable salts; although other salts may be used in the preparation of the inventive compound or of the pharmaceutically acceptable salt thereof. By pharmaceutically acceptable salt is meant those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.

Suitable pharmaceutically acceptable salts of the compounds of the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid.

Suitable pharmaceutically acceptable salts of the compounds of the present invention therefore include acid addition salts.

For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1-19. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting o the free base function with a suitable organic acid. Representative acid addition salts WO 00/40585 PCT/AU00/00003 9 include acetate, adipate, alginate, ascorbate, asparate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts. and the like. Representative alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

The radiolabelling of compounds of formula and salts thereof can be is accomplished by using procedures recognised in the art. For example, radiolabelling of the chelator with 6 7 Cu can be achieved by adding copper in an aqueous acetate solution to a compound of formula in an aqueous solution and incubating at room temperature.

Alternatively, the radiolabelling of a compound of formula with technetium, for example, may be achieved by adding a reducing agent such as stannous salts typically stannous chloride, to an aqueous solution of a compound of formula followed by reaction with aqueous sodium pertechnetate solution (Na 99 mTcO 4 The order of mixing these three components is believed not to be critical. However, typically the reducing agent is added to the chelator of formula Other suitable reducing agents comprise alkali metal dithionites such as sodium dithionite, sodium borohydride, hydrochloric acid, hydrobromic acid, other soluble dithionites such as potassium dithionite or ammonium dithionite, a soluble bisulfite or metabisulfite such as sodium bisulfite, potassium bisulfite, lithium bisulfite, ammonium bisulfite, sodium metabisulfite, potassium metabisulfite, lithium metabisulfite or ammonium metabisulfite, or an aqueous solution of sulfur dioxide.

Technetium-99m in the form of an aqueous solution of sodium pertechnetate is readily obtainable from commercially available molybdenum-99/technetium-99m generators or alternatively, instant 99 mTc may be used. Cu-64 is commercially available from Australian Nuclear Science Technology Organisation and 67 Cu from the US Department of Energy, Brookhaven, USA.

The conjugate compounds of the eighth embodiment of this invention may be formed by the reaction of a radiolabelled metal complex of a compound of Formula (I) together with a molecular recognition unit. The radionuclides which are useful for complexing with the compounds of Formula typically comprise metal ions which have at least two oxidation states, most typically an oxidation state of +2 or In a typical embodiment, the radiolabelled metal complex is selected from 64 Cu, 6 7 Cu and 9 9 mTc, Sm, Ho, Re, Sc, Cu, Pd, Rh, and Y. The most typical metal ions comprise 64 Cu, 67 Cu, 99 mTc.

WO 00/40585 PCT/AUOO/00003 These radiolabelled metal complexes are then reacted with a molecular recognition unit.

The radiolabelled molecular recognition unit so formed is useful for diagnostic, therapeutic and radioimaging applications.

Alternatively a conjugate of a compound of formula may be first prepared, and then it may be radiolabelled.

Thus, radiolabelling of molecular recognition units such as proteinaceous materials using compounds of formula can be conducted in two ways, namely: prelabelling of a compound of Formula with a suitable radionuclide, followed by conjugation of the resultant radiocomplexed compound to proteinaceous or other material, or conjugating the compound of formula to proteinaceous or other material for subsequent radiolabelling.

The formation of a conjugate compound of formula is usually achieved by the reaction of the functionalised linkage group with a thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group present in the molecular recognition unit. For example, an amino or hydroxy group of the functionalised linkage group may be reacted with a free carboxyl group of the molecular recognition unit, or vice versa. Suitably a coupling agent such as a carbodiimide may be employed to facilitate the coupling reaction.

The conjugate compounds according to the eighth embodiment of the present invention may contain more than one molecule of a compound of formula to any one molecular recognition unit. The metal complexing and radiolabelling of compounds of formula and pharmaceutically acceptable salts thereof can be accomplished by using procedures recognised in the art. For example, the radiolabelling of the conjugate compounds with 6Cu may be achieved by adding an aqueous acetate solution of 64 Cu to the conjugate compound in an aqueous solution and incubating for 5 minutes at room temperature. A composition comprising an uncomplexed conjugate in accordance with the invention may also be supplied to radio-chemists, technicians, radiopharmacists, doctors or the like in the form of a kit for radiolabelling immediately prior to use.

In a typical form of this invention, the kit comprises a first container that contains a radiolabelling metal ion, usually in solution, and a second container that contains a conjugate compound as described in the eighth embodiment of the present invention. The kit, in use, then involves mixing the contents of said first and second containers to obtain the radiolabelled conjugate compound.

Typically, the compounds of Formula or the metal complex, radiolabelled complex or pharmaceutically acceptable salt thereof are useful for labelling molecular recognition units for use in methods of diagnosis and therapy of disease. In particular, the typical radiolabelled molecular recognition units are monoclonal antibodies and fragments thereof, peptides, oligonucleotides, oligosaccharides or liposome or a part of a specific binding pair.

WO 00/40585 PCT/AU00/00003 11 The applications of radiolabelled molecular recognition units comprise diagnosis, imaging and therapy of disease such as cancer. Typically, the compounds of formula and their metal complexes and/or salts thereof have a diagnostic use as imaging agents in vitro and in vivo. The method of diagnosis using the aforesaid imaging agents will result from the localisation of the radiolabelled conjugate compounds on specific organs and tissues in a subject.

The method of diagnosis will typically involve first the administration of an effective amount of a radiolabelled compound of Formula to a subject; and then monitoring the subject after a suitable period of time in order to ascertain the presence or absence of a cancer for example as evidenced by localisation of the radiolabel at a particular site in the subject. Typically, the monitoring step shall provide information regarding the location of any cancer if it is present. The effective amount or dosage of the radiolabelled compound of Formula will depend upon the desired amount of radioactivity required for the diagnostic application balanced with the safety requirement of not exposing the subject, in particular their organs and tissues, to harmful amounts of radiation. Appropriate dosages for any given application may be determined by persons skilled in the relevant art by no more than routine experimentation, given the teaching herein.

The method of therapy will typically involve compounds of formula or the metal complexes, radiolabelled complexes and/or pharmaceutically acceptable salts thereof which are useful as cytotoxic agents. In a typical embodiment, the therapy of disease comprises treatment of cancer, abnormal cell disorders and the treatment of tumrnours. In such applications, the radiolabelled compound of formula is typically conjugated to a molecular recognition unit which is capable of binding specifically to the tumour or abnormal cell. Examples of such molecular recognition units comprise one part of specific binding pairs and are known to persons skilled in the relevant art and typically comprise antibody/antigen pairs and the like.

The method of therapy will typically involve the administration of an effective amount of a radiolabelled compound of Formula to a subject. The effective amount or dosage will depend upon the desired amount of radioactivity required for the diagnostic application balanced with the safety requirement of not exposing the subject, in particular their organs and tissues, to harmful amounts of radiation. Appropriate dosages for any given application may be determined by persons skilled in the relevant art by no more than routine experimentation, given the teaching herein.

Typically the treatment would be for the duration of the condition, and contact times would typically be for the duration of the condition.

Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages of a compound of the present invention will be determined by the nature and extent of-the condition being treated, the form, route and site of administration, and the nature of the particular vertebrate being treated. Also, such optimum conditions can be determined by conventional techniques.

