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Title:
COMPOUNDS FOR IMAGING AND THERAPY
Document Type and Number:
WIPO Patent Application WO/2008/025941
Kind Code:
A3
Abstract:
The invention provides metal complexes of formula (I), wherein M is a transition metal or a p-block metal, and a, b, c, n, L, X, X', Y, Y', L1, L1', R1, R1', R2 and R2' are defined herein. Such complexes are useful in medical imaging and therapy, in particular, in the treatment of conditions characterised by undesirable cellular proliferation. The invention further provides processes for producing the complexes of the invention.

Inventors:
PASCU SOFIA LOANA (GB)
WAGHORN PHILIP ALAN (GB)
CHURCHILL GRANT CHARLES (GB)
SIM ROBERT BRAIDWOOD (GB)
Application Number:
PCT/GB2007/002950
Publication Date:
April 17, 2008
Filing Date:
August 03, 2007
Export Citation:
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Assignee:
ISIS INNOVATION (GB)
PASCU SOFIA LOANA (GB)
WAGHORN PHILIP ALAN (GB)
CHURCHILL GRANT CHARLES (GB)
SIM ROBERT BRAIDWOOD (GB)
International Classes:
C07C337/08; C07F3/00
Domestic Patent References:
WO2005084168A22005-09-15
Foreign References:
US20030208067A12003-11-06
US20030187007A12003-10-02
US3382266A1968-05-07
Other References:
J.A. MCCLEVERTY, ET AL.: "Complexes of transition metals with Schiff bases and the factors influencing their redox properties. I. Nickel and copper complexes of some diketone bis-thiosemicarbazones", JOURNAL OF THE CHEMICAL SOCIETY, SECTION A: INORGANIC, PHYSICAL, THEORETICAL, no. 17, 1970, ROYAL SOCIETY OF CHEMISTRY LETCHWORTH, GB, pages 2829 - 2836, XP002459283
G. R. GUMMERUS, ET AL.: "Über isomere Dithiosemicarbazone von Methylcyclopentenolonen", ACTA CHEMICA SCANDINAVICA, vol. 10, no. 3, 1956, MUNKSGAARD, COPENHAGEN, DK, pages 459 - 465, XP002459284
DATABASE CAPLUS CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; XP002459285, Database accession no. 1967:463894
Attorney, Agent or Firm:
KEEN, Celia Mary . et al. (14 South SquareGray's Inn,London, WC1R 5JJ, GB)
Download PDF:
Claims:
CLAIMS

1. A metal complex of formula (I):

(D

for use in the treatment of a condition characterised by undesirable cellular proliferation, wherein:

M is a transition metal or a p-block metal; either (i) n is 0, b is a bond and c is not a bond; (ii) n is 1, b is not a bond and c is a bond; or (iii) n is 1, b is a bond and c is not a bond;

L is a ligand comprising an electron donor group; a is a C 5-10 carbocyclic, Cs -10 heterocyclic, Cs -1O aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a Cs -10 carbocyclic ring, a C 5-1O heterocyclic ring, a C 5-10 aryl ring and a Cs -10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P;

Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk-

C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7)- 5 -alk-N(R7)-, -N(R7)C(O), -alk- N(R7)C(0)-, -C(O)N(R7) 5 -alk-C(0)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)- 5 -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(0)0-, -alk-N(R7)C(O)O- 5 - OC(O)N(R7)- 5 -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7)- 5 -alk-N(R7)C(O)N(R7)-, -

N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl ', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C^ 10 alkylamino, di(Ci- 10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-2O heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-2 O alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene; Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-I0 alkylamino, di(C 1 - 10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-2O heteroaryl, aryl, aryloxy, -alk-C 3 . 20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(SJ), O, S or arylene; -alk- is unsubstituted or substituted Ci -20 alkylene which is optionally interrupted by

N(R7), O, S or arylene; and

R7 is H, Ci-6 alkyl, C3.1 0 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll', Rl' and R2' may form, together with the N atom to which Ll' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-2O heteroaryl group.

2. A metal complex of formula (I) :

(I)

for use in a method of treatment of the human or animal body by therapy or in a diagnostic method practised on the human or animal body, wherein:

M is a transition metal or a p-block metal; either (i) n is 0, b is a bond and c is not a bond; (ii) n is 1 , b is not a bond and c is a bond; or (iii) n is 1 , b is a bond and c is not a bond;

L is a ligand comprising an electron donor group; a is: (i) a Cs -10 carbocyclic, Cs -10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring which is part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a Cs -10 carbocyclic ring, a C 5-1O heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted; (ii) an unsubstituted or substituted C 5-I0 aryl ring; or (iii) an unsubstituted or substituted C 5-10 heteroaryl ring;

X and X', which are the same or different, are independently selected from N and P;

Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(0), -alk-

N(RT)C(O)-, -C(O)N(RT), -alk-C(O)N(RT), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(RT>, - alk-C(S)N(RT>, -N(RT)C(S)-, -alk-N(RT)C(S)- 5 -S(O) 2 N(R7)- 5 -JUk-S(O) 2 N(RT)-, - N(RT)S(O) 2 -, -alk-N(RT)S(0) 2 -, -S(O) -, -alk-S(O)-, -N(RT)C(O)O-, -alk-N(RT)C(O)O-, - OC(O)N(RT)-, -alk-OC(O)N(RT)-, -N(RT)C(O)N(RT)-, -alk-N(RT)C(O)N(RT)-, - N(RT)C(S)N(RT)-, -alk-N(RT)C(S)N(R7)-, -N=C(RT)-, -alk-N=C(RT)-, -C(RT)=N-, -alk- C(RT)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z 5 L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C1.20 alkyl, C 2-20 alkenyl, C 2-2 O alkynyl, amino, C 1-I0 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3 . 20 heteroaryl, aryl, aryloxy, -alk-C 3 . 20 carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3 . 20 heteroaryl, and -atk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1 ^o alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3 _ 2 o carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3 . 20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT) 5 O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and

RT is H 5 C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll , Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll' , Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3 . 20 heteroaryl group.

3. A metal complex of formula (I) :

(D

wherein:

M is a transition metal or a p-block metal; either (i) n is 0, b is a bond and c is not a bond; (ii) n is 1 , b is not a bond and c is a bond; or (iii) n is 1, b is a bond and c is not a bond;

L is a ligand comprising an electron donor group; a is: (i) a C 5-10 carbocyclic, Cs -10 heterocyclic, Cs -10 aryl or C 5-10 heteroaryl ring which is part of a fused bi- 5 tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5 - 10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein at least one ring of said fused bi-, tri-, tetra- or polycyclic ring system is a C 5-10 aryl ring or a Cs-iQ heteroaryl ring, and wherein the ring system is unsubstituted or substituted; (ii) an unsubstituted or substituted C 5-10 aryl ring; or (iii) an unsubstituted or substituted C 5-10 heteroaryl ring;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S 5 O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(O), -alk-

N(RT)C(O)-, -C(O)N(RT), -alk-C(O)N(RT), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)- 5 -N(RT)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(RT)-, -alk-S(O) 2 N(R7)- 5 - N(RT)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(RT)C(O)O-, -alk-N(R7)C(0)0-, - OC(O)N(RT)-, -alk-0C(0)N(R7)-, -N(RT)C(O)N(RT)-, -alk-N(RT)C(O)N(RT)-, - N(R7)C(S)N(R7)- 5 -alk-N(RT)C(S)N(RT)-, -N=C(RT)-, -alk-N=C(R7)-, -C(RT)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-2 O alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1 . 1 o)alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -a\k-C^. 2 o heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll \ and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2 . 2 o alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fiuorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-1 o)alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(R7), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll , Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl' andR2' may form, together with the N atom to whichLl' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group.

4. A metal complex according to claim 1 or claim 2 wherein at least one ring of said fused bi-, tri-, tetra- or polycyclic ring system is a C 5-1O aryl ring or a Cs -I0 heteroaryl ring.

5. A metal complex according to any one of the preceding claims, wherein a is part of said fused bi-, tri-, tetra- or polycyclic ring system.

6. A metal complex according to any one of the preceding claims, wherein a is a C 5-6 carbocyclic ring, which ring is part of said fused tri- or tetracyclic ring system, wherein the ring system is selected from the groups of the following formulae (Ha), (lib), (lie) and (Hd), which groups may be substituted or unsubstituted at any one or more available positions in the ring system:

7. A metal complex of formula (Id) :

(Id) wherein M 5 b, c, n, L, X', X, Y', Y, R2 5 R2\ Ll, Ll', Rl and Rl' are as defined in claim 1.

8. A metal complex according to any one of the preceding claims wherein L is selected from: an unsubstituted or substituted C 5-1O heterocyclic ring; an unsubstituted or substituted C 5-1O heteroaryl ring; an unsubstituted or substituted organic radical; a halo group; DMSO; water; a macromolecule; a C 1-20 hydrocarbon molecule, which hydrocarbon molecule comprises one or more heteroatoms, is unsubstituted or substituted and is optionally interrupted by O, S, arylene or N(R7); or a compound comprising from 2 to 20 unsubstituted or substituted Cs -I0 heterocyclic rings, wherein each of said heterocyclic rings is linked to another of said heterocyclic rings by a group A, wherein A is a covalent bond, substituted or unsubstituted arylene, or substituted or unsubstituted C 1 ^ 0 alkylene, which compound is either (i) complexed to M", wherein M" is a transition metal or a main group metal, or (ii) uncomplexed, and wherein said C 1 ^o alkylene is optionally interrupted by by N(R7), O, S or arylene.

9. A metal complex according to any one of the preceding claims wherein said electron donor group of L is an O, S, N or P atom, or wherein L is a halo group.

10. A metal complex according to any one of the preceding claims wherein L is selected from a halo group; l,4-diaza-bicyclo[2,2,2]octane; a porphyrin; a porphyrin complexed to M"; a nitrogenous base selected from guanine, adenine, cytosine, uracil and thymine; a nucleoside; a nucleotide; a deoxynucleoside; a deoxynucleotide; an oligonucleotide; a

polynucleotide; an oligopeptide; a polypeptide; and a compound of any one of the following formulae (HIa) 5 (HIb), (IIIc), (HId), (HIe) 5 (HIf) 5 (HIg) 5 (IHh) 5 (HIi) 5 (IVa) and (IVb):

wherein M" is a transition metal or a main group metal.

11. A metal complex according to any one of the preceding claims wherein: X and X' are both N 5 and wherein Y and Y' are both S.

12. A metal complex according to any one of the preceding claims wherein: Ll and Ll ' are both covalent bonds;

Rl and Rl ', which are the same or different, are each independently selected from hydrogen, and a substituted or unsubstituted group selected from Ci. 2O alkyl, C 2 . 2 o alkenyl, C 2- 20 alkynyl, amino, Ci -10 alkylamino, di(C 1-1 o)alkylamino, C 3 - 2 0 carbocyclyl, C 3-2O heterocyclyl, C 3-2O heteroaryl, aryl, aryloxy, -8Ik-C 3-2 O carbocyclyl, -alk-C3- 20 heterocyclyl, -a.k-C3.20 heteroaryl, and -alk-aryl, wherein said C 1-20 alkyl, C 2-2O alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene; and

R2 and R2', which are the same or different, are each independently selected from H, substituted or unsubstituted C 1-6 alkyl and substituted or unsubstituted phenyl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted Cs -7 heterocyclyl group or an unsubstituted or substituted C 5-7 heteroaryl group; and provided that Ll ', Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 5-7 heterocyclyl group or an unsubstituted or substituted C 5-7 heteroaryl group.

13. A metal complex according to any of claims 1 to 11 wherein either Rl or Rl ' is selected from Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from Ci -2O alkyl, C 2-20 alkenyl, C 2-2 O alkynyl, amino, C 1-10 alkylamino, di(C 1 . 1 o)alkylamino, C 3-20 carbocyclyl, C3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-2 o carbocyclyl, -alk-C 3 . 2 0 heterocyclyl, -alk-C 3 . 2 o heteroaryl, and -alk-aryl, which group is further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined for Ll and Ll' in claim 1, and wherein said Ci -2O alkyl, C 2-2 O alkenyl and C 2-2 O alkynyl are optionally interrupted by N(R7), O, S or arylene.

14. A metal complex according to any one of the preceding claims wherein Z is a biologically active molecule, an amino acid, a peptide, an oligopeptide, or a polypeptide.

15. A metal complex according to any one of the preceding claims wherein the biologically active molecule is a monoclonal antibody, an antibody fragment, octreotide or folic acid.