WO 00/40585 PCT/AU00/00003 12 It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the compound of the present invention given per dav for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Also included within the scope of the present invention are prodrugs of the inventive compound. Typically, prodrugs will be functional derivatives of a compound of Formula (I) in accordance with the first embodiment of the invention, which are readily converted in vivo to the required compound for use in the present invention as described herein. Typical procedures for the selection and preparation of prodrugs are known to those of skill in the art and are described, for instance, in H. Bundgaard Design of Prodrugs, Elsevier, 1985.

When used in the treatment of disease, the compound of Formula in accordance with the first embodiment of the invention or a metal complex, radiolabelled complex or a pharmaceutically acceptable salt thereof, may be administered alone. However, it is generally preferable that these compounds be administered in conjunction with other chemotherapeutic treatments conventionally administered to patients for treating disease.

For example, a tumour may be treated conventionally with surgery, and the compound of Formula in accordance with the first embodiment of the invention or a metal complex, radiolabelled complex or a pharmaceutically acceptable salt thereof, to extend the dormancy of micrometastases and to stabilise and inhibit the growth of any residual primary tumour.

Typically, when used in the treatment of solid tumours, compounds of the present invention may be administered with chemotherapeutic agents such as: adriamycin, taxol, fluorouricil, melphalan, cisplatin, alpha interferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone),

PROMACE/MOPP

(prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine), vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG and the like. Other chemotherapeutic agents include alkylating agents such as nitrogen mustards including mechloethamine, melphan, chlorambucil, cyclophosphamide and ifosfamide; nitrosoureas including carmustine, lomustine, semustine and streptozocin; alkyl sulfonates including busulfan; triazines including dacarbazine; ethyenimines including thiotepa and hexamethylmelamine; folic acid analogues including methotrexate; pyrimidine analogues including 5-fluorouracil, cytosine arabinoside; purine analogues including 6-mercaptopurine and 6-thioguanine; antitumour antibiotics including actinomycin D; the anthracyclines including doxorubicin, bleomycin, mitomycin C and methramycin; hormones and hormone antagonists including tamoxifen and cortiosteroids and miscellaneous agents including cisplatin and brequinar.

WO 00/40585 PCT/AU00/00003 13 When used in the treatment of disease, the compound of Formula in accordance with the first embodiment of the invention or a metal complex, radiolabelled complex or a pharmaceutically acceptable salt thereof, may be administered alone. However, it is generally preferable that they be administered as pharmaceutical formulations. In general pharmaceutical formulations of the present invention may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.

These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral intravenous, intraspinal, subcutaneous or intramuscular) route. In addition, the compound of Formula in accordance with the first embodiment of the invention or a metal complex, radiolabelled complex or a pharmaceutically acceptable salt thereof, may be incorporated into biodegradable polymers allowing for sustained release, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of for example, a tumour or implanted so that the active agents are slowly released systemically.

Osmotic minipumps may also be used to provide controlled delivery of high concentrations of the active agents through cannulae to the site of interest, such as directly into for example, a metastatic growth or into the vascular supply to that tumour.

The carriers, diluents and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

Examples of pharmaceutically and veterinarily acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

WO 00/40585 PCT/AU00/00003 14 Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.

The radiolabelled molecular recognition units especially radiolabelled antibodies are particularly useful in medicine, for example, in locating specific tissue types and in the therapy of cell disorders. The radiolabelled antibodies can also be used to target metal ions to a specific tissue type, both in vitro and in vivo A typical use of the radiolabelled compounds of Formula is to radiolabel monoclonal antibodies specific for colon, ovarian, lymphoma, breast and/or bladder cancer, with beta emitter radionuclides of metals such as Sm, Ho, Re, Sc, Cu, Pd, Pb, Rh and Y for therapy of above mentioned cancer. A further typical use is in the radiolabelling of a monoclonal antibody specific for metastasis of colon cancer for diagnosis and therapy.

In another typical embodiment, the antibody in the conjugate compound may be a complete antibody molecule or a fragment thereof or an analogue of either of these, provided that the antibody comprises a specific binding region. The antibody may be a humanised monoclonal or a fragment thereof. The antibody may also be a recombinant antibody. The antibody may be specific for any number of antigenic determinants, but is typically specific for one antigenic determinant.

In another typical embodiment, there is provided radiolabelling of monoclonal antibodies with 6 7 Cu (beta and gamma emitter) and 6Cu (positron and beta emitter), for combined radioimmunoscintography (RIS) (SPECT and PET) and radioimmunotherapy (RIT). Other radionuclides comprise Auger emitting agents where the compound of Formula is coupled to the monoclonal antibody and labelled with auger emitting isotope such as Fe-59 or Cu-64.

In still another typical embodiment, there is provided a two step pretargeted radioimmunotherapy where a monoclonal antibody with a first marker molecule attached thereto is injected into a subject. Once the antibody has cleared from the system and localised to the tumour, a second injection is administered to the subject. This second injection typically involves the radiolabelled complex of Formula attached to a second marker molecule which recognises the first marker molecule on the targeted antibody.

Alternatively, the second injection may typically be the second marker molecule alone and when cleared from the system, the radiolabelled complex of Formula attached to the first marker molecule is administered to the subject. Both procedures provide amplification of the target site and reduce exposure of the radiolabelled complex to normal tissue. Still typically, the first marker molecule is biotin and the second marker molecule is avidin or streptavidin. Still more typically, the first marker molecule is smaller than the targeted antibody.

WO 00/40585 PCT/AU00/00003 The invention also provides a two step procedure which involves the administration of an antibody-DNA conjugate or antibody-oligonucleotide conjugate followed by targeting with a radiolabelled complementary DNA or complementary oligonucleotide. This procedure also provides amplification of the target site and reduces exposure complex of the radiolabelled to normal tissue.

The invention also provides a use of compounds of Formula or metal complexes, radiolabelled complexes, conjugate compounds or pharmaceutically acceptable salts thereof as Magnetic Resonance Imaging (MRI) agents. In this form of the invention, there is typically formed a complex of compound of formula or a conjugate of the eighth embodiment, with a paramagnetic metal ion, typically Fe (III), Mn(II), which may be used as a contrast agent to enhance images. Further, complexes such as these may be employed in the form of a pharmaceutical formulation where the complex is present with a pharmaceutically acceptable carrier, excipient or vehicle therefor.

The pharmaceutical formulations described for the different embodiments of this invention typically comprise a formulation in the form of a suspension, solution or other suitable formulation. Physiologically acceptable suspending media together with or without adjuvants may be used. Still typically, the pharmaceutical formulations are in a liquid form and still more typically are in an injectable form. Still more typically, the injectable formulations are dissolved in suitable physiologically acceptable carriers which are recognised in the art.

The industrial use of the compounds of formula further comprises their attachment to solid surfaces such as polymers, for use in the concentration of metal ions and purification of water or attached to an electrode surface for detection of specific metal ions.

Brief Description Of The Drawings The present invention will be further illustrated, by way of example only, with reference to the accompanying figures; in which: Figures la and lb are graphical representations of the serum stability of 6 7 Cu complex of compound at 0 and 7 days respectively; Figure 2 is a graphical representation of the effect of pH on the complexation of 6 4 Cu with compound Figure 3 is a graphical representation of the biodistribution of a 64 Cu complex of compound Figure 4 is a graphical representation of the biodistribution of a 64Cu complex of compound Figure 5 is a graphical representation of the biodistribution of a 64 Cu complex of compound and Figure 6 is a graphical representation of the biodistribution of 64 Cu labelled conjugate of B72.3 conjugated with compound in Tumour Bearing Nude Mice.

WO 00/40585 PCT/AU00/00003 16 Figure 7 is a graphical representation of the radiotherapeutic effect of 30MBq of

C

u labelled conjugate of B72.3 conjugated with compound in Tumour Bearing Nude Mice.

Examples The following examples serve only to illustrate the invention and should not be construed as limiting the generality of the above description.

Example 1: Preparation of Compound (3) Compound (3) Preparation of compound is illustrated in Schemes 1 and 2.

NH NH H7N-- ,N NH -NH 2

NO

2 O -CHO in EtOH +NaBH 3

CN

+CH

3

COOH

-NH

2 WO 00/40585 PCT/AU00/00003 Scheme 1.

NO2

NH

2 Schiff Base Condensation reaction of compound with pnitrobenzaldeheyde Pd/C NaBH 4

-NH

2 Scheme 2. Reduction of compound to compound A. Preparation of Compound (2) Compound is prepared by Schiff base reaction of compound with pnitrobenzaldehyde. Synthesis of compound is described in Aust. J. Chem (1994) 47, 143-179. A copper complex of(l) is dissolved in dry ethanol (2.4 mmoles in 100 mis) and p-nitrobenzaldehyde (2.2 mmoles) is added. The reaction mixture is evaporated to dryness and reconstituted (with dry ethanol) twice to remove any water. The reaction mixture is once again reconstituted in dry ethanol and stirred for 30 mins under nitrogen gas. Sodium cyanoborohydride (25 mmoles), glacial acetic acid (2 mmoles) and activated 3 A molecular sieves are added and the reaction is allowed to stir overnight. The mixture is filtered, evaporated to dryness and extracted with chloroform and water (100 ml 200 ml). The water layer is diluted to 2 L, sorbed onto SP Sephadex C25 and eluted with 0.3 M sodium acetate. (Scheme 1).

WO 00/40585 PCT/AU00/00003 18 B. Preparation of Compound (3) To palladium/C catalyst (20 mg) in water (0.5 ml) is added sodium borohydride mg) in water (0.5 ml) under nitrogen gas. To this mixture is then added compound mg) dissolved in approximately 0.1 M sodium hydroxide (0.5 ml). The mixture is left to stir at room temperature for a further 30 mins or until the solution becomes clear. A 2 ml glass vial is cooled on ice. The mixture is 0.22 [im filtered into the cooled vial to remove the suspended palladium/C catalyst. To this cooled filtrate is added concentrated hydrochloric acid dropwise until all excess sodium borohydride is quenched until gas evolution on addition of acid ceases). The quantity of product is determined by titration with a known concentration of 64Cu as described in Example 2. Product is stored frozen at pH 1 in a rubber-stoppered vial under nitrogen gas. Yield: 95% (Scheme The final product is characterised by IH-NMR in D 2 0 at 298K (Bruker Avance DPX 400). 1 H NMR 3.28 ppm, m. 6H, CH 2 (cage); 3.39 ppm, m, 6H, CH 2 (cage); 3.59 ppm, s, 6H, CH 2 (cage); 3.71 ppm, s. 6H. CH 2 (cage); 4.45 ppm, s, 2H, CH 2 7.51 ppm, d, 2H, Ar; 7.70 ppm, d, 2H, Ar.

Example 2 Complexation of 64 Cu by Compound (3) The effect of pH on complexation of 64 Cu by compound was investigated.

Compound was diluted into buffers ofpH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0. A sufficient amount of 64 Cu was added and the rate of complexation was monitored at (t 1, 2.5, 5, 60 and 90 mins) (see method below) by Instant Thin Layer Chromatography (ITLC-SG).

Complete complexation 98 was achieved within 1 min for all pH 4.0. The rate of complexation at pH 3.0 was slower. (Figure 2).

Monitoring Complexation by ITLC-SG ITLC-SG strips (10 cm X 0.8 cm) were spotted with 1 pL of reaction mixture 1 cm from the bottom of the strip (origin) and were developed in a solvent containing 0.1 M sodium acetate (pH 4.5) ethanol 9:1. 64 Cu-compound remains at the origin (Rf= 0.0), and "free" 64 Cu appears at the solvent front (Rf A typical method of radiolabelling the ligand is achieved by adjusting the pH of an aqueous solution of the ligand to pH 5.0. Sufficient volume of 64 6 7 Cu solution (usually in 0.02 M HCI or diluted into 0.1 M sodium acetate buffer pH 5.0) is added to form a 1:1 complex. The efficacy of labelling is determined by ITLC-SG as described above. One main radiochemical species is observed.

Example 3 Serum Stability of 67 Cu complex of compound (4) Serum stability studies were conducted by incubating a 6 7 Cu complex of related species in human plasma at 37°C.

WO 00/40585 PCT/AU00/00003 19 NH NH H NH NH H NH NH [4] At various time intervals, the complex was separated from the plasma by size exclusion chromatography and the complex breakdown was assessed. Results indicated that s no more than 2% of the copper is lost from the chelator during the first 174 hours at 37 0

C.

(Figure la and Ib) Example 4 Synthesis of B72.3 conjugated with using 1-Ethyl-3-(3dimethylamino-propyl)carbodiimide

(EDC)

A typical method for radiolabelling an antibody (such as B72.3) is to incubate the antibody with the ligand (such as compound in the presence of EDC at pH 5.0 for mins at 37 0 C. The unreacted ligand and EDC by-products are removed by washing with buffer (pH 5.0) on a size exclusion ultra filtration membrane. The purified immunoconjugate is exposed to a slight excess of 64 6 7 Cu. The reaction is allowed to proceed at room temperature, and labelling is complete in less than 5 mins. Excess 64 6 7 Cu is removed by washing with 0.1 M EDTA in PBS (pH 7.2) on a size exclusion ultra filtration membrane or by separation on a size exclusion column (sephadex G25, eluted with PBS pH (Scheme 3) The conditions for radiolabelling B72.3 were optimised over incubation time mins), reaction pH concentration of B72.3 (5 mg/ml) and the molar ratio of B72.3:EDC:ligand (1:1000:250).

WO 00/40585 PCT/AU00/00003 Protein EDC+ Compound (3) at least 30 mins 370C Purify on size exclusion ultra filtration membrane Fitrate protein-(3) unbound cryptate and EDC 64Cu and Analyse radiochemical purity by ITLC-SG Purify on size exclusion ultra filtration membrane or sephadex G25 column Column/ Filtrate Colunreacte u F e 64Cu labelled protein unreacted 64 Cu Scheme 3. Synthesis of protein conjugated with compound using EDC.

Example 5 Biodistribution of Cu complexes of(1), and in Balb/C mice The biodistribution of the 6Cu complexes of compounds and (0.10 ml intravenous injection) were evaluated in balb/c mice (5 animals per time point) at 3, 5, 20 and 30 minute time intervals. Biodistribution studies were performed in duplicate.

Biodistribution of the radiolabel is presented in Tables 1, 2 and 3, and is illustrated diagrammatically in Figures 3, 4 and Example 6 Evaluation of 6Cu-labelled conjugate of B72.3 with compound in Tumour Bearing Nude mice The B72.3 antibody recognises the TAG-72 antigen which is expressed on colorectal and ovarian tumours. The animal model used in the present study uses LS174t cells which also express TAG-72 antigen. The biodistribution of the 6Cu immunoconjugate (0.10 ml intravenous injection) was evaluated in LS174t tumour bearing nude mice (5 animals per time point) at 1, 3, 5, 24 and 48 hour time intervals. Biodistribution studies were performed in duplicate. Biodistribution of the radiolabel is presented in Table 4, and is illustrated diagrammatically in Figure 6a; Biodistribution of complex of compounds and (3) show that if the ligand actually detaches from the antibody in any form it will clear rapidly from the system and not release the 64 Cu.