16. A complex according to any one of claims 1 to 13 wherein Z is a Cu or Zn complex of a bis(thiosemicarbazone) or of a thiosemicarbazone, thereby creating a dimer wherein two

complexes are linked by -Ll-Rl-Ll-, -Ll-alk-Rl-Ll-, -Ll-Rl -alk-Ll- or -Ll-alk-Rl-alk-Ll- wherein Ll and Rl are as defined in any one of claims 1 to 11.

17. A metal complex according to any one of the preceding claims wherein M is Cu, Zn, Ni, Ga 5 In, Zr or Tc.

18. A metal complex according to any one of the preceding claims wherein M is 99m Tc, 111 In, 67 Ga 5 89 Zr 5 60 Cu 5 61 Cu 5 62 Cu or 64 Cu.

19. A metal complex according to any one of the preceding claims which is hypoxic selective.

20. A pharmaceutical composition comprising a metal complex as defined in claim 2 or claim 3 and a pharmaceutically acceptable carrier.

21. Use of a metal complex as defined in any one of claims 1 to 19 in the manufacture of a medicament for use in the treatment of a condition characterised by undesirable cellular proliferation.

22. A method of treating a condition characterised by undesirable cellular proliferation, which method comprises administering to a patient in need of such treatment an effective amount of a metal complex as defined in any one of claims 1 to 19.

23. A diagnostic agent or medical imaging agent which comprises a metal complex as defined in any one of claims 1 to 19.

24. An agent according to claim 23 which is suitable for both radioactive imaging and non-radioactive optical imaging, wherein the metal complex comprises a radionuclide.

25. Use of a metal complex as defined in any one of claims 1 to 19 in the manufacture of a medicament for use as a diagnostic agent, an imaging agent or a therapeutic agent.

26. Use according to claim 25 wherein the medicament is for use as a combined imaging and therapeutic agent.

27. Use according to claim 25 or claim 26 wherein the imaging agent is suitable for both radioactive imaging and non-radioactive optical imaging, wherein the complex comprises a radionuclide.

28. An agent according to claim 24 or use according to claim 27 wherein said radioactive imaging is either Positron Emission Tomography or Single Photon Emmission Computerised Tomography, and said non-radioactive optical imaging is fluorescence imaging.

29. A method of imaging a cell or in vitro biopsy sample, which method comprises: (a) contacting the cell or in vitro biopsy sample with a metal complex as defined in any one of claims 1 to 18; and (b) imaging the cell or in vitro biopsy sample.

30. A method of imaging a patient in need thereof, which method comprises: (a) administering to the patient a metal complex as defined in any one of claims 1 to 19; and (b) imaging the patient.

31. A process for producing a metal complex of formula (Ia):

wherein:

M is a transition metal or a p-block metal; a is a C 5-10 carbocyclic, C 5-10 heterocyclic, C 5-1O aryl or C 5-1O heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is

independently selected from a Cs -10 carbocyclic ring, a C 5-10 heterocyclic ring, a Cs-I 0 aryl ring and a Cs -10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, Ci-C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-0C(O)- 5 -0-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(0), -alk-

N(R7)C(O)-, -C(O)N(R7), -alk-C(0)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(RT)C(O)CK -alk-N(R7)C(0)0-, - OC(O)N(RT)-, -alk-0C(0)N(R7)-, -N(RT)C(O)N(RT)-, -alk-N(R7)C(O)N(R7>, - N(RT)C(S)N(RT)-, -alk-N(R7)C(S)N(R7)-, -N=C(RT)-, -alk-N=C(R7)-, -C(RT)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl ', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-2 O alkenyl, C 2-20 alkynyl, amino, C 1-10 atkylamino, di(Ci.io)alkylamino, C 3-2O carbocyclyl, Cs -20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3 . 20 carbocyclyl, -alk-C 3- 2 o heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from Ci -20 alkyl, C 2-20 alkenyl, C 2 . 20 alkynyl, amino, Ci -10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -3Ik-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which Ci -20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

-alk- is unsubstituted or substituted C 1 ^ 0 alkylene which is optionally interrupted by N(R7), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll , Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll' , Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating, in the presence of a solvent, a salt of M with either:

(i) a compound of following formula (X), a compound of the following formula (Y) and a compound of the following formula (Z):

(X) , (Y) , (Z) ; or (ii) a compound of the following formula (V) and a compound of the following formula (Z):

(V) (Z)

32. A process for producing a metal complex of the following formula (Ia) by transmetallation:

wherein:

M is a transition metal or a p-block metal;- a is a C 5-10 carbocyclic, C 5-1O heterocyclic, C 5-10 aryl or C 5-1O heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-I0 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O,

N(R5) and P(Ri), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7>, -alk-N(R7)-, -N(R7)C(0), -alk- N(R7)C(O>, -C(0)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S>, -S(O) 2 N(R7>, -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(O)O-, -alk-N(R7)C(O)O- 5 - OC(O)N(R7)- 5 -alk-OC(O)N(R7>, -N(R7)C(O)N(R7>, -alk-N(R7)C(O)N(R7)-, -

N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-1 o)alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3 - 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by

L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-2 O alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O 5 S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-1 o)alkylamino, C 3 . 20 carbocyclyl, C 3-20 heterocyclyl, C 3-2 O heteroaryl, aryl, aryloxy, -alk-C 3-2 o carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-2O heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating a salt of M, in the presence of a solvent, with a metal complex of the following formula (W):

wherein M' is a metal other than M and is either (i) a transition metal or (ii) a main group metal; and a, X', X, Y', Y 5 R2, R2', Rl 5 Rl', Ll and Ll' are as defined above for the metal complex of formula (Ia).

33. A process for producing a metal complex of the following formula (Ib):

(Ib) wherein: M is a transition metal or a p-block metal; either (i) b is a bond and c is not a bond, or (ii) c is a bond and b is not a bond; L is a ligand comprising an electron donor group; a is a C 5-10 carbocyclic, C 5-1O heterocyclic, C 5-10 aryl or C 5-I0 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5 - 10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-1O aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(0), -alk-

N(R7)C(O)- 5 -C(O)N(R7), -alk-C(O)N(R7) 5 -C(S)-, -alk-C(S)-, -S-, -alk-S- 5 -C(S)N(R7)- 5 -

alk-C(S)N(R7)-, -N(RT)C(S)-. -alk-N(R7)C(S)- 5 -S(O) 2 N(RJ)-, -alk-S(O) 2 N(R7)- 5 - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(O)O- 5 -alk-N(R7)C(O)O- 5 - OC(O)N(R7)- 5 -alk-OC(O)N(R7)- 5 -N(R7)C(O)N(R7)-, -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)- 5 -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1 . 1 o)alkylamino, C 3-20 carbocyclyl, C 3 . 20 heterocyclyl, C 3 . 2 o heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3 . 2 o heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-1 o)alkylamino, C 3-20 carbocyclyl, C 3 . 20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-2 o heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(R7), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3 . 10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3 . 20 heterocyclyl group or an unsubstituted or substituted C 3 . 20 heteroaryl group; and provided that Ll ', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3 . 20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; the process comprising treating a compound of formula (Ia) as defined in claim 31 or claim 32, with either:

G) L; or

(ii) a precursor compound comprising L; resence of a solvent, wherein L is said ligand comprising an electron donor group.

Description:

COMPOUNDS FOR IMAGING AND THERAPY

Field of the Invention The present invention relates to metal complexes of thiosemicarbazone derivatives and the uses of those complexes in medical imaging and therapy. The present invention also relates to processes for producing the complexes of the invention and to pharmaceutical compositions comprising them.

Background to the Invention

Molecular imaging is a growing area of research. There is a wide interest in designing novel imaging probes for biological targets. Targets can then be imaged in vivo with a range of molecular imaging devices to attain research and clinical objectives.

Non-invasive techniques such as fluorescence imaging, PET (Positron Emission Tomography) and SPECT (Single Photon Emission Computerised Tomography), can be used to follow the mechanism of uptake and distribution of metal complexes of interest in terms of therapeutic and imaging applications. Fluorescence imaging can also be used to follow such molecules in living cells in vitro.

Bis(thiosemicarbazone) complexes of transition metals have been known for nearly 50 years. Despite demonstrations of biological activity, the mechanism of action of such complexes at the cellular level remains unknown. There is therefore an ongoing interest to synthesise such complexes and study their biological uptake, by, for example, fluorescence imaging. Indeed, there has been great interest in Cu(II) as a result of its role in biology, and the versatility in its radioactive isotopes. However, Cu(II) is d 9 paramagnetic and as a result tends to quench the fluorescence of adjacent fluorophores. This generally prevents direct fluorescent observation of the copper complexes of interest. By direct substitution with zinc, however, the ability to image via fluorescence is a practical possibility. Despite this, fluorescent, molecular zinc complexes are not well known.

There is therefore an ongoing interest to address these problems, to synthesise biologically active metal complexes for use as imaging probes and/or as therapeutic agents, and to study their mechanism of action at the cellular level.

Summary of the Invention

The inventors have shown that a certain class of metal complexes are taken up into a range of human cancer cell lines and exhibit significant cytotoxicity towards those cell lines. The metal complexes are therefore candidates for use as therapeutic agents, in particular for use in treating cancers and other conditions characterised by undesirable cellular proliferation.

Furthermore, it is a finding of the invention that certain complexes have the potential to be hypoxic selective and may therefore be suitable for therapy and/or imaging of hypoxic tumours. Additionally, or as an alternative to hypoxic selectivity, the complexes may be conjugated to a biologically active molecule which serves to target the complex to the desired site in vivo.

Accordingly, the invention provides metal complex of formula (I):

(I)

for use in the treatment of a condition characterised by undesirable cellular proliferation, wherein:

M is a transition metal or a p-block metal; either (i) n is 0, b is a bond and c is not a bond; (ii) n is 1 , b is not a bond and c is a bond; or (iii) n is 1 , b is a bond and c is not a bond; L is a ligand comprising an electron donor group; a is a Cs -10 carbocyclic, C 5-10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, terra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, terra- or polycyclic ring system is independently selected from a C 5-1 O carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-1O aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O 5 N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk~, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(RJ)-, -alk-N(R7)-, -N(R7)C(0), -alk- N(R7)C(0)-, -C(0)N(R7), -alk-C(0)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S- 5 -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, -

N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(0)0-, -alk-N(R7)C(O)O-, - 0C(0)N(R7)-, -alk-OC(O)N(R7)- 5 -N(R7)C(O)N(R7)- 5 -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl ' , which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylammo, C 3-20 carbocyclyl, Cs -20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-1 o)alkylamino, C 3-2O carbocyclyl, C 3 . 20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(R7), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group.

The invention further provides:

- use of a metal complex as defined above in the manufacture of a medicament for use in the treatment of a condition characterised by undesirable cellular proliferation.

- a method of treating a condition characterised by undesirable cellular proliferation, which method comprises administering to a patient in need of such treatment an effective amount of a metal complex as defined above.

- a pharmaceutical composition for use in treating a condition characterised by undesirable cellular proliferation, comprising a pharmaceutically acceptable carrier or diluent and a metal complex as defined above.

- an agent for the treatment of a condition characterised by undesirable cellular proliferation, comprising a metal complex as defined above.

The inventors have also found that the complexes defined above, including copper complexes, have enhanced fluorescence compared with known metal thiosemicarbazone compounds, and that the complexes possess intrinsic fluorescence. The uptake of the intrinsically fluorescent species, into a range of human cancer cell lines, was observed using fluorescence imaging. The fluorescent properties of the complexes allows the distribution of the complexes within cells to be monitored. In addition, the complexes defined above which are employed in the present invention may be radiolabeled with metastable metal radionuclides which are useful in medical imaging techniques such as PET (Positron Emission Tomography) and SPECT (Single Photon Emmission Computerised Tomography).

Thus, the complexes are candidates for use as imaging agents, using either a radioactive imaging technique such as PET or SPECT, or a non-radioactive optical technique, such as fluorescence. The radiolabelled complexes may be suitable for both non-radioactive and radioactive imaging, which may be used in combination. When combined with

fluorescence imaging, radioactive imaging techniques such as PET and SPECT could provide an extremely powerful tool in the clinical diagnosis and treatment of disease.

Furthermore, the complexes are candidates for use as dual therapy and imaging agents, for example in the treatment and imaging of cancer tumours and other conditions characterised by undesirable cellular proliferation.

Accordingly, the present invention further provides: a diagnostic agent or medical imaging agent which comprises a metal complex as defined above. use of a metal complex as defined above in the manufacture of a medicament for use as a diagnostic agent, an imaging agent and/or a therapeutic agent. a method of imaging a cell or in vitro biopsy sample, which method comprises: (a) contacting the cell or in vitro biopsy sample with a metal complex of the invention as defined above; and (b) imaging the cell or in vitro biopsy sample. a method of imaging a patient in need thereof, which method comprises: (a) administering to the patient a metal complex of the invention as defined above; and (b) imaging the patient.