TABLE 1.

64 Cu-labelled compound (4) INJECTED DOSE PER GRAM 10 MIN 15 MIN 20 MIN MEAN S.D MEAN S.D MEAN S.D TIMI.i 3 MIN 5 MIN ORGAN MEAN S.D. MEAN S.D LIVER 1.81 0.30 1.59 0.12 SPLEEN 2.26 0.43 2.17 0.29 KIDNEY 20.08 4.98 18.62 4.09 MUSCLE 1.76 0.23 2.10 0.59 SKIN 5.98 0.31 6.46 0.56 BONE 1.60 0.25 1.56 0.24 LUNGS 5.85 0.03 5.28 0.46 HEART 3.25 0.34 2.66 0.14 BLOOD 8.25 0.32 7.08 0.22 BLADDER 13.15 6.81 28.05 18.33 STOMACH 3.53 0.56 3.04 0.29 GIT 2.62 0.08 2.16 0.20 1.16 1.28 14.79 1.24 4.21 1.28 2.84 1.60 4.41 69.91 1.84 1.49 0.06 0.14 6.26 0.27 0.68 0.23 1.09 0.36 0.78 54.03 0.55 0.31 0.64 0.67 7.52 1.73 2.76 1.60 2.09 1.08 2.71 8.17 0.89 1.07 0.06 5.01 0.55 0.15 0.52 0.08 0.54 25.99 3.24 1.32 0.36 0.06 0.10 1.73 0.45 0.41 0.89 0.51 0.24 1.13 0.30 0.18 0.51 0.08 0.57 1.40 0.12 4.96 16.38 20.77 0.37 0.70 0.14 0.18 0.89 0.05 30 MIN MEAN S.D 5.76 0.58 0.35 0.07 30.26 4.50 1.27 1.43 1.42 0.42 0.65 0.10 0.92 0.15 0.47 0.17 1.07 0.21 25.38 35.78 0.65 0.10 0.80 0.04 TABLE 2.

64 Cu-labelled compound (1) INJECTED DOSE PER GRAM "T1Ml 3 MIN 5 MIN 10 MIN 15 MIN 20 MIN 30 MIN 00 (ORGAN NMI:AN S.D. MEAN S.D MEAN S.D MEAN S.D MEAN S.D MEAN S.D ,o LIVER 2.29 0.22 1.50 0.17 1.42 0.22 0.82 0.19 0.69 0.15 0.50 0.08 SPLEEN 2.09 0.52 1.48 0.21 1.54 0.25 1.44 0.76 0.69 0.12 0.52 0.16 KIDNEY 23.18 11.73 11.50 1.34 12.80 1.83 6.77 1.40 7.77 0.67 4.95 0.92 MUSCLE 1.81 0.32 1.81 0.59 2.39 1.14 2.28 1.66 1.17 1.02 1.33 1.59 SKIN 5.82 0.42 5.59 0.59 5.48 1.07 2.61 0.44 2.96 0.72 1.63 0.08 BONE 2.25 0.45 1.87 0.47 1.79 0.29 1.23 0.12 1.25 0.44 0.78 0.23 LUNGS 6.51 1.28 4.45 0.54 3.91 1.98 2.06 0.32 2.73 0.61 0.85 0.31 -HEART 3.11 0.38 2.25 0.10 2.27 0.43 0.89 0.12 1.23 0.71 0.40 0.10 BLOOD 8.68 1.35 6.32 0.70 5.45 0.81 2.48 0.31 2.63 0.29 1.14 0.25 BLADDER 24.36 16.90 31.12 19.21 27.75 21.59 15.17 8.53 23.29 15.56 122.93 71.11 STOMACH 3.50 0.64 2.73 0.36 2.08 0.80 1.50 0.46 1.16 0.06 0.74 0.04 GIT 2.75 0.59 2.21 0.08 1.85 0.38 1.11 0.06 1.05 0.17 1.16 0.72 TABLE 3.

64Cu-ilalelled compound (3) INJCIITED DOSE PER GRAM

TIME

ORGAN

3 MIN 5 MIN 10 MIN

MEAN

15 MIN

MEAN

20 MIN 30 MIN

LIVER

SPLEEN

KIDNEY

MUSCLE

SKIN

BONE

LUNGS

HEART

BLOOD

BLADDER

STOMACH

GIT

MEAN S.D. MEAN S.D 1.94 0.17 2.00 0.52 1.96 0.14 2.01 0.16 22.41 5.77 18.99 2.98 1.98 0.48 3.17 2.34 5.63 1.05 6.18 1.17 1.68 0.04 2.11 0.60 5.48 0.61 5.74 1.07 2.79 0.36 3.00 0.59 7.74 0.43 8.19 1.53 11.36 4.02 12.31 4.09 2.69 0.51 3.11 0.79 2.66 0.29 2.88 0.46

S.D

S.D MEAN 1.46 1.26 13.41 1.73 4.86 1.29 4.00 2.03 5.23 14.36 2.35 1.80 0.39 0.20 3.04 0.28 1.14 0.43 0.79 0.58 1.28 9.79 0.65 0.38 1.24 1.09 10.26 2.40 4.49 1.36 3.25 1.56 4.39 10.54 1.91 2.92

S.D

0.23 0.25 1.76 1.39 1.03 0.24 0.64 0.29 0.59 9.64 0.45

MEAN

1.04 0.93 9.98 2.13 3.41 1.11 2.59 1.24 3.05 13.45 1.54

S.D

0.08 0.13 1.73 1.35 1.10 0.08 0.50 0.05 0.44 16.47 0.33

MEAN

0.72 0.39 5.95 0.50 1.45 0.72 1.13 0.50 1.25 2.74 0.60

S.D

0.02 0.05 0.27 0.01 0.13 0.10 0.01 0.11 0.02 2.02 0.04 2.46 1.05 0.22 0.55 0.03 TABLE 4.

64 Cu-lab.llcd conjugate of B72.3 wilh compound (3) INJECTED I)DOSE PER GRAM TIME 1 HR 3 HR 5 HR 16 HR 24 HR 48 HR ORGAN MEAN SD MEAN SD MEAN SD MEAN SD MEAN SD MEAN SD LIVER 19.09 1.05 18.44 0.58 19.54 2.34 16.38 1.59 15.52 2.95 13.76 3.21 SPLEEN 11.51 1.13 8.35 1.19 10.17 0.98 10.08 1.75 8.47 1.89 8.44 1.57 KIDNEY 10.64 1.47 9.94 0.15 10.41 1.10 9.16 1.77 7.39 0.65 7.59 1.35 MUSCLE 1.45 0.52 1.48 0.23 1.45 0.16 1.98 0.27 2.54 0.54 1.72 0.42 SKIN 2.83 0.52 5.18 0.26 5.99 1.38 8.64 1.16 8.30 1.30 6.95 0.16 BONE 4.42 0.55 3.82 0.49 3.21 0.30 3.93 0.49 4.28 0.72 3.39 0.38 LUNGS 13.32 1.52 11.43 1.44 9.78 0.86 11.03 1.93 7.76 0.87 6.81 1.09 HEART 10.56 2.29 8.58 1.21 9.04 3.30 6.77 0.92 5.14 0.72 5.20 0.43 BLOOD 47.98 5.60 37.94 1.52 33.41 3.66 26.41 1.48 21.50 1.81 18.69 2.01 BLADDER 2.33 0.77 2.93 0.62 3.15 0.47 7.00 5.09 5.41 2.37 4.87 0.92 STOMACH 1.41 0.29 1.69 0.33 2.32 0.51 1.48 0.74 1.58 0.31 1.43 0.32 GIT 2.23 0.35 2.60 0.20 2.87 0.23 2.28 0.14 2.20 0.20 1.86 0.31 TAIL 5.03 0.73 4.96 0.83 5.64 1.82 6.14 2.47 5.18 1.20 4.08 0.30 TUMOUR 5.98 0.80 11.10 0.76 12.31 1.68 23.77 2.46 29.43 4.44 38.43 4.79

,,J

trable Iliodistribu tin of"131172.3 in nu/nu miiice Time (hourIIS) ORZGAkN 0

SD

LIV'ER

SPLEEN

KIDNEY

MIUSC LE

SKIN

BONE

LUNGS

HEART

BLOOD

URINE

BLADDER

STOMACI I

GIT

Ti JYRolI)

TUM'OUR

%ID TH-YROID TU MO UR:BLOOD

TUMOUR:LIVER

TUMOUR: IDNEY

KIDNEY:BLOOD

KIDNEY:LIVER

Ni can 11.89 10.17 11.20 1.69 4.36 5.4.3 14.28 14.03 5 5.6 3 3.96 -49 1.80 2.35 8.76 9.61 0.08 0.2 0.8 0.9 2.79 0.77 0.42 1.85 0.54 2.97 6.26 0.58 1.15 0.23 0.26 3.60 2.04 0.02 0.0 0.3 0.2 8.93) 6.92 8.89 2.40 5.96 4.43 12.84 10.11 44.78 13.29 5.10 2.94 2.74 17.29 16.46 0.19 0.4 1.8 1.9 1.24 0.92 1.40 0.43 0.94 0.69 2.89 1.16 4.03 5.00 1. 63 0.44 0.29 6.24 1.61 0.03 0.1 0.4 0.5 SD M~ean 6 MIea n 7.45 6.27 9.24 3.04 7.79 11.00 9.46 42.20 18.31 4.85 4.06 2.74 38.66 -31.43 0.42 0.7 4.2 3.4 16 SD Mean 24 SD Mean 48 SD Mean 1.51 0.79 0.53 0.59 0.76 0.43 1.57 1.59 6.92 0.91 0.50 0.39 7.92 8.39 0.11 0.3 2.0 1.1 5.61 5.20 5. 8 3 2.55 7.94 3.19 7.39 7.28 28.27 7.28 4.74 2.64 1.84 29.47 31.37 0.40 1.1 5.6 5.4 0.64 0.95 0.88 0.65 0.95 0.78 1.62 2.50 .3.00 4.17 1.24 0.90 0.33 9.54 6.54 0.18 0.3 1.8 1.9 5.03 4. 33 5.40 2.70 7.96 2.58 6.64 5.78 28.73 6.92 5.70 3.06 1.65 85.96 44.67 1.24 1.6 8.9 8.3 0.82 1.34 0.98 0.42 0.78 0.58 1.57 0.62 2.23 1.11 1. 13- 0.99 0.26 44.73 8.50 0.64 0.4 4.92 4.58 4.76 2.48 7.41 2.54 6.47 4.87 22.73 6.87 6.63 3.05 1.51 5 49. 83 46.17 2.59 2.0 9.4 9.7 0.90 1.25 0.78 0.33 1.44 0.36 0.64 1.10 2.50 2.11 1.23 0.72 0.07 353.44 7.25 1.13 3.2 3.1 0.0 0.2 0.2 1.0 0.2 0.0 1.2 0.3 Table 6.

Biodistrihution of "'In-DTPA-B72.3 in nu/nu mice.

ID/g Time (hours)

ORGAN

0 o

SD

LIVER

SPLI:EN

KIDNEY

MUSCLE

SKIN

BONE

LUNGS

HEART

BLOOD

URINE

BLADDER

STOMACH

GIT

TAIL

THYRIOD

TUMOUR

%1D THYROID Mecan 13.4 12.52 15.41 2.08 4.17 5.9 18.58 11.9 56.19 11.82 2.43 2.12 9.37 18.66 8.87 0.07 S Mean SD Mean 6 SD Mean 16 SD Mean 24 SD Mean 1.72 2.66 1.57 0.81 1.98 1.12 4.72 3.88 9.04 9.4 0.8 0.34 5.68 8.1 2.39 0.01 10.54 9.56 12.04 1.96 7.54 4.59 13.14 9.59 39.03 5.23 5.59 2.49 2.44 8.39 25.91 17.95 0.06 1.41 1.96 1.67 0.42 1.53 0.53 4.58 1.65 4.66 0 2.85 0.66 0.51 4.38 17.2 4.93 0.03 11.2 10.05 14.13 2.56 9.31 3.85 13.03 11.71 38.23 3.96 8.31 2.2 2.33 8.53 8.09 21.24 0.07 1.2 2.55 1.83 0.32 1.41 0.97 2.65 4.12 3.64 1.16 19.94 0.9 0.42 5.27 3.14 6.11 0.01 7.27 6.47 15.59 2.44 9.48 5.81 8.06 6.25 26.51 4.46 7.68 1.53 2.17 4.98 7.37 38.25 0.07 0.86 1.39 0.82 1.21 1.12 1.89 0.88 1.72 1.68 2.97 0.48 0.25 1.17 1.62 8.71 0.02 8.43 7.32 13.97 2.28 8.44 5.52 6.58 4.99 21.78 4.46 6.88 1.47 2.04 4.69 8.51 35 0.06 48 SD Mean 2.28 1.43 1.64 0.26 0.42 0.58 1.4 0.39 2.36 1.12 1.87 0.46 0.2 0.96 2.21 5.21 0.03 7.59 4.48 15.04 1.84 7.54 5.23 7.07 3.95 17.04 4.43 4.28 1.47 2.33 3.79 4.45 48.99 0.06 0.72 2.28 1.57 0.51 0.75 0.9 1.93 0.45 0.89 1.56 0.39 0.44 0.2 1.37 0.91 4 0.01

TUMOUR:BLOOD

TUMOUR:LIVER

TUMOUR:KIDNEY

KIDNEY:BLOOD

KIDNEY:LIVER

0.4 1.1 V 0.6 0.1 0.4 tj 0.3 0.1 1.2 0.3 WO 00/40585 PCT/AU00/00003 27 It is to be understood that the term "SarAr" as used in the following tables and description refers to compound as shown in example 1.