Many of the metal complexes employed in the present invention are novel compounds.

Accordingly, the invention further provides a metal complex of formula (I):

(I) wherein:

M is a transition metal or a p-block metal; either (i) n is 0, b is a bond and c is not a bond; (ii) n is 1, b is not a bond and c is a bond; or (iii) n is 1, b is a bond and c is not a bond;

L is a ligand comprising an electron donor group; a is: (i) a C 5-10 carbocyclic, C 5-10 heterocyclic, C 5-I0 aryl or C 5-10 heteroaryl ring which is part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein at least one ring of said fused bi-, tri-, tetra- or polycyclic ring system is a C 5-10 aryl ring or a C 5-10 heteroaryl ring, and wherein the ring system is unsubstituted or substituted; (ii) an unsubstituted or substituted C 5-10 aryl ring; or (iii) an unsubstituted or substituted C 5-10 heteroaryl ring; X and X', which are the same or different, are independently selected from N and P;

Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached; Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)~, -alk-N(R7)-, -N(R7)C(0), -alk- N(R7)C(O)-, -C(O)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(O)O-, -alk-N(R7)C(O)O-, - OC(O)N(R7)- 5 -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7>, -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3 . 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a

leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )aIkylammo, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-2 O heteroaryl, aryl, aryloxy, -alk-C 3-2 o carbocyclyl, -alk-C 3-2 o heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O 5 S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll' , Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3 _ 20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group.

The invention further provides a pharmaceutical composition comprising a metal complex of the invention as defined above and a pharmaceutically acceptable carrier. The invention further provides a metal complex of formula (I):

(I)

for use in a method of treatment of the human or animal body by therapy or in a diagnostic method practised on the human or animal body, wherein:

M is a transition metal or a p-block metal; either (i) n is O 5 b is a bond and c is not a bond; (ii) n is 1 , b is not a bond and c is a bond; or (iii) n is 1, b is a bond and c is not a bond;

L is a ligand comprising an electron donor group; a is: (i) a C 5-10 carbocyclic, C 5-10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring which is part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi- 5 tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted; (ii) an unsubstituted or substituted C 5 . 10 aryl ring; or (iii) an unsubstituted or substituted C 5-10 heteroaryl ring;

X and X', which are the same or different, are independently selected from N and P;

Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(O), -alk- N(R7)C(0)-, -C(O)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, -

N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(0)0-, -alk-N(R7)C(O)O-, - OC(O)N(R7>, -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7)-, -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-2 O alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, ditQ.^alkylamino. C 3-20 carbocyclyl, C3 -20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may.be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a

leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from Ci -20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, Ci -I0 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3 . 2 o carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group.

The invention further provides a pharmaceutical composition comprising a metal complex as defined above and a pharmaceutically acceptable carrier. The invention further provides processes for producing the metal complexes employed in the present invention.

Thus, the invention provides a process for producing a metal complex of the following formula (Ia):

wherein:

M is a transition metal or a p-block metal; a is a C 5-10 carbocyclic, C 5-10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system,

wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-1O heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O,

N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(0), -alk- N(R7)C(0)-, -C(O)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)- 5 -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(0)0-, -alk-N(R7)C(O)O- 3 - OC(O)N(R7)- 5 -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7)-, -alk-N(R7)C(O)N(R7)-, -

N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C^)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl ', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-2O carbocyclyl, C 3-20 heterocyclyl, C 3-2O heteroaryl, aryl, aryloxy, -alk-C 3-2 o carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene; Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylammo, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3 . 20 heteroaryl,

and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O 5 S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating, in the presence of a solvent, a salt of M with either:

(i) a compound of following formula (X), a compound of the following formula (Y) and a compound of the following formula (Z):

(U) * i compound of the following formula (V) and a compound of the following formula (Z) :

(V) , (Z) The invention further provides a process for producing a metal complex of the following formula (Ia) by transmetallation:

wherein:

M is a transition metal or a p-block metal; a is a C 5-10 carbocyclic, C 5-1 o-heterocyclic, C 5-10 aryl or C 5-I0 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi- 5 tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O,

N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms, from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7)-, -alk-N(R7)- 5 -N(R7)C(0), -alk- N(R7)C(O)-, -C(O)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(0)0-, -alk-N(R7)C(0)0-, - OC(O)N(R7)-, -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7>, -alk-N(R7)C(O)N(R7)-, -

N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)- 5 -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, Ci -10 alkylamino, di(Ci -10 )alkylarnino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-2 o carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by

L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-2O alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1 J 20 alkyl, C 2 -20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-2 o heterocyclyl, C 3-2 O heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll , Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3 . 20 heteroaryl group; and provided that Ll', Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating a salt of M, in the presence of a solvent, with a metal complex of the following formula (W):

wherein M' is a metal other than M and is either (i) a transition metal or (ii) a main group metal; and a, X', X, Y', Y 5 R2, R2\ Rl 3 Rl', Ll and Ll' are as defined above for the metal complex of formula (Ia).

The invention further provides a process for producing a metal complex of the following formula (Ib):

(Ib) wherein: M is a transition metal or a p-block metal; either (i) b is a bond and c is not a bond, or (ii) c is a bond and b is not a bond; L is a ligand comprising an electron donor group; a is a C 5-1O carbocyclic, C 5-10 heterocyclic, Cs -10 aryl or C 5-1 O heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, terra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5 . 10 aryl ring and a C 5-1O heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S 5 O 5 N(R5) and P(R5), wherein each R5 is independently selected from H 5 C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll ', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(0), -alk-

N(RT)C(O)-, -C(O)N(RT), -alk-C(0)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(RT)-, -

alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)- 5 -alk-S(O) 2 N(R7)- 5 - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 - 5 -S(O) -, -alk-S(O)-, -N(R7)C(O)O- 5 -alk-N(R7)C(O)O-, - OC(O)N(R7)- 5 -alk-OC(O)N(R7)- 5 -N(R7)C(O)N(R7)- 5 -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl ', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C3 -20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3 . 20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2 _ 20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1 . 1 o)alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(R7), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; the process comprising treating a compound of formula (Ia) as defined above, with either:

(i) L; or

(ii) a precursor compound comprising L; in the presence of a solvent, wherein L is said ligand comprising an electron donor group.

Brief Description of the Figures

Fig. Ia is a graph of fluorescence intensity (y axis) versus wavelength in units of nm (x axis) for (a) the acenaphthenequinone-derived Zn complex 14 (solid line) and (b) the corresponding butadiene-derived complex Zn(ATSPh) (dotted line) in DMSO at concentrations of O.OlmM. Fig. Ib is a graph of fluorescence intensity (y axis) versus wavelength in units of nm

(x axis) for (a) the acenaphthenequinone-derived Zn complex 12 (solid line) and (b) the corresponding butadiene-derived complex Zn(ATSM) (dashed line) in DMSO at concentrations of O.OlmM.

Fig. 2 is a graph of fluorescence intensity (y axis) versus wavelength in units of nm (x axis) for compound 13 dissolved in (a) DMSO at a concentration of O.OlmM (solid line), and (b) a 5:95 DMSOrwater mix at a concentration of 0. ImM (dashed line).

Fig. 3 is an energy diagram showing the relative energies in eV (y axis) of the DFT- calculated frontier orbitals (HOMO(-l), HOMO and LUMO) for Zn complex 12, and the nature of the electron density on those frontier orbitals. Fig. 4 is a graph of absorbance (y axis) versus wavelength in units of nm (x axis) for

Zn complex 12 (solid line) and Zn(ATSM) (dashed line).

Fig. 5 shows an IGROV cell fluorescence uptake image (a) and the corresponding bright field image (b) for compound 12, and an IGROV cell fluorescence uptake image (c) and the corresponding bright field image (d) for compound 14. Fig. 6 is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 12 in IGROV cells.

Fig. 7 shows IGROV cell fluorescence uptake images for compound 13, both without lysotracker (a) and with lysotracker (b), and the corresponding bright field image (c).

Fig. 8 is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 13 in IGROV cells.

Fig. 9 shows S W620 cell fluorescence uptake images (left) and the corresponding bright field images (right) for compounds 12 (a) 13 (b) and 14 (c).

Fig. 10a is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 13 in SW620 cells.

Fig. 10b is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 14 in SW620 cells. Fig. 11 shows A431 cell fluorescence uptake images (left) and the corresponding bright field images (right) for compounds 12 (a) 13 (b) and 14 (c).

Fig. 12a is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 13 in A431 cells.

Fig. 12b is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 14 in A431 cells.

Fig. 13 shows a T24 cell fluorescence uptake image (a) and the corresponding bright field image (b) for compound 13, and a T24 cell fluorescence uptake image (c) and the corresponding bright field image (d) for compound 14.

Fig. 14a is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 13 in T24 cells.

Fig. 14b is a graph of fluorescence uptake intensity (y axis) versus time in units of minutes (x axis) for compound 14 in T24 cells.

Fig. 15 is a histogram showing the cytotoxic effect of compounds 12, 13 and 14 on U937 cells with respect to a 1% DMSO standard and medium standard. The histogram shows the number of live U937 cells after 48 hours in units of 10 s cells/ml (y axis) after incubation (a) with medium alone, (b) with 1 :99 DMSO:medium solution, (c) with lOOμM compound 12 in 1:99 DMSO medium, (d) with lOOμM compound 13 in 1:99 DMSO:medium and (e) with lOOμM compound 14 in 1:99 DMSO:medium.

Fig. 16 is a histogram showing the cytotoxic effect of compound 13 on SW620 cells over a range of concentrations, with respect to a 1 % DMSO standard and medium standard. The histogram shows the number of live SW620 cells after 48 hours in units of 10 5 cells/ml (y axis) after incubation (a) with medium alone, (b) with 1:99 DMSO:medium solution, (c) with lOOμM compound 13 in 1:99 DMSO:medium, (d) with 50μM compound 13 in 1:99 DMSO:medium, (e) with 25μM compound 13 in 1 :99 DMSO:medium and (f) with 12.5μM compound 13 in 1 : 99 DMSO :medium.

Fig. 17 is a histogram showing the cytotoxic effect of compound 13 on T24 cells over a range of concentrations, with respect to a 1% DMSO standard and medium standard. The histogram shows the number of live T24 cells after 48 hours in units of 10 5 cells/ml (y axis)

after incubation (a) with medium alone, (b) with 1 :99 DMSO:medium solution, (c) with 50μM compound 13 in 1:99 DMSOmedium, (d) with 25μM compound 13 in 1:99 DMSO:medium, (e) with 12.5μM compound 13 in 1:99 DMSOmedium and (f) with 6.5μM compound 13 in 1 :99 DMSO:medium. Fig. 18 is a histogram showing the cytotoxic effect of compound 13 on A431 cells over a range of concentrations, with respect to a 1% DMSO standard and medium standard. The histogram shows the number of live A431 cells after 48 hours in units of 10 5 cells/ml (y axis) after incubation (a) with medium alone, (b) with 1 :99 DMSO:medium solution, (c) with lOOμM compound 13 in 1:99 DMSOmedium, (d) with 50μM compound 13 in 1:99 DMSOmedium, (e) with 25μM compound 13 in 1 :99 DMSOmedium and (f) with 12.5μM compound 13 in 1:99 DMSOmedium.

Fig. 19 is a histogram comparing the cytotoxic effects of compounds 13, 16 and cis platin, at a range of concentrations, on IGROV cells with respect to a 1% DMSO standard and medium standard. The histogram shows the number of live IGROV cells after 48 hours in units of 10 5 cells/ml (y axis) after incubation (a) with medium alone, (b) with 1 :99

DMSOmedium solution, (c) with lOOμM compound 13 in 1:99 DMSOmedium, (d) with 50μM compound 13 in 1:99 DMSOmedium, (e) with 25 μM compound 13 in 1:99 DMSOmedium, (f) with 12.5μM compound 13 in 1:99 DMSOmedium, (g) with lOOμM cis platin in 1 :99 DMSOmedium, (h) with 50μM cis platin in 1 :99 DMSOmedium, (i) with 25μM cis platin hi 1 :99 DMSOmedium, Q) with 12.5μM cis platin in 1 :99 DMSOmedium, (k) with lOOμM compound 16 in 1:99 DMSOmedium, (1) with 50μM compound 16 in 1:99 DMSOmedium, (m) with 25μM compound 16 in 1:99 DMSOmedium, (n) with 12.5μM compound 16 in 1 :99 DMSOmedium.

Fig. 20 shows (a) a full voltammagram sweep for compound 15 and (b) the enlarged reduction wave of the Cu(II)/Cu(I) couple, at a scan rate of 100mV/s. In each case voltage (V) is given on the y axis and current (A) is given on the x axis.