Table 7 Maximum Radiation Dose Estimates for 6Cu-SarAr-B72.3 Organ Total Dose Total Dose mGy/MBq Rad/mCi Adrenals 0.030 0.111 Brain 0.018 0.068 Breasts 0.021 0.078 Gallbladder Wall 0.036 0.133 LLI Wall 0.094 0.347 Small Intestine 0.047 0.175 Stomach 0.029 0.107 ULI Wall 0.069 0.255 Heart Wall 0.075 0.278 Kidneys 0.129 0.477 Liver 0.152 0.563 Lungs 0.062 0.230 Ovaries 0.026 0.095 Muscle 0.013 0.047 Pancreas 0.030 0.110 Red Marrow 0.029 0.107 Bone Surfaces 0.030 0.111 Skin 0.019 0.069 Spleen 0.088 0.327 Thymus 0.023 0.086 Testes 0.019 0.072 Thyroid 0.020 0.074 Bladder Wall 0.019 0.070 Uterus 0.029 0.109 Total Body 0.024 0.090 Tumour Mass (g) 0.1 S factor rad/uCi-h 2.25 0.48 0.25 0.13 Dose Gy 17.80 3.80 1.97 1.00 Dose rad 1779.75 380.47 196.96 99.67 WO 00/40585 WO 0040585PCT/AUOO/00003 Table 8 Maximum Radiation Dose Estimate for 3 1 1-372.3 Organ Total Dose Total Dose mGy/MBq radlmCi Adrenals 0.821 3.038 Brain 0.514 1.902 Breasts 0.603 2.23 1 Gallbladder Wall 0.913 3.378 LLI Wall 2.24 8.288 Small Intestine 1.3 4.8 Stomach 1.15 4.255 ULI Wall 1.69 6.253 Heart Wall 1.82 6.734 Kidneys 2.41 8.917 Liver 1.92 7.104 Lungs 1.55 5.735 Muscle 0.473 1.750 Ovaries 0.768 2.842 Pancreas 0.858 3.175 Red Marrow 0.783 2.897 Bone Surfaces 0.678 2.509 Skin 0.547 2.024 Spleen 2.04 7.548 Testes 0.595 2.202 Thymus 0.717 2.653 Thyroid 44.6 165.020 Bladder Wall 0.755 2.794 Uterus 1.13 4.181 Total Body 0.715 2.646 Effective Dose 3.45 mSv/MBq 12.765 rem/mCi (Tumour not included) Tumnour Mass (g) 0.1 S factor 3.6 0.759 0.393 Dose per mCi rad 982 207 107 98275 20720 10728 0.2 54 56 5460 WO 00/40585 PCT/AU00/00003 29 Contribution to organ dose from activity in tumour It is assumed that the tumour is a small source located in the lower trunk which will make a contribution to all other organ doses. So that the computer software program known as MIRDOSE3 can be used, the activity is assumed to be located in the ovaries.

The ovary dose given in above was calculated separately. It is not the tumour dose.

Bladder Residence Time The activity excreted via the bladder is almost insignificant.

The total bladder and urine residence times in Table 5 was used as the urine activity in the dose calculation. No excretion model or assumed voiding time were used.

Table 9. Maximum Organ Adrenals Brain Breasts Gallbladder Wall LLI Wall Small Intestine Stomach ULI Wall Heart Wall Kidneys Liver Lungs Muscle Ovaries Pancreas Red Marrow Bone Surfaces Skin Spleen Testes Thymus Thyroid Bladder Wall Uterus Total Body Radiation Dose Estimates for 90

Y

Total Dose Total Dose mGy/MBq rad/mCi 0.57 2.109 0.57 2.109 0.57 2.109 0.57 2.109 3.47 12.839 1.36 5.032 0.709 2.623 2.25 8.325 1.8 6.660 7.87 29.119 2.89 10.693 1.88 6.956 0.232 0.858 0.57 2.109 0.57 2.109 0.964 3.567 1.03 3.811 0.57 2.109 2.32 8.584 0.57 2.109 0.57 2.109 1.72 6.364 0.444 1.643 0.57 2.109 0.645 2.387 Tumour Mass (g) 0.1 1 2

S

rad/uCi-h 8.97 2.51 1.4 0.758 Dose per mCi Gy 546 152.7 85.2 46.1 rad 54585 15274 8519 4613 WO 00/40585 PCT/AU00/00003 Contribution to organ dose from activity in tumour It is assumed that the tumour is a small source located in the lower trunk which will make a contribution to all other organ doses. So that the computer software program known as MIRDOSE3 can be used, the activity is assumed to be located in the ovaries. The ovary dose given in above was calculated separately. It is not the tumour dose.

Bladder Residence Time: The activity excreted via the bladder is almost insignificant.

The total bladder and urine residence times in Table 5 was used as the urine activity in the dose calculation. No excretion model or assumed voiding time were used.

Table 10. Radiation Dose Estimate for 31 1-B72.3 (7 days) Organ Total Dose Total Dose mGy/MBq rad/mCi Adrenals 0.377 1.395 Brain 0.24 0.888 Breasts 0.276 1.021 Gallbladder Wall 0.418 1.547 LLI Wall 1.02 3.774 Small Intestine 0.587 2.172 Stomach 0.52 1.924 ULI Wall 0.769 2.845 Heart Wall 0.863 3.193 Kidneys 1.13 4.181 Liver 0.888 3.286 Lungs 0.727 2.690 Muscle 0.212 0.784 Ovaries 0.35 1.295 Pancreas 0.393 1.454 Red Marrow 0.358 1.325 Bone Surfaces 0.309 1.143 Skin 0.25 0.925 Spleen 0.951 3.519 Testes 0.272 1.006 Thymus 0.328 1.214 Thyroid 17.8 65.860 Bladder Wall 0.338 1.251 Uterus 0.497 1.839 Total Body 0.323 1.195 WO 00/40585 PCT/AU00/00003 31 Tumour Dose to 24 hour Mass rad/mCi 0.1 2991 631 1 327 2 166 Table 11. Radiation Dose Estimates for 90 Y (7 days) Organ Total Dose Total Dose mGy/MBq rad/mCi Adrenals 0.5 1.850 Brain 0.365 1.351 Breasts 0.5 1.850 Gallbladder Wall 0.5 1.850 LLI Wall 2.87 10.619 Small Intestine 1.14 4.218 Stomach 0.603 2.231 ULI Wall 1.87 6.919 Heart Wall 1.53 5.661 Kidneys 6.5 24.050 Liver 2.41 8.917 Lungs 1.58 5.846 Muscle 0.194 0.718 Ovaries 0.4 1.480 Pancreas 0.5 1.850 Red Marrow 0.808 2.990 Bone Surfaces 0.858 3.175 Skin 0.5 1.850 Spleen 1.97 7.289 Testes 0.5 1.850 Thymus 0.5 1.850 Thyroid 1.43 5.291 Bladder Wall 0.38 1.406 Uterus 0.5 1.850 Total Body 0.546 2.020 Tumour rad/mCi at Mass 24 hour 0.1 6925 1938 1 1081 2 585 WO 00/40585 PCT/AUOO/00003 32 Radiotherapeutic Study Radiotherapeutic Study was conducted in two parts.

Part A where the theoretical doses to target and non-target organs were calculated for the analogous radioimmunoconjugates.

Part B where the various radioactive levels of 6 Cu-SarAr-B72.3 was injected into tumour bearing mice and the therapeutic effect of the product was monitored as an extension of animal survival time.

Radiotherapeutic Study Part A Biodistribution studies of 1231- and radiolabelled B72.3 were conducted in 1o LS174t tumour bearing nude mice (see Table 5,6) Standard calculations were performed using computer software MIRDOSE 3 which was used to compare target to non-target dose of their analogous therapeutic counterparts and 13'I respectively) with "Cu-SarAr-B72.3 (see Table 7,8,9) Theoretical maximum accumulated dose (which is equivalent to 10 half life decay) for each radioimmunoconjugate was calculated. Total body dose for 6Cu-SarAr-B72.3 was significantly lower (0.09 rad/mCi) than analogous products 2.64 rad/mCi; 9Y- B72.3, 2.387 rad/mCi). Comparative maximum doses to tumours of various sizes was calculated. Doses for and 90Y-B72.3 appear to be better than for 6Cu-SarAr-B72.3.

However, the lack of stability of the radioimmunoconjugate at the tumour site in real biological systems prevents the tumour from receiving the maximum accumulated dose.

Hence doses to target and non-target organs were re-calculated assuming the radioimmunoconjugates and were stable for approximately 24 hours at the tumour site and the non-target organs were dosed for up to 7 days allowing for natural biological clearance. MIRDOSE 3 was used to re-calculate dose to various organs under these conditions (see Table 10,11 Most relevant resultant target to non-target ratios are given in Table 12.

Table 12 Ratio of Target:Non-Target Doses for each Radioimmunoconjugate 64Cu 131 I Tumour (0.1 g):Kidneys 2094 716 288 Tumour (0.1g):Liver 1795 906 776 Total Body Dose for 7 days 0.09 1.195 2.020 *MIRDOSE 3 was used to estimate human organ doses assuming the residence times in man are the same as the animal model. It is acknowledged that this affects the accuracy of the dose estimates.

Radiotherapeutic Study Part B.

WO 00/40585 PCT/AU00/00003 33 In order to assess therapeutic effect of 6Cu, in a real biological system, nude mice bearing LS174t colorectal carcinoma were injected with various doses 10, 20, 30, MBq) of "Cu-SarAr-B72.3.

Results are given in Table 13 and a typical profile of a study is given in Figure 1.

For all activity levels greater than 20 MBq a significant extension in mouse life was achieved. Experimental details follow.

Table 13 Extension of mouse life relative to "Cu-SarAr-B72.3 Part B Experiment Survival' (Days) Control 2 20-25 MBq 30-35 MBq 60-70 MBq 40-45 MBq 30-35 'Greater than 30 of animals; 2 Greater than 50 control animals; Figure 1. Radiotherapeutic effect of 30 MBq of64Cu-SarAr-B72.3 in tumour bearing mice.

Experimental for Radiotherapeutic Study Part B Animal model: LS174t tumour tissue from nude mice was transplanted into nude mice for each experiment.

Animal selection: Only animals bearing tumours 3.5 5.5 mm 7 days post transplantation were selected for each study. Up to 10 animals per dose.

Injection: The product was injected into the nude mice on Day 7 after transplantation of tumour tissue.

Doses: Various activities of Cu-64-SarAr-B72.3 was injected into the mice. Control animals receive only antibody.

Monitoring of animals: Animals were monitored for changes in tumour size Animal mass Behavioural and physical abnormalities movement gait, food intake and hunching) (For any of the above characteristic, frequency of weighing increased) Histology, Haematolog and Biochemistry: Animals were sacrificed at pre-determined time points (2 days, 1, 2, 3, 4 weeks, 2, 3, 4, 5 and 6 months).

Radiotoxic effects were monitored.

(conducted by external pathologist of Department of Veterinary Anatomy and Pathology, University of Sydney) Endpoint of the study: 1) Body weight loss 20 WO 00/40585 PCT/AU00/00003 34 2) Rapid weight loss of> 10% overnight.

3) Ulceration of tumour 4) Limitation of normal behaviour ability to feed or drink).

Tumour size 10 x 10 mm (UK Cancer Council) Example 7 In accordance with the description of the invention provided above specific preferred pharmaceutical compositions of the present invention may be prepared, and examples of which are provided below. The following specific formulations are to be construed as merely illustrative examples of formulations and not as a limitation of the scope of the present invention in any way.

A compound of Formula may be administered alone, although it is preferable that it be administered as a pharmaceutical formulation.

Example 7(a) Composition for Parenteral Administration A pharmaceutical composition of the present invention for intramuscular injection could be prepared to contain 1 mL sterile isotonic saline, and 1 mg of compound of Formula Similarly, a pharmaceutical composition for intravenous infusion may comprise 250 ml of sterile Ringer's solution, and 5 mg of compound of Formula Example 7(b) Injectable Parenteral Composition A pharmaceutical composition of this invention in a form suitable for administration by injection may be prepared by mixing 1% by weight of compound of Formula in 12% by volume propylene glycol and isotonic saline. The solution is sterilised by filtration.

Modifications and variations such as would be apparent to a skilled addressee are deemed to be within the scope of the present invention. It is to be understood that the present invention should not be limited to the particular embodiment(s) described above.

Throughout the specification, unless the context clearly indicates otherwise, the word, "comprise", "comprises", "comprising" or other variations thereof shall be understood as meaning that the stated integer is included and does not exclude other integers from being present even though those other integers are not explicitly stated.

Further, the present invention relates to all steps, compounds, intermediates as well as final products.

Claims (33)

14. FEB. 2005 16:30 SPRUSON FERGUSON NO. 7044 P. 28 The claims defining the invention are as follows: 1. A compound which is capable of chelating a metal ion or a metal ion radionuclide, of general Formula /(CR4RS)n. W Z4 C R 5 (1) in which n represents an integer from 2 to 4, wherein each R 4 and R 5 is independently selected from H, CH 3 COOH, NO 2 H 2 PO 4 HS03, CN, C(=O)NH2 and CHO; X and Y are the same or different and are selected from the group consisting of C-R, N, P and C-Z in which R is selected from hydrogen, halogen, hydroxyl, nitro, o. nitroso, amino, optionally substituted alkyl, optionally substituted aryl, optionally rsubstituted aralkyl, cyano, -COOR', COCOOR', NH-COCH 2 Br, -NH-CO-CH=CH- S. *o COOR' in which R' is a hydrogen atom or alkyl group, wherein at least one of X and Y is C-Z; W is selected from the group consisting of NH and S; Z is a functionalised group which is capable of binding said compound of Formula to a molecular recognition unit, selected from S C H2 -NH-(CH2)p-Ar-NH2 and -NH-(CH 2 )p-Ar-NCS, wherein p is an integer from I to 4 and Ar is optionally substituted phenyl; wherein said optional substituents are selected from the group consisting of amino, 20 halogen, hydroxy, mercapto, nitro, cyano, thiocyano, alkyl, alkoxy, halogenoalkyl, acyl, acylamino, acyloxy, carboxyl, alkoxycarboxyl, carbamoyl, pyridoylamino, carboxyalkyl- carbamoyl, N-carboxylalkylcarbamoyl, sulfo, sulfamoyl, mono- or di-alkylated or phenylated sulfamoyl optionally having one or more alkyl substituents, alkylsulfonyl, alkoxysulfonyl, optionally hydroxy-containing phenylsulfonyl or phenoxy sulfonyl; or a pharmaceutically acceptable salt thereof 2. A compound of Formula according to claim 1, wherein W is NH. 3. A compound of Formula according to claim 1 or 2, wherein Z is -NH-(CH 2 ),-Ar-NH2 or -NH-(CH2)p-Ar-NCS, and p is 1 or 2. [IL UBMHI404 AU l ra inu9,20S .dtc:.NXL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:30 SPRUSON FERGUSON NO. 7044 P. 29 36 4. A compound of Formula according to claim 1, wherein W is NH and Z is NH-(CH 2 )p-Ar-NH 2 A compound of Formula according to claim 1, wherein W is NH and Z is -NH-(CH 2 )p-Ar-NCS. s 6. A compound of Formula according to claim 4 or 5, wherein Ar is phenyl. 7. A compound of Formula according to any one of claims 1 to 6, wherein W is NH and n is 2. 8. A compound of Formula according to claim 1 which is NH NH NH 2 NH NH NH-- -C2--OH 2 NH 2 N H N H 9. A compound according to any one of claims 1 to 8, wherein the molecular recognition unit is selected from the group consisting of an antibody, protein, peptide, carbohydrate, nucleic acid, oligonucleotide, oligosaccharide and liposome. 10. A compound according to claim 9, wherein the molecular recognition unit is an antibody. s 11. A compound according to any one of claims I to 10, wherein said compound is complexed with a metal ion selected from the group consisting of Cu, Tc, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti. i* 12. A compound according to claim 11, wherein the metal ion is selected from the group consisting of Cu, Tc, Ga, In, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti. 13. A compound of Formula according to claim 11, wherein the metal ion is a radionuclide selected from the group consisting of Cu. Tc, In, Ga, Fe, Ti and Re. 14. A compound of Formula according to claim 13, wherein the radionuclide is selected from 6 Cu and 67Cu.