Fig. 21 shows (a) a full voltammagram sweep for compound 16 and (b) the enlarged reduction wave of the Cu(II)/Cu(I) couple, at a scan rate of 100mV/s. In each case voltage (V) is given on the y axis and current (A) is given on the x axis. Fig. 22 shows CV coupled EPR spectra for complex 16 measured at (1) E = OV, (2) E

= -1.2V, (3) E = OV and (4) E = +1V. In each spectrum the y axis represents intensity (xlOOO) and the x axis represents G (xlOOO).

Fig. 23 is a graph of fluorescence intensity (y axis) versus wavelength in units of nm (x axis) for (a) the Cu complex 15 (solid line), and (b) the acenaphthenequinone-derived Zn complex 12 (dashed line) in 100% DMSO at concentrations of 0. ImM.

Fig. 24 shows an IGROV cell fluorescence uptake image (a) and the corresponding bright field image (b) for compound 16.

Fig. 25 is an HPLC trace of complex 13 after radiolabelling with 64 Cu. The y axis represents counts per second and the x axis represents time in units of minutes.

Fig. 26 is a graph of fluorescence intensity (y axis) versus wavelength in units of nm (x axis) for complexes 17, 18 and 21 (λ ex 480 nm, DMSO 100 μM). Fig. 27 is a graph of fluorescence intensity (y axis) versus wavelength in units of nm

(x axis) for the Zn complex 18 in DMSO. The fluorescence intensity was monitored over 90 minutes and the decay in intensity was observed at 9 minutes (largest second peak), 15 minutes (second-largest second peak), 30 minutes (third-largest second peak), 45 minutes (fourth-largest second peak), 60 minutes (fifth-largest second peak) and 90 minutes (smallest second peak) (λex=480 nm, 0.10 mM).

Figs. 28a and 28b show epifluorescence imaging of 18 in HeLa cells; Fig. 28a is an image taken 60 minutes after loading of 18; Fig. 28b is a bright field image (scale bar ca. 150 μm).

Figs. 29a, 29b, 29c and 29d show confocal fluorescence imaging of 18 in MCF-7 (breast cancer carcinoma) cells. Fig. 29a shows cells incubated with 18 at 4° C for 3h. Fig. 29b shows cells incubated with 18 at 37° C for 3h. Fig. 29c shows a bright field image of cells incubated with 18 at 4° C for 3h (scale bar ca. 200 μm). Fig 29d shows a bright field image of cells incubated with 18 at 37° C for 3h (scale bar ca. 200 μm).

Figs. 30a, 30b and 30c relate to confocal fluorescence imaging of 18 in IGROV cells. Fig. 30a is an image taken 90 minutes after loading with 18 at room temperature. Fig. 30b is a bright field image (scale bar ca. 200 μm). Fig. 30c is a graph of fluorescence intensity (y axis) versus time in units of minutes (x axis), and shows three uptake profiles of 18 in three different IGROV cells from 0 to 90 minutes (photobleaching occurred after 60 min in each case). Fig. 31 is a histogram comparing the cytotoxic effects of compounds 18, 21 and cis platin, at a range of concentrations, on MCF-7 cells with respect to a DMS O -.medium standard and a medium standard. The histogram shows the number of live MCF-7 cells after 48 hours

in units of 10 s cells/ml (y axis) after incubation (A) with medium alone, (B) with 1:99 DMSO:medium solution, (C) with 100μM compound 18 in 1:99 DMSO:medium, (D) with 50μM compound 18 in 1:99 DMS O medium, (E) with 25μM compound 18 in 1:99 DMSOmedium, (F) with 12.5μM compound 18 in 1:99 DMSO:medium, (G) with 100μM compound 21 in 1 :99 DMSO:medium, (H) with 50μM compound 21 in 1 :99 DMSO:medium, (I) with 25μM compound 21 in 1 :99 DMSOmedium, (J) with 12.5μM compound 21 in 1 :99 DMSOmedium, (K) with 100μM cis platin in 1:99 DMSOmedium, (L) with 50μM cis platin in 1 :99 DMSOmedium, (M) with 25 μM cis platin in 1 : 99 DMSOmedium, and (N) with 12.5μM cis platin in 1:99 DMSOmedium. Fig. 32 shows (a) a radio HPLC trace, and (b) a UV HPLC trace of complex 18 after radiolabelling with 64 Cu by transmetallation. Graph (a) is of intensity in units of counts per second (y axis) versus time in units of minutes (x axis). Graph (b) is of intensity in units of mV (y axis) versus time in units of minutes (x axis).

Fig. 33 shows two graphs (a) and (b) of fluorescence intensity (y axis) versus wavelength in units of nm (x axis) for (a) the Zn complex 18 and compound D at lOOμM in DMSO, and (b) the Zn complex 18 and compound D at lμM in DMSO.

Fig. 34 shows an electrochemical in-situ X-band (9.435 GHz) EPR spectrum of 16 taken in [NH 4 ][BF 4 ] electrolyte at room temperature upon applying different potentials between CV electrodes after a given equilibration time; E = OV (top spectrum), E = -1.2V (right spectrum), E = OV (bottom spectrum) and E = +1 V (left spectrum). In each spectrum the x axis represents B in units of mT and the y axis represents amplitude (a.u.).

Fig. 35 shows an X-band (9.450 GHz) cw-EPR spectrum of 16 (powder; 295 K) and its simulation. The x axis represents B in units of mT.

Fig. 36 shows a graph of fluorescence intensity (y axis) versus wavelength in units of nm (x axis) for the copper complexes 15, 16 and 21.

Fig. 37 is a histogram comparing the cytotoxic effects of compounds 13, 16 and cis platin, at a range of concentrations, on MCF-7 cells with respect to a DMSOmedium standard and a medium standard. The histogram shows the number of live MCF-7 cells after 48 hours in units of 10 5 cells/ml (y axis) after incubation (A) with medium alone, (B) with 1 :99 DMSOmedium solution, (C) with 1 OOμM compound 13 in 1 :99 DMSOmedium, (D) with 50μM compound 13 in 1 :99 DMSOmedium, (E) with 25μM compound 13 in 1 :99 DMSOmedium, (F) with 12.5μM compound 13 in 1:99 DMSOmedium, (G) with lOOμM compound 16 in 1:99 DMSOmedium, (H) with 50μM compound 16 in 1:99 DMSOmedium,

(I) with 25μM compound 16 in 1:99 DMSOrmedium, (J) with 12.5μM compound 16 in 1:99 DMSO:medium, (K) with lOOμM cis platin in 1:99 DMSO:medium, (L) with 50μM cis platin in 1:99 DMSO:medium 5 (M) with 25μM cis platin in 1:99 DMSO:medium, and (N) with 12.5μM cis platin in 1 :99 DMSO:medium. Fig. 38 shows a MCF-7 cell fluorescence uptake image (a) and the corresponding bright field image (b) for MCF-7 cells incubated at 4° C for 3h with compound 13 (Zn Et).

Fig. 39 shows a MCF-7 cell fluorescence uptake image (a) and the corresponding bright field image (b) for MCF-7 cells incubated at 37° C for 3h with compound 13 (Zn Et).

Fig. 40 shows a MCF-7 cell fluorescence uptake image (a) and the corresponding bright field image (b) for MCF-7 cells incubated at 37° C for 12 h with compound 13 (Zn Et). Uptake in the nucleus as well as in the cytoplasm was observed. Advanced cell death was observed.

Fig. 41 shows a MCF-7 cell fluorescence uptake image (a) and the corresponding bright field image (b) for MCF-7 cells incubated at 4° C for 3h with compound 18 (Zn allyl). Fig. 42 shows a MCF-7 cell fluorescence uptake image (a) and the corresponding bright field image (b) for MCF-7 cells incubated at 37° C for 3h with compound 18 (Zn allyl). Fig. 43 shows a schematic diagram of a possible mechanism of uptake and localisation of the compounds in cells, as evidenced by Example 41 below, in which 1 represents endocytosis, 2 represents localisation to mitochondria, 3 represents mitochondrial disruption and 4 represents nuclear localisation.

Fig. 44 is a graph of fluorescence intensity (y axis) versus wavelength in units of run (x axis) for the Ga complex 25 in 100% DMSO at a concentration of 20 μM, at room temperature (λ ex = 400 nm).

Detailed Description of the Invention

A C 1-2O alkyl group is an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical. Typically it is C 1-10 alkyl, for example methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl, or C 1-6 alkyl, for example methyl, ethyl, propyl, butyl, pentyl or hexyl, or C M alkyl, for example methyl, ethyl, i-propyl, n-propyl, t- butyl, s-butyl or n-butyl. When an alkyl group is substituted it typically bears one or more (e.g. one to four) substituents selected from substituted or unsubstituted C 1-20 alkyl; substituted or unsubstituted aryl; substituted or unsubstituted aralkyl; cyano; amino; C 1-10 alkylamino; di(C 1-1 o)alkylamino; arylamino; diarylamino; arylalkylamino; nitro; amido;

acylamido; hydroxyl; keto; halo; carboxy; ester; acyl; acyloxy; C 1-10 alkoxy; aryloxy; haloalkyl; sulfhydryl (i.e. thiol, -SH); C 1-I o alkylthio; arylthio; phosphoric acid; phosphate ester; phosphonic acid; phosphonate ester; -N=N-C 1-20 alkyl, which C 1-20 alkyl is unsubstituted or substituted; -N=N-aryl, which aryl is unsubstituted or substituted; -S(O)IU; -S(O) 2 R e ; -S(O) 2 OR 6 ; -S(O)NR s R 6 ; -S(O) 2 NR e R 6 ; -0(CReRe)NR 6 R 6 ; -C(O)NR 6 R 6 ; -CO 2 (CR 6 R e )CONR 6 R 6 ; -OC(O)NR 6 R 6 ; -NR 6 C(O)OR 6 ; -R 6 C(O)NR 6 R 6 ; -CR 6 (N-OR 6 ); -N 2 + ; -CFH 2 ; -CF 2 H; or -CF 3 ; wherein Re is H, C 1-6 alkyl, C 1-6 carbocyclyl or aryl and if two or more Re groups are present these may be the same or different.

Examples of substituted alkyl groups include haloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl and alkaryl groups. The term alkaryl, as used herein, pertains to a C 1-20 alkyl group in which at least one hydrogen atom (e.g., 1, 2, 3) has been replaced with an aryl group. Examples of such groups include, but are not limited to, benzyl (phenylmethyl, PhCH 2 -), benzhydryl (Ph 2 CH-), trityl (triphenylmethyl, Ph 3 C-), phenethyl (phenylethyl, Ph-CH 2 CH 2 -), styryl (Ph-CH=CH-), cinnamyl (Ph-CH=CH-CH 2 -). A C 2-20 alkenyl group or moiety is a straight or branched group or moiety, which contains from 2 to 20 carbon atoms. One or more double bonds may be present in the alkenyl group or moiety, typically one double bond. A C 2-20 alkenyl group or moiety is typically ethenyl or a C 3-10 alkenyl group or moiety. A C 3-10 alkenyl group or moiety is typically a C 3-6 alkenyl group or moiety, for example allyl, propenyl, butenyl, pentenyl or hexenyl. A C 2-4 alkenyl group or moiety is ethenyl, propenyl or butenyl. An alkenyl group may be unsubstituted or substituted by one to four substituents, the substituents, unless otherwise specified, being selected from those listed above for C 1-20 alkyl groups. Where two or more substituents are present, these may be the same or different.

A C 2-20 alkynyl group or moiety is a straight or branched group or moiety which, unless otherwise specified, contains from 2 to 20 carbon atoms. One or more triple bonds, and optionally one or more double bonds may be present in the alkynyl group or moiety, typically one triple bond. A C 2-20 alkynyl group or moiety is typically ethynyl or a C 3-10 alkynyl group or moiety. A C 3-1O alkynyl group or moiety is typically a C 3-6 alkynyl group or moiety, for example propynyl, butynyl, pentynyl or hexynyl. A C 2-4 alkynyl group or moiety is ethynyl, propynyl or butynyl. An alkynyl group may be unsubstituted or substituted by one to four substituents, the substituents, unless otherwise specified, being selected from those listed above for C 1 ^o alkyl groups. Where two or more substituents are present, these may be the same or different.

A C 5-10 carbocyclic ring is a closed ring of from 5 to 10 covalently linked carbon atoms, which ring is saturated or unsaturated. Typically, the C 5-10 carbocyclic ring is not an aromatic ring. Typically the C 5-10 carbocyclic ring is a C 5-6 carbocyclic ring. The carbocyclic ring may be saturated or unsaturated. Thus, the term C 5-10 carbocyclic ring includes the sub- classes C 5-10 cycloalkyl ring, C 5-1O cycloalkyenyl ring and C 5-10 cycloalkynyl ring. When a C 5- 10 carbocyclic ring is substituted it typically bears one or more substituents selected from those listed above for C 1-20 alkyl groups. Examples of C 5-10 carbocyclic rings include, but are not limited to: cyclopentane (C 5 ), cyclohexane (C 6 ), cycloheptane (C 7 ), methylcyclopropane (C 4 ), dimethylcyclopropane (C 5 ), methylcyclobutane (C 5 ), dimethylcyclobutane (C 6 ), methylcyclopentane (C 6 ), dimethylcyclopentane (C 7 ), methylcyclohexane (C 7 ), dimethylcyclohexane (C 8 ), menthane (C 1O ), cyclopentene (C 5 ), cyclopentadiene (C 5 ), cyclohexene (C 6 ), cyclohexadiene (C 6 ), methylcyclopropene (C 4 ), dimethylcyclopropene (C 5 ), methylcyclobutene (C 5 ), dimethylcyclobutene (C 6 ), methylcyclopentene (C 6 ), dimethylcyclopentene (C 7 ), methylcyclohexene (C 7 ), dimethylcyclohexene (C 8 ).

A C 5-10 heterocyclic ring is a closed ring of from 5 to 10 covalently linked atoms, which ring is saturated or unsaturated, wherein at least one of the ring atoms is a multivalent ring heteroatom, for example, nitrogen, phosphorus, silicon, oxygen, or sulfur (though more commonly nitrogen, oxygen, or sulfur). Typically, the C 5-1 O heterocyclic ring is not an aromatic ring. Typically, the C 5-10 heterocyclic ring has from 1 to 4 heteroatoms, the remainder of the ring atoms are carbon. Typically, the C 5-10 heterocyclic ring is a C 5-6 heterocyclic ring in which from 1 to 4 of the ring atoms are ring heteroatoms, and the remainder of the ring atoms are carbon atoms. In this context, the prefixes C 5-10 and C 5-6 denote the number of ring atoms, or range of number of ring atoms. When a C 5-10 heterocyclic ring is substituted it typically bears one or more substituents selected from those listed above for C 1-2O alkyl groups.

Examples of monocyclic C 5-10 heterocyclic rings include, but are not limited to: N 1 : pyrrolidine (tetrahydropyrrole) (C 5 ), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C 5 ), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C 5 ), piperidine (C 6 ), dihydropyridine (C 6 ), tetrahydropyridine (C 6 ), azepine (C 7 );

O 1 : oxolane (tetrahydrofuran) (C 5 ), oxole (dihydrofuran) (C 5 ), oxane (tetrahydropyran) (C 6 ), dihydropyran (C 6 ), pyran (C 6 ), oxepin (C 7 );

S 1 : thiolane (tetrahydrothiophene) (C 5 ), thiane (tetrahydrothiopyran) (C 6 ), thiepane

(C 7 );

O 2 : dioxolane (C 5 ), dioxane (C 6 ), and dioxepane (C 7 );

O 3 : trioxane (C 6 ); N 2 : imidazolidine (C 5 ), pyrazolidine (diazolidine) (C 5 ), imidazoline (C 5 ), pyrazoline

(dihydropyrazole) (C 5 ), piperazine (C 6 );

N 1 O 1 : tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ), dihydroisoxazole (C 5 ), morpholine (C 6 ), tetrahydrooxazine (C 6 ), dihydroøxazine (C 6 ), oxazine (C 6 ); N 1 S 1 : thiazoline (C 5 ), thiazolidine (C 5 ), thiomorpholine (C 6 );

N 2 O 1 : oxadiazine (C 6 );

O 1 S 1 : oxathiole (C 5 ) and oxathiane (thioxane) (C 6 ); and,

N 1 O 1 S 1 : oxathiazine (C 6 ).

A C 5-10 aryl ring is an aromatic ring of from 5 to 10 covalently linked carbon atoms. Typically, the C 5-10 aryl ring is a C 5-6 aryl ring, examples of which include cyclopentadienyl (Cp) and phenyl.

A C 5-10 heteroaryl ring is a heteroaromatic ring of from 5 to 10 covalently linked atoms including one or more heteroatoms. The one or more heteroatoms are typically selected from nitrogen, phosphorus, silicon, oxygen and sulfur (more commonly from nitrogen, oxygen and sulfur). A C 5-1O heteroaryl ring is typically a 5- or 6-membered ring (i.e. a C 5-6 heteroaryl ring) containing at least one heteroatom selected from nitrogen, phosphorus, silicon, oxygen and sulfur (more commonly selected from nitrogen, oxygen and sulfur). It may contain, for example, 1, 2 or 3 heteroatoms. Examples of heteroaryl rings include pyridine, pyrazine, pyrimidine, pyridazine, furan, thiofuran, pyrazole, pyrrole, oxazole, oxadiazole, isoxazole, thiadiazole, thiazole, isothiazole, imidazole and pyrazole. hi this context, the prefixes C 5-10 and C 5-6 denote the number of ring atoms, or range of number of ring atoms.

A C 3-2 O carbocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic ring of a carbocyclic compound, which moiety has from 3 to 20 carbon atoms (unless otherwise specified), including from 3 to 20 ring atoms. The carbocyclyl ring may be saturated or unsaturated. Thus, the term "carbocyclyl" includes the sub-classes cycloalkyl, cycloalkyenyl and cycloalkynyl. Preferably, each ring has from 5 to 7 ring atoms. Examples of groups of

C 3-20 carbocyclyl groups include C 3-10 carbocyclyl, C 5-7 carbocyclyl and C 5-6 carbocyclyl. When a C 3-20 carbocyclyl group is substituted it typically bears one or more substituents selected from those listed above for C 1-20 alkyl groups.

Examples of C 3-10 carbocyclyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds: cyclopropane (C 3 ), cyclobutane (C 4 ), cyclopentane (C 5 ), cyclohexane (C 6 ), cycloheptane (C 7 ), methylcyclopropane (C 4 ), dimethylcyclopropane (C 5 ), methylcyclobutane (C 5 ), dimethylcyclobutane (C 6 ), methylcyclopentane (C 6 ), dimethylcyclopentane (C 7 ), methylcyclohexane (C 7 ), dimethylcyclohexane (C 8 ), menthane (C 10 ); unsaturated monocyclic hydrocarbon compounds : cyclopropene (C 3 ), cyclobutene (C 4 ), cyclopentene (C 5 ), cyclopentadiene (C 5 ), cyclohexene (C 6 ), cyclohexadiene (C 6 ), methylcyclopropene (C 4 ), dimethylcyclopropene (C 5 ), methylcyclobutene (C 5 ), dimethylcyclobutene (C 6 ), methylcyclopentene (C 6 ), dimethylcyclopentene (C 7 ), methylcyclohexene (C 7 ), dimethylcyclohexene (C 8 ); saturated polycyclic hydrocarbon compounds : thujane (C 10 ), carane (C 10 ), pinane (C 10 ), bornane (C 10 ), norcarane (C 7 ), norpinane (C 7 ), norbornane (C 7 ), adamantane (C 10 ), decalin (decahydronaphthalene) (C 10 ); unsaturated polycyclic hydrocarbon compounds: camphene (C 1O ), limonene (C 10 ), pinene (C 10 ); polycyclic hydrocarbon compounds having an aromatic ring: indene (C 9 ), indane (e.g., 2,3-dihydro-lH-indene) (C 9 ), tetraline

(1,2,3,4-tetrahydronaphthalene) (C 10 ), acenaphthene (C 12 ), fluorene (C 13 ), phenalene (C 13 ), acephenanthrene (C 15 ), aceanthrene (C 16 ), cholanthrene (C 20 ).

A C 3-1O cycloalkyl group or moiety is a 3- to 10- membered group or moiety, typically a 3 -to 6-membered group or moiety, which may be a monocyclic ring or which may consist of two or more fused rings. Examples OfC 3-10 cycloalkyl groups or moieties include cyclopropane (C 3 ), cyclobutane (C 4 ), cyclopentane (C 5 ), cyclohexane (C 6 ), cycloheptane (C 7 ), methylcyclopropane (C 4 ), dimethylcyclopropane (C 5 ), methylcyclobutane (C 5 ), dimethylcyclobutane (C 6 ), methylcyclopentane (C 6 ), dimethylcyclopentane (C 7 ), methylcyclohexane (C 7 ), dimethylcyclohexane (C 8 ), menthane (C 10 ), thujane (C 10 ), carane (C 1O ), pinane (C 10 ), bornane (C 10 ), norcarane (C 7 ), norpinane (C 7 ), norbornane (C 7 ), adamantane (C 10 ) and decalin (decahydronaphthalene) (C 10 ).

A C 3- 2o heterocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which

moiety has from 3 to 20 ring atoms (unless otherwise specified), of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. When a C 3-20 heterocyclyl group is substituted it typically bears one or more substituents selected from those listed above for C 1-20 alkyl groups. Examples of groups of heterocyclyl groups include C 3- 2oheterocyclyl,

C 5-20 heterocyclyl, C 3-15 heterocyclyl, C 5-15 heterocyclyl, Cs-^heterocyclyl, Cs.nheterocyclyl, Cs-ioheterocyclyl, Cs -10 heterocyclyl, C 3-7 heterocyclyl, C 5-7 heterocyclyl, and Cs-eheterocyclyl. Examples of (non-aromatic) monocyclic C 3-20 heterocyclyl groups include, but are not limited to, those derived from: N 1 : aziridine (C 3 ), azetidine (C 4 ), pyrrolidine (tetrahydropyrrole) (C 5 ), pyrroline (e.g.,

3-pyrroline, 2,5-dihydropyrrole) (C 5 ), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C 5 ), piperidine (C 6 ), dihydropyridine (C 6 ), tetrahydropyridine (C 6 ), azepine (C 7 );

O 1 : oxirane (C 3 ), oxetane (C 4 ), oxolane (tetrahydrofuran) (C 5 ), oxole (dihydrofuran) (Cs), oxane (tetrahydropyran) (C 6 ), dihydropyran (C 6 ), pyran (C 6 ), oxepin (C 7 ); S 1 : thiirane (C 3 ), thietane (C 4 ), thiolane (tetrahydrothiophene) (C 5 ), thiane

(tetrahydrothiopyran) (C 6 ), thiepane (C 7 );

O 2 : dioxolane (C 5 ), dioxane (C 6 ), and dioxepane (C 7 ); O 3 : trioxane (C 6 );

N 2 : imidazolidine (C 5 ), pyrazolidine (diazolidine) (C 5 ), imidazoline (C 5 ), pyrazoline (dihydropyrazole) (C 5 ), piperazine (C 6 );

N 1 O 1 : tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ), dihydroisoxazole (C 5 ), morpholine (C 6 ), tetrahydrooxazine (C 6 ), dihydrooxazine (C 6 ), oxazine (C 6 );

N 1 S 1 : thiazoline (C 5 ), thiazolidine (C 5 ), thiomorpholine (C 6 ); N 2 O 1 : oxadiazine (C 6 );

O 1 S 1 : oxathiole (C 5 ) and oxathiane (thioxane) (C 6 ); and, N 1 O 1 S 1 : oxathiazine (C 6 ).

An aryl group is a substituted or unsubstituted, monocyclic, bicyclic or tricyclic aromatic group which typically contains from 6 to 14 carbon atoms, preferably from 6 to 10 carbon atoms in the ring portion. Examples include phenyl, naphthyl, indenyl, indanyl and anthracenyl groups. An aryl group is unsubstituted or substituted. When an aryl group as defined above is substituted it typically bears one or more substituents selected from those listed above for C 1-20 alkyl groups. Typically it carries 0, 1, 2 or 3 substituents. The term

aralkyl as used herein, pertains to an aryl group in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with a C^ 20 alkyl group, which C 1-20 alkyl group is unsubstituted or substituted and optionally interrupted by N(R7), O, S or arylene, wherein R7 is as defined above. Examples of such groups include, but are not limited to, tolyl (from toluene), xylyl (from xylene), mesityl (from mesitylene), and cumenyl (or cumyl, from cumene), and duryl (from durene).

Alternatively, the ring atoms may include one or more heteroatoms, as in a heteroaryl group. A heteroaryl group is a substituted or unsubstituted mono- or bicyclic heteroaromatic group which typically contains from 6 to 10 atoms in the ring portion including one or more heteroatoms. It is generally a 5- or 6-membered ring, containing at least one heteroatom selected from O, S, N, P, Se and Si. It may contain, for example, 1, 2 or 3 heteroatoms. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, quinolyl and isoquinolyl. A heteroaryl group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically it carries 0, 1, 2 or 3 substituents.

A C 1 - K ) alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. Typically it is C 1-10 alkylene, for instance C 1-6 alkylene. Preferably it is C 1-4 alkylene, for example methylene, ethylene, i- propylene, n-propylene, t-butylene, s-butylene or n-butylene. It may also be pentylene, hexylene, heptylene, octylene and the various branched chain isomers thereof. An alkylene group may be unsubstituted or substituted as specified above for C 1-20 alkyl.

In this context, the prefixes (e.g., C 1-4 , C 1-7 , C 1-20 , C 2-7 , C 3-7 , etc.) denote the number of carbon atoms, or range of number of carbon atoms. For example, the term "C 1-4 alkylene," as used herein, pertains to an alkylene group having from 1 to 4 carbon atoms. Examples of groups of alkylene groups include C 1-4 alkylene ("lower alkylene"), C 1-7 alkylene, C 1-10 alkylene and C 1-20 alkylene.

Examples of linear saturated C 1-7 alkylene groups include, but are not limited to, -(CH 2 ) n - where n is an integer from 1 to 7, for example, -CH 2 - (methylene), -CH 2 CH 2 - (ethylene), -CH 2 CH 2 CH 2 - (propylene), and -CH 2 CH 2 CH 2 CH 2 - (butylene).

Examples of branched saturated C 1-7 alkylene groups include, but are not limited to, -CH(CH 3 )-, -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 CH 2 -,

-CH 2 CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, -CH(CH 2 CH 3 )-, -CH(CH 2 CH 3 )CH 2 -, and -CH 2 CH(CH 2 CH 3 )CH 2 -.

Examples of linear partially unsaturated C 1-7 alkylene groups include, but is not limited to, -CH=CH- (vinylene), -CH=CH-CH 2 -, -CH 2 -CH=CH 2 -, -CH=CH-CH 2 -CH 2 -, -CH=CH-CH 2 -CH 2 -CH 2 -, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH 2 -, -CH=CH-CH=CH- CH 2 -CH 2 -, -CH=CH-CH 2 -CH=CH-, and -CH=CH-CH 2 -CH 2 -CH=CH-.

Examples of branched partially unsaturated C 1-7 alkylene groups include, but is not limited to, -C(CH 3 )=CH-, -C(CH 3 )=CH-CH 2 -, and -CH=CH-CH(CH 3 )-.

Examples of alicyclic saturated C 1-7 alkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-l,3-ylene), and cyclohexylene (e.g., cyclohex-l,4-ylene).

Examples of alicyclic partially unsaturated C 1-7 alkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-l,3-ylene), cyclohexenylene (e.g., 2-cyclohexen- 1 ,4-ylene; 3 -cyclohexen- 1 ,2-ylene; 2,5-cyclohexadien- 1 ,4-ylene).

An arylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, which moiety has from 5 to 14 ring atoms (unless otherwise specified). Typically, each ring has from 5 to 7 or from 5 to 6 ring atoms. An arylene group may be unsubstituted or substituted, for instance, as specified above for aryl.

In this context, the prefixes (e.g., C 5-20 , C 6-20 , C 5-14 , Cs_ 7 , C 5-6 , etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C 5-6 arylene," as used herein, pertains to an arylene group having 5 or 6 ring atoms. Examples of groups of arylene groups include C 5-20 arylene, C 6-20 arylene, C 5-14 arylene, C 6-14 arylene, C 6-10 arylene, C 5-12 arylene, C 5-10 arylene, C 5-7 arylene, C 5-6 arylene, C 5 arylene, and C 6 arylene. The ring atoms may be all carbon atoms, as in "carboarylene groups" (e.g., C 6-20 carboarylene, C 6-14 carboarylene or C 6-10 carboarylene).

Examples of C 6-20 arylene groups which do not have ring heteroatoms (i.e., C 6-20 carboarylene groups) include, but are not limited to, those derived from the compounds

discussed above in regard to aryl groups, e.g. phenylene, and also include those derived from aryl groups which are bonded together, e.g. phenylene-phenylene (diphenylene) and phenylene-phenylene-phenylene (triphenylene) .

Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroarylene groups" (e.g., C 5-10 heteroarylene).

Examples of C 5-10 heteroarylene groups include, but are not limited to, those derived from the compounds discussed above in regard to heteroaryl groups.

As used herein the term halo is a group selected from -F, -Cl, -Br, and -I. As used herein the term keto represents a group of formula: =0 As used herein the term nitro represents a group of formula: -NO 2

As used herein the term acyl represents a group of formula: -C(=0)R, wherein R is an acyl substituent, for example, a substituted or unsubstituted C 1-20 alkyl group, a substituted or unsubstituted C 3-20 heterocyclyl group, or a substituted or unsubstituted aryl group. Examples of acyl groups include, but are not limited to, -C(=O)CH 3 (acetyl), -C(O)CH 2 CH 3 (propionyl), -C(=O)C(CH 3 ) 3 (t-butyryl), and -C(=O)Ph (benzoyl, phenone).

As used herein the term acylamido represents a group of formula: -NR C(=0)R , wherein R 1 is an amide substituent, for example, hydrogen, a C^oalkyl group, a C 3-20 heterocyclyl group, an aryl group, preferably hydrogen or a C 1-20 alkyl group, and R 2 is an acyl substituent, for example, a C 1-20 alkyl group, a C 3-20 heterocyclyl group, or an aryl group, preferably hydrogen or a C 1-20 alkyl group. Examples of acylamido groups include, but are not limited to, -NHC(=0)CH 3 , -NHC(=O)CH 2 CH 3 , -NHC(=O)Ph, -NHC(=O)C 15 H 31 and -NHC(=O)C 9 Hi9.

As used herein the term acyloxy (or reverse ester) represents a group of formula: -OC(=O)R, wherein R is an acyloxy substituent, for example, substituted or unsubstituted C 1-20 alkyl group, a substituted or unsubstituted C 3-20 heterocyclyl group, or a substituted or unsubstituted aryl group, typically a C 1-6 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(=O)CH 3 (acetoxy), -OC(=O)CH 2 CH 3 , -OC(=O)C(CH 3 ) 3 , -OC(=O)Ph, and -OC(=O)CH 2 Ph.

As used herein the term ester (or carboxylate, carboxylic acid ester or oxycarbonyl) represents a group of formula: -C(K))OR, wherein R is an ester substituent, for example, a C 1-6 alkyl group, a C 3-20 heterocyclyl group, or an aryl group (typically a phenyl group). Examples of ester groups include, but are not limited to, -C(=O)OCH 3 , -C(=O)OCH 2 CH 3 , -C(=O)OC(CH 3 ) 3 , and -CC=O)OPL

As used herein the term phosphonic acid represents a group of the formula: -P(^O)(OH) 2 . As would be understood by the skilled person, a phosphonic acid group can exist in protonated and deprotonated forms (i.e. -P(=O)(OH) 2 , -P(=O)(O " ) 2 and -P(=O)(OH)(O " )) all of which are within the scope of the term "phosphonic acid". As used herein the term phosphonic acid salt represents a group which is a salt of a phosphonic acid group. For example a phosphonic acid salt may be a group of the formula - Pt=O)(OH)(O-X + ) wherein X is a monovalent cation. X + may be an alkali metal cation. X + may be Na + or K + , for example.

As used herein the term phosphonate ester represents a group of one of the formulae: -P(=O)(OR) 2 and -P(=O)(OR)O " wherein each R is independently a phosphonate ester substituent, for example, -H, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 3-2O heterocyclyl, C 3-20 heterocyclyl substituted with a further C 3-20 heterocyclyl, substituted or unsubstituted C 1-20 alkylene-C 3-2 o heterocyclyl, substituted or unsubstituted C 3-25 cycloalkyl, substituted or unsubstituted C 1-20 alkylene-C 3-25 cycloalkyl, aryl, substituted or unsubstituted C 1-20 alkylene-aryl. Examples of phosphonate ester groups include, but are not limited to, -P(=O)(OCH 3 ) 2 , -P(=O)(OCH 2 CH 3 ) 2 , -P(^O)(O 4 Bu) 2 , and -PC=O)(OPh) 2 ,

As used herein the term phosphoric acid represents a group of the formula: -OP(=O)(OH) 2 . As used herein the term phosphate ester represents a group of one of the formulae:

-OP(=O)(OR) 2 and -OP(=O)(OR)O " wherein each R is independently a phosphate ester substituent, for example, -H, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C3-20 heterocyclyl, C 3-20 heterocyclyl substituted with a further C 3-20 heterocyclyl, substituted or unsubstituted C 1-20 alkylene-C 3-20 heterocyclyl, substituted or unsubstituted C 3-25 cycloalkyl, substituted or unsubstituted C 1-20 alkylene-C 3-25 cycloalkyl, aryl, substituted or unsubstituted C 1-20 alkylene-aryl. Examples of phosphate ester groups include, but are not limited to, -OP(=O)(OCH 3 ) 2 , -OP(=O)(OCH 2 CH 3 ) 2 , -OP(=O)(O-t-Bu) 2 , and -OP(=O)(OPh) 2 .

As used herein the term amino represents a group of formula -NH 2 . The term C 1 -C 1O alkylamino represents a group of formula -NHR' wherein R' is a C 1-10 alkyl group, preferably a Ci-6 alkyl group, as defined previously. The term di(C 1-1 o)alkylamino represents a group of formula -NR'R" wherein R' and R" are the same or different and represent C 1-10 alkyl groups, preferably C 1-6 alkyl groups, as defined previously. The term arylamino represents a group of

formula -NHR' wherein R' is an aryl group, preferably a phenyl group, as defined previously. The term diarylamino represents a group of formula -NR'R" wherein R' and R" are the same or different and represent aryl groups, preferably phenyl groups, as defined previously. The term arylalkylamino represents a group of formula -NR 'R" wherein R' is a C 1-10 alkyl group, preferably a Ci- 6 alkyl group, and R" is an aryl group, preferably a phenyl group.

As used herein the term amido represents a group of formula: -C(=O)NR'R", wherein R and R are independently amino substituents, as defined for di(C 1-1 o)alkylamino groups. Examples of amido groups include, but are not limited to, -CC=O)NH 2 , -C(=O)NHCH 3 , -C(=O)N(CH 3 ) 2 , -C(=O)NHCH 2 CH 3 , and -C(=O)N(CH 2 CH 3 ) 2 , as well as amido groups in which R and R , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

A C 1-10 alkylthio group is an unsubstituted or substituted C 1-10 alkyl group, preferably a C 1-6 alkyl group, attached to a thio group. An arylthio group is an unsubstituted or substituted aryl group, preferably a phenyl group, attached to a thio group.

A C 1-10 alkoxy group is a said C 1-10 alkyl group attached to an oxygen atom. A C 1-6 alkoxy group is a said C 1-6 alkyl group attached to an oxygen atom. A C 1-4 alkoxy group is a C 1-4 alkyl group attached to an oxygen atom. Examples of C 1-4 alkoxy groups include, -OMe (methoxy), -OEt (ethoxy), -0(nPr) (n-propoxy), -O(iPr) (isopropoxy), -0(nBu) (n-butoxy), - O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy). An aryloxy group is an unsubstituted or substituted aryl group, preferably a phenyl group, attached to an oxygen atom. An example of an aryloxy group is -OPh (phenoxy).

As used herein, the terms "carboxy", "carboxyl" and "carboxylic acid" each represent a group of the formula: -C(=O)OH. C 1-20 alkylene, C 1-20 alkyl, C 2-20 alkenyl and C 2 . 20 alkynyl groups as defined herein are either uninterrupted or interrupted by one or more heteroatoms or heterogroups, such as -S-, - O- or -N(R7)- wherein R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl (typically phenyl), or by one or more arylene (typically phenylene) groups. The phrase "optionally interrupted" as used herein thus refers to a C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl or C 1-20 alkylene group, as defined above, which is uninterrupted or which is interrupted between adjacent carbon atoms by a heteroatom such as oxygen or sulfur, by a heterogroup such as N(R7) wherein R7 is H, aryl or C 1 -C 6 alkyl. C 3-10 cycloalkyl or by an arylene group.

For instance, a C 1-20 alkyl group such as n-butyl may be interrupted by the heterogroup N(RT) as follows: -CH 2 N(R7)CH 2 CH 2 CH 3 , -CH 2 CH 2 N(R7)CH 2 CH 3 , or -CH 2 CH 2 CH 2 N(R7)CH 3 . Similarly, an alkylene group such as n-butylene may be interrupted by the heterogroup N(RT) as follows: -CH 2 N(R7)CH 2 CH 2 CH 2 -, -CH 2 CH 2 N(R7)CH 2 CH 2 -, or -CH 2 CH 2 CH 2 N(T^)CH 2 -.

Typically the overall charge on a metal complex of formula (I) as defined herein is zero, in which case the complex is neutral and is not associated with a counter-ion. However, the metal complex of formula (I) may be catonic, and associated with one or more counter- anion(s). The complex may be a monocation or a dication, for example. The counter-anion or -anions may be selected from halide, hexafluorophosphate, chlorate or tetrafluoroborate anions, for example.

In the metal complexes of formula (I) defined herein, M is a transition metal or a p- block metal. In one embodiment, M is a transition metal, hi another embodiment, M is a p- block metal. The term "transition metal" as used herein means any one of the three series of elements arising from the filling of the 3d, 4d and 5d shells, and situated in the periodic table following the alkaline earth metals. This definition is used in N.N. Greenwood and A. Earnshaw "Chemistry of the Elements", First Edition 1984, Pergamon Press Ltd., at page 1060, first paragraph, with respect to the term "transition element". The same definition is used herein for the term "transition metal". Thus, the term "transition metal", as used herein, includes for instance Sc, Y, La, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd and Hg. Accordingly, in the complexes of the invention, M is typically selected from Sc, Y, La, Ti, Zr 5 Hf, V 5 Nb 5 Ta, Cr, Mo 5 W, Mn, Tc, Re, Fe 5 Ru, Os 5 Co, Rh, Ir, Ni, Pd 5 Pt, Cu 5 Ag, Au, Zn, Cd and Hg. More typically, M is a first row transition metal. Thus, more typically, M is selected from Sc, Ti, V, Cr, Mn 5 Fe, Co, Ni 5 Cu and Zn. Even more typically, M is Ni 5 Cu, Zr, Tc or Zn. Even more typically, M is Ni 5 Cu or Zn.

The term "p-block metal" as used herein means any metal in the p-block of the periodic table. Thus, the term "p-block metal", as used herein, includes for instance Al, Ga, In, Tl, Ge, Sn, Pb, Sb, Bi and Po. Accordingly, in the complexes of the invention, M is typically selected from Al, Ga, In, Tl, Ge, Sn 5 Pb, Sb 5 Bi and Po. More typically, in this embodiment, M is Ga or In.

In one embodiment the present invention provides complexes as defined above wherein M is Cu. By "Cu", herein, is meant a stable isotope or radioisotope of Cu. Thus, when M is Cu, M may, for instance, be 60 Cu, 61 Cu, 62 Cu, 64 Cu or 67 Cu.

In one embodiment the present invention provides complexes as defined above wherein M is Zn. By "Zn", herein, is meant a stable isotope or radioisotope of Zn. Thus, when M is Zn, M may, for instance, be 60 Zn, 61 Zn, 62 Zn, 63 Zn, 65 Zn, 69 Zn, 71 Zn or 72 Zn.

In one embodiment the present invention provides complexes as defined above wherein M is Ni.

In one embodiment the present invention provides complexes as defined above wherein M is Ga. By "Ga", herein, is meant a stable isotope or radioisotope of Ga. Thus, when M is Ga, M may, for instance, be 65 Ga, 66 Ga, 67 Ga, 68 Ga, 69 Ga, 70 Ga, 71 Ga, 72 Ga, 73 Ga or 74 Ga. In one embodiment, M is 67 Ga.

In one embodiment the present invention provides complexes as defined above wherein M is In. By "In", herein, is meant a stable isotope or radioisotope of In. Thus, when M is In, M may, for instance, be 107 In, 108 In, 109 In, 110 In, 111 In, 112 In, 113 In, 114 In, 115 In, 116 In or 117 In. In one embodiment, M is 111 In. In another embodiment the present invention provides complexes as defined above wherein M is Zr. By "Zr", herein, is meant a stable isotope or radioisotope of Zr. Thus, when M is Zr, M may, for instance, be Zr, Zr, Zr, Zr, Zr, 89 Zr, 90 Zr, 91 Zr, 92 Zr, 93 Zr, 94 Zr, 95 Zr, 96 Zr or 97 Zr. In one embodiment, M is 89 Zr. In one embodiment the present invention provides complexes as defined above wherein M is Tc. By "Tc", herein, is meant a stable isotope or radioisotope of Tc. Thus, when M is Tc, M may, for instance, be 99m Tc or 95m Tc. In one embodiment, M is 99m Tc. hi one embodiment M is Ni, Cu, Zn, Ga, In, Zr or Tc. In another embodiment, M is Zn, Zr, Ga, In, Tc or Cu. Thus, for instance, M may be 99m Tc, 111 In, 67 Ga, 89 Zr, 60 Cu, 61 Cu, 62 Cu or 64 Cu. More typically, M is Zn, Ga, In, Zr or Cu. Thus, for instance, M may be 111 In, 67 Ga, 89 Zr, 60 Cu, 61 Cu, 62 Cu or 64 Cu. Even more typically, M is Zn or Cu. In one embodiment M is 60 Cu, 61 Cu, 62 Cu and 64 Cu. In one embodiment, M is 64 Cu. hi the definition of R5 for the complexes of formula (I), an electron donor group is an atom or group which bears either a lone pair of electrons or an overall negative charge. It is, for instance, an O, S or N atom, or an anionic group. The electron donor group is separated by two carbon atoms from the atom to which R5 is attached; this is so that it may adopt the correct position for coordinating to the central metal atom of the complex. Typically the two carbon atoms are connected to each other by a single (saturated) bond, since this allows free

rotation and thus further assists with the optimum positioning of the electron donor group. When present, the group R5 is effectively a fifth "pendant" ligand within the complex and its electron donor group becomes part of the metal co-ordination sphere. Example of suitable groups R5 include -CH 2 CO 2 Ro, -CH 2 CO 2 " , -CH 2 CH 2 N(Ro) 2 and -CH 2 CH 2 N " (R6), wherein R6 is H or alkyl, typically H or methyl, typically H; and a group of formula (FV):

wherein ring a is a 5- or 6-membered N-containing heteroaromatic ring which is monocyclic or which is fused to a second aromatic heterocyclic ring or to a benzene ring. An example of a group of formula (IV) is a (pyrid-2-yl)methyl group. Alternatively, R5 may be H, phenyl or Ci-C 6 alkyl. In particular, R5 may be H or C 1 -C 6 alkyl. For instance R5 may be H, methyl or ethyl.

Typically, in the complexes of formula (I) as defined herein, a is a C 5-10 carbocyclic ring or a C 5-10 heterocyclic ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted.

Alternatively, a is typically a C 5-6 carbocyclic, C 5-6 heterocyclic, C 5-6 aryl or C 5-6 heteroaryl ring, which ring is unsubstituted, substituted or part of said fused bi-, tri-, tetra- or polycyclic ring system. More typically, a is a C 5-6 carbocyclic ring or a C 5-6 heterocyclic ring, which ring is unsubstituted, substituted or part of said fused bi-, tri-, tetra- or polycyclic ring system. More typically, a is a C 5-6 carbocyclic or a C 5-6 aryl ring, which ring is unsubstituted, substituted or part of said fused bi-, tri-, tetra- or polycyclic ring system. Thus, a may be a substituted or unsubstituted C 5-6 carbocyclic ring selected from the following groups, which may be unsubstituted (as shown below) or substituted at any one or more available positions in the C 5-6 carbocyclic ring:

Substituents are typically selected from those listed above for C 1 ^ 0 alkyl. Thus, for example, a may be a methyl substituted C 5 carbocyclic ring, as follows:

Typically, in the complexes of formula (I) as defined herein a is part of a fused bi-, tri- , tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring.

The ring system is substituted or unsubstituted. Thus, one or more available positions in the ring system are substituted or unsubstituted. Substituents are typically selected from those listed above for C 1-20 alkyl.

Typically, the ring system is a fused bi-, tri- or tetracyclic ring system.

Thus, in one embodiment, the ring system is a fused bicyclic ring system.

When the ring system is a fused bicyclic ring system, the ring system is typically a group of the following formula, which group may be unsubstituted (as shown below) or substituted at any one or more available positions in the bicyclic ring:

In another embodiment, the ring system is a fused tricyclic ring system.

When the ring system is a fused tricyclic ring system, the ring system is typically selected from groups of the following formulae, which groups may be unsubstituted (as shown below) or substituted at any one or more available positions in the tricyclic ring:

More typically, the fused tricyclic ring system is selected from the substituted and unsubstituted fused tricyclic ring systems of the diketone compounds of formula (X) below (under the heading "Preparation of complexes employed in the present invention"). hi another embodiment, the ring system is a fused tetracyclic ring system.

When the ring system is a fused tetracyclic ring system, the ring system is typically selected from groups of the following formulae, which groups may be unsubstituted (as shown below) or substituted at any one or more available positions in the tricyclic ring:

More typically, the fused tetracyclic ring system is selected from the substituted and unsubstituted fused tetracyclic ring systems of the diketone compounds of formula (X) below (under the heading "Preparation of complexes employed in the present invention").

Alternatively, the ring system is a fused polycyclic ring system. As used herein, "fused polycyclic ring system" means a fused ring system consisting of from 5 to 50 fused rings.

Thus, when a is part of a fused polycyclic ring system, that fused ring system consists of ring a and from 4 to 49 further fused rings, wherein each of said further fused rings is independently selected from a Cs -10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring.

One or more available positions in the polycyclic ring system are substituted or unsubstituted. Substituents are typically selected from those listed above for C 1-20 alkyl.

When the ring system is a fused polycyclic ring system, the ring system is typically selected from groups of the following formulae, which groups may be unsubstituted (as shown below) or substituted at any one or more available positions in the polycyclic ring system:

More typically, the fused polycyclic ring system is selected from the substituted and unsubstituted fused polycyclic ring systems of the diketone compounds of formula (X) below (under the heading "Preparation of complexes employed in the present invention").

In one embodiment, a is a C 5-6 carbocyclic ring or C 5-6 aryl ring, more typically a C 5-6 carbocylic ring, which ring is part of said fused tri- or tetracyclic ring system. Typically, the ring system is selected from the groups of the following formule (Ha), (lib), (lie) and (Hd):

(Ha) (Hb) (lie)

(Hd)

The groups (Ha), (lib), (lie) and (Hd) may be substituted or unsubstituted at any one or more available positions in the ring system.

More typically, the ring system is selected from the groups of formule (Ha), (lie) and (Hd) above, which groups may be substituted or unsubstituted at any one or more available positions in the ring system. Typically, the groups are unsubstituted.

In one embodiment, a is a C 5-6 carbocyclic ring or C 5-6 aryl ring, more typically a C 5-6 carbocylic ring, which ring is part of said fused tri- or tetracyclic ring system. Typically, the ring system is selected from the groups of the following formule (Ha) 5 (lib) and (lie):

(Ha) , (lib) , (lie)

More typically, the ring system is the group of formula (Ha), which group may be substituted or unsubstituted at any one or more available positions in the ring system.

Substituents for the fused ring systems in the complexes of formula (I) as defined herein are typically selected from those listed above for C 1-20 alkyl. The skilled person will understand that the chelating ligand of the complexes of formula (I) as defined herein may exist in more than one resonance form, due to the presence of multiple double bonds in that ligand, particularly within the ring a, between the ring a and the atoms X and X', and within the fused ring systems of which a may be part. Such resonance forms arise from conjugation of those double bonds. Similarly, the chelating ligand of the complexes of formula (I) as defined herein may exist in more than one tautomeric form. Indeed, the double bond between the X or X' atom and the ring a may become a single bond if the atom X or X' becomes protonated. This may occur particularly when the X or X' atom in question is N. The source of the proton may, for example, be the complex itself. Alternatively, if the complex is hi solution, for instance, or in contact with another compound, the source of the proton may be the solution in which the complex is dissolved or that other compound with which the complex is in contact. Thus, one or both of the double bonds between the ring a and the X and X' atoms may become single bonds through protonation of X and X' respectively. It is to be understood that all of such forms are within the scope of the present invention, and references to any of the complexes of formula (I) as defined herein include all such forms.

Typically, at least one ring of the fused ring systems in the complexes of formula (I) as defined herein is a C 5-10 aryl ring or a C 5-10 heteroaryl ring. More typically, a is part of said fused tri-, tetra- or polycyclic ring system and at least two rings of said ring system are selected from a C 5-10 aryl ring and a Cs -10 heteroaryl ring. Without wishing to be bound by theory, the pi-conjugated aromatic systems of such C 5-10 aryl and C 5-10 heteroaryl rings may

contribute to the intrinsic fluorescence of complexes of formula (I) as defined herein. In other words, the ligand backbone may be acting as a fluorophore.

Even more typically, the ring system is an unsubstitued group of the formula (Ha). Accordingly, in one embodiment, the invention provides a metal complex of the following formula (Id):

(id) wherein M, b, c, n, L, X', X, Y', Y, R2, R2', Ll, Ll ', Rl and Rl ' are as defined above for the metal complex of formula (I).

In another embodiment, the ring system is an unsubstitued group of the formula (lie). Accordingly, in one embodiment, the invention provides a metal complex of the following formula (If):

(If) wherein M, b, c, n, L, X', X, Y', Y, R2, R2\ Ll, Ll', Rl and Rl' are as defined above for the metal complexes of formula (I).

In another embodiment, the ring system is an unsubstitued group of the formula (lie). Accordingly, in one embodiment, the invention provides a metal complex of the following formula (Ig):

(Ig) wherein M, b, c, n, L, X', X, Y', Y, R2, R2', Ll, Ll', Rl and Rl' are as defined above for the metal complexes of formula (I).

In one embodiment, n is 0, b is a bond and c is not a bond. Accordingly, in one embodiment the invention provides a metal complex of formula (Ia):

(Ia) wherein M, a, X 5 X', Y, Y', Ll, Ll', Rl, Rl', R2 and R2' are as defined above for the metal complexes of formula (I).

In another embodiment, n is 1 and either: (i) b is a bond and c is not a bond, or (ii) c is a bond and b is not a bond. Accordingly, in one embodiment, the invention provides a metal complex of formula (Ib):

(Ib) wherein: either (i) b is a bond and c is not a bond, or (ii) c is a bond and b is not a bond; and

M, a, X 5 X', Y 5 Y', Ll 5 Ll', Rl 5 Rl', R2 and R2' are as defined above for the metal complexes of of formula (I).

Examples of metal complexes of formula (Ib) are compounds 19 and 20, whose molecular structures were determined by X-ray crystallography (see Examples 19 and 20 below). The X-ray structures of those compounds show that, in each case, M is not bonded to X 5 but instead to N. Thus, in compounds 19 and 20 b is not a bond and c is a bond. However, crystal structures of other bis(thiosemicarbazone) complexes having an apical ligand have revealed that in other cases the reverse is true, i.e. c is not a bond and b is a bond. Typically, however, when n is 1 , b is not a bond and c is a bond.

Accordingly, in one embodiment, the invention provides a metal complex of formula (Ic):

wherein M, a, X 5 X' 5 Y 5 Y', Ll 5 Ll', Rl, Rl', R2, R2' and L are as defined above for the metal complex of formula (I). Typically, in this embodiment, M is Zn.

The person of skill in the art will appreciate that depending on the metal present in the centre of a complex of formula (I) as defined above, it may be appropriate to have one or more further ligands bound to the central metal. For example, in one embodiment a donor group features in the group R5 of the complex of formula (I) as defined above. In another embodiment, where necessary, the complex may also feature one or more further ligands. Additional ligands can be neutral or anionic and can be any suitable small molecule. Typical examples include halogen and water. Hydroxyl is a further example. For example, when M represents Zn(II), then typically an apical ligand, such as chloride, methanol, water or (O- bound) DMSO, is present. The d 10 configuration of zinc does not confer stabilisation energy to any particular geometry, and instead the conformation adopted by zinc in its complexes is a function of the steric and conformational demands of the ligands. Considering, on the other hand, copper and nickel, both of these show a preference for tetradentate planar complexes.

In the complexes of formula (I), the ligand L may be any donor entity. Thus, the ligand may be any molecule that comprises an electron donor group. In the complexes of formula (I) in which L is present, said electron donor group is bonded to M. Li this context, an electron donor group is an atom or group which bears either a lone pair of electrons, a radical electron, or an overall negative charge. The electron donor group is, for instance, an O, S, N or P atom, an anionic group (e.g. a halide group), or a carbon atom of an organic radical. Typically, the electron donor group is an O, S, N or P atom, or L is a halo group, for instance chloro, bromo or iodo. More typically, the electron donor group is an O 5 S, N or P atom, even more typically an O or N atom. In one embodiment, L is a halo group. Thus L may be chloro, bromo or iodo. In one embodiment, L is Cl, Br or I. More typically, L is Cl. L may be an unsubstituted or substituted C 5 - I0 heterocyclic ring; an unsubstituted or substituted C 5-10 heteroaryl ring; a C 1-20 hydrocarbon molecule, which hydrocarbon comprises one or more heteroatoms, is unsubstituted or substituted, and is optionally interrupted by N(R7), O, S or arylene, wherein R7 is as defined above; an unsubstituted or substituted organic radical; a macromolecule; a halo group; water; DMSO; or a compound comprising from 2 to 20 unsubstituted or substituted C 5-10 heterocyclic rings, wherein each of said heterocyclic rings is linked to another of said heterocyclic rings by a group A, wherein A is a covalent bond, substituted or unsubstituted arylene, or substituted or unsubstituted C 1-20 alkylene, which compound is either (i) complexed to M", wherein M" is a' transition metal or a main group metal, or (ii) uncomplexed, and wherein said C 1-20 alkylene is optionally interrupted by by N(R7), O, S or arylene.

Typically, L is a C 5-10 heterocyclic ring. More typically, L is a nitrogen-containing C 5- io heterocyclic ring. For instance, L may be l ; 4-diaza-bicyclo[2,2 5 2]octane (also known as DABCO), whose structure is as follows:

l,4-Diaza-bicyclo[2.2.2]octane Alternatively, L may be a compound comprising from 2 to 20 unsubstituted or substituted C 5-1O heterocyclic-rings, wherein each of said heterocyclic rings is linked to another of said heterocyclic rings by a group A, wherein A is a covalent bond, substituted or unsubstituted arylene, or substituted or unsubstituted C 1-20 alkylene, wherein said C 1-20 alkylene is optionally interrupted by by N(R7), O, S or arylene. Typically, in such compounds the electron donor group is an O or N atom, more typically an N atom. Such compounds include porphyrins, porphyrins complexed to M" (wherein M" is a transition metal or a main group metal), bipyridyl compounds and terpyridyl compounds. An example of a porphyrin which can act as a ligand L is shown below as formula (IVa). An example of a porphyrin complexed to M" is shown below as formula (IVb). Examples of bipyridyl and terpyridyl compounds are given below as formulae (Ilia), (DIb), (HIc) 5 (Did), (Die), (HIf), (Dig), (HIh) and (DH):

(IVa) (IVb)

M" is either a transition metal (as defined above for M) or a main group metal. When M" is a main group metal, M" may be an alkali metal, an alkaline earth metal, or a p-block metal. For instance, M" may be selected from Li, Na, K, Be, Mg, Ca, Sr, Ba, Al, Ga, In 3 Tl, Ge, Sn, Pb, Sb, Bi, Po. When M" is a main group metal, M" is typically Sn, Li, Na, K, Be, Mg or Ca.

It is believed that the complexes of formula (I) are able to interact with cellular nucleic acids, and in particular with DNA, via an interaction between the metal atom M of the complex and a nitrogen atom of the guanosine nucleoside of the nucleic acid. Without wising to be bound by theory, such an interaction could account, at least in part, for the cytotoxicity observed for complexes of formula (I). Other possible mechanisms that could account for cytotoxicity are DNA intercalation or DNA groove binding.

Accordingly, it is believed that the cytotoxicity of the complexes of formula (I) could be modulated, for instance enhanced, if the ligand L of the complex is a molecule which modulates, e.g. enhances, the ability of the complexes to interact with nucleic acids (e.g. DNA). Thus, L may be an unsubstituted or substituted C 5-10 heterocyclic or heteroaryl ring which ring is a nitrogenous base. Typically, the nitrogenous base is selected from guanine, adenine, cytosine, uracil and thymine, whose structures are shown below. Thus, L may be guanine, adenine, cytosine, uracil or thymine. Further, L may be a nucleoside, nucleotide, deoxynucleoside or deoxynucleotide. Furthermore, L may be a macromolecule. Typically, the macromolecule comprises a nucleic acid, for instance an oligonucleotide or polynucleotide. Thus, L may comprise DNA or RNA. Additionally or alternatively, the macromolecule may comprise a peptide (e.g. an oligopeptide or a polypeptide). Thus L may comprise a protein or an enzyme. Where L comprises a peptide, the electron donor group is

typically an O 5 N or S atom of the peptide. Where L comprises a nucleic acid, a nitrogenous base, a nucleoside, a nucleotide, a deoxynucleoside or a deoxynucleotide, the electron donor group is typically a nitrogen atom.

When L is an organic radical, the organic radical may be selected from the following groups, for instance, which may be unsubstituted or substituted: C 1-20 alkyl, C 1-10 alkoxy, C 1-10 alkylthio, C 2-20 alkenyl, C 2-20 alkynyl, acyl, acylamido, acyloxy, ester, aryl heteroaryl, C 3-20 heterocyclyl and C 3-20 carbocyclyl.

Typically, L is selected from a halo group; l,4-diaza-bicyclo[2,2,2]octane; a porphyrin; a porphyrin complexed to M"; a nitrogenous base selected from guanine, adenine, cytosine, uracil and thymine; a nucleoside; a nucleotide; a deoxynucleoside; a deoxynucleotide; an oligonucleotide; a polynucleotide; an oligopeptide; a polypeptide; and a compound of any one of the above formulae (Ilia), (HIb), (IIIc), (Did), (Die), (HIf), (Dig), (DUi), (DH), (IVa) and (IVb).

In another embodiment, L may be selected from l,4-diaza-bicyclo[2,2,2]octane; a porphyrin; a porphyrin complexed to M"; a nitrogenous base selected from guanine, adenine, cytosine, uracil and thymine; a nucleoside; a nucleotide; a deoxynucleoside; a deoxynucleotide; an oligonucleotide; a polynucleotide; an oligopeptide; a polypeptide; and a compound of any one of the above formulae (Ilia), (DIb), (IIIc), (Did), (Die), (HIf), (Dig), (DIh), (DH), (IVa) and (IVb).

Typically, in the complexes of formula (I), X is N. Typically, X' is N.

Usually, X and X' are the same. Thus, typically X and X' are both N. Alternatively, however, X and X' may both be P.

Typically, Y is S. Typically, Y' is S.

Usually, Y and Y' are the same. Thus, Y and Y' may both be S, O 5 N(R5) or P(R5). More typically, both Y and Y' are S.

In one embodiment the complex of formula (I) is water soluble. Thus, in one embodiment, the present invention provides complexes which are water soluble. Control of this aspect of the complex can be achieved by manipulation of the -N(R2)-L1-R1 and -

N(R2')-L1 '-Rl ' groups. For example, where a water soluble complex is desired, these groups can be chosen such that the complex has one or more polar groups, for instance sugar moieties, attached. On the other hand, high solubility in non-polar organic solvents can be conferred by selecting these groups to include one or more long chain aliphatic groups, such as a C 6 -C 2O alkyl group.

In the complexes of formula (I) as defined above, it is to be understood that each of the linker groups, Ll 5 Ll ' and L3 can be arranged either way around, ie. head-to-tail or tail- to-head, when linking two groups together. Thus, for example, the linking group -alk- S(O) 2 NR 7 - may link the Rl and N groups together in either of the following arrangements: N-alk-S(O) 2 NR 7 -Rl and N-NR 7 S(O) 2 -alk-Rl .

Functional substituents, such as biologically active molecules and labelled groups, may be conjugated to the metal complexes of formula (I) in such a way as to maximise the potential biomedical use of those complexes. Typically the complex is linked via an exocyclic nitrogen, which may have the advantage of causing minimum perturbation in any potential hypoxic selectivity of the molecules.

Typically, neither or only one of Rl and Rl ' features a Z group. In one embodiment, neither Rl nor Rl' features a Z group. In another embodiment, one of Rl and Rl' features a Z group.

Thus in one embodiment, either Rl or Rl' is selected from Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1 _ 1 o)alkylamino, C3.20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -3Ik-C 3-20 heteroaryl, and -alk-aryl, which group is further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene.

When Z is a fluorophore, this group may be any group, part or all of which can be excited by radiation to fluorescence. In one embodiment, the fluorophore is a zinc bis(thiosemicarbazone) complex, hi another embodiment, the fluorophore is a 2-(4-

aminophenyl)benzothiazole or derivative thereof. Examples of such suitable derivatives include those in the table below.

Complexes of formula (III) which incorporate a fluorphore Z may be synthesised by reaction of a fluorophore containing an aldehyde, ketone, carboxylic acid or acid halide fluorophore with a precursor complex containing an amino group. The precursor complex may be a complex of formula (I) in which Rl or Rl ' contains an amino group, for example a complex of formula (I) in which Rl or Rl ' is