15. A compound of Formula according to claim 1, wherein n is 3 or 4 and the compound is complexed with a metal ion selected from the lanthanide group of elements in the Periodic Table.

16. A compound of Formula as defined in claim 1, substantially as hereinbefore described with reference to Example 1. 0o 17. A radiolabelled complex of a compound of Formula as defined in claim 1, substantially as hereinbefore described with reference to Example 2 or 3. (RALUB )4 14642AUtn dtC I ;m9205.doc:NXL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:31 SPRUSON FERGUSON NO. 7044 P. 37

18. A phannaceutical composition for radioimaging or diagnosis comprising a compound of Formula according to any one of claims I to 16, a phannaceutically acceptable salt thereof, a metal ion complex thereof, or a radiolabelled complex thereof, together with a pharmaceutically acceptable carrier.

19. Use of a radiolabelled complex of a compound of Formula according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for diagnosing a disease, radioimaging, or radiotherapy of a disease. The use according to claim 19, wherein said disease is cancer. 0o 21. The use according to claim 19 or 20, wherein said medicament is for diagnosing a disease and the metal ion is a radionuclide selected from the group consisting of Cu, Tc, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti. e 22. The use according to claim 21, wherein the metal ion is a radionuclide is selected from the group consisting of Cu, Tc, In, Ga, Fe, Ti and Re.

23. The use according to claim 22, wherein the radionuclide is selected from 64 Cu and 6 7 Cu.

24. The use according to claim 19 or 20, wherein said medicament is for S*radioimaging a disease and said metal ion is a radionuclide selected from the group consisting of Cu, Tc, Ga, In, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti.

25. The use according to claim 24, wherein the metal ion is a radionuclide selected from the group consisting of Cu, Tc, In, Ga, Fe, Ti and Re.

26. The use according to claim 25, wherein the radionuclide is selected from 6Cu 25 and 6 7 Cu.

27. The use according to claim 19 or 20, wherein said medicament is for radiotherapy of a disease, wherein said metal ion is a radionuclide selected from the group consisting of Cu, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Pt, Zn, Cd, Mn, Ru, Pd, Hg and Ti.

28. The use according to claim 27, wherein the metal ion is a radionuclide selected from the group consisting of Cu, In, Ga, Fe, Ti and Re.

29. The use according to claim 28, wherein the radionuclide is selected from 64 Cu and 67 Cu. (R:\UtlB )4 04 :AU IincUldodIcl& 920 3.do; NXL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:31 SPRUSON FERGUSON NO. 7044 P. 31 38 The use according to claim 19, wherein n is 3 or 4 and the compound is complexed with a radiolabelled metal ion selected from the lanthanide group of elements in the Periodic Table.

31. A method for diagnosing a disease, radioimaging, or radiotherapy of a disease in a mammal, comprising administering to said mammal an effective amount of a radiolabelled complex of a compound of Formula according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 18.

32. The method according to claim 31, wherein said disease is cancer.

33. The method according to claim 31 or 32, wherein said method is for diagnosing a disease and the metal ion is a radionuclide selected from the group consisting of Cu, Tc, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti. 1

34. The method according to claim 33, wherein the metal ion is a radionuclide Is selected from the group consisting of Cu, Tc, In, Ga, Fe, Ti and Re. C 35. The method according to claim 34, wherein the radionuclide is selected from S~ 64Cu and 67 Cu. I k

36. The method according to claim 31 or 32, wherein said medicament is for radioimaging a disease and said metal ion is a radionuclide selected from the group S 20 consisting ofCu, Tc, Ga, In, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti.

37. The method according to claim 36, wherein the metal ion is a radionuclide selected from the group consisting ofCu, Tc, In, Ga, Fe, Ti and Re. •38. The method according to claim 37, wherein the radionuclide is selected from 25 64Cu and 6 7 Cu.

39. The method according to claim 31 or 32, wherein said medicament is for radiotherapy of a disease, wherein said metal ion is a radionuclide selected from the group consisting ofCu, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Pt, Zn, Cd, Min, Ru, Pd, Hg and Ti.

40. The method according to claim 39, wherein the metal ion is a radionuclide selected from the group consisting of Cu, In, Ga, Fe, Ti and Re.

41. The method according to claim 40, wherein the radionuclide is selected from 64 Cu and 67 Cu. JR UJOII)4O442!AUffnwdtWwn05. doaNXL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:31 SPRUSON FERGUSON NO. 7044 P. 32 39

42. The method according to claim 31, wherein n is 3 or 4 and the compound is complexed with a radiolabellcd metal ion selected from the lanthanide group of elements in the Periodic Table.

43. A conjugate, compound comprising at least one compound' of Formula (I) s according to any one of claims 1 to 16, or a metal complex, or radiolabelled complex, or a pharmaceutically acceptable salt thereof, bonded to at least one molecular recognition unit comprising an antibody, protein, peptide, carbohydrate, oligonucleotide or oligosaccharide.

44. A conjugate compound according to claim 43, wherein the molecular to recognition unit is an antibody. A conjugate compound as defined in claim 43, substantially as hereinbefore described with reference to Example 4. I 46. A pharmaceutical composition comprising a conjugate compound according @00 Ito any one of claims 43 to 45, or metal complex thereof, together with a pharmaceutically 1is acceptable carrier.

47. Use of a radiolabelled metal ion complex of a conjugate compound according to any one of claims 43 to 45, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for diagnosing a disease, radioimaging, or radiotherapy of a disease. z0 48. The use according to claim 47, wherein said disease is cancer.

49. The use according to claim 47 or 48, wherein said medicament is for diagnosing a disease and the metal ion is a radionuclide selected from the group consisting of Cu, Tc, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti.

50. The use according to claim 49, wherein the metal ion is a radionuclide :selected from the group consisting of Cu, Tc, In, Ga, Fe, Ti and Re.

51. The use according to claim 50, wherein the radionuclide is selected from 6Cu and 67 Cu.

52. A method for diagnosing a disease, radioimaging, or radiotherapy of a disease in a mammal, comprising administering to said mammal an effective amount of a radiolabelled metal ion complex of a conjugate compound according to any one of claims 43 to 45, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 46.

53. The method according to claim 52, wherein said disease is cancer. IM440423AUs mndedcims9205.doc:NXL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14 14. FEB. 2005 16:32 SPRUSON FERGUSON NO. 7044 P. 33

54. The method according to claim 52 or 53, wherein said method is for diagnosing a disease and the metal ion is a radionuclide selected from the group consisting of Cu, Tc, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg and Ti.

55. The method according to claim 54, wherein the metal ion is a radionuclide selected from the group consisting of Cu, Tc, In, Ga, Fe, Ti and Re.

56. The method according to claim 55, wherein the radionuclide is selected from 64 Cu and 67 Cu. Dated 14 February, 2005 Australian Nuclear Science and Technology Organisation The Australian National University Patent Attorneys for the Applicants/Nominated Persons SPRUSON FERGUSON oi oo i ILU 4H4 4CfAUaCfitCggmal ,CiWFo: NPAL COMS ID No: SBMI-01119416 Received by IP Australia: Time 16:37 Date 2005-02-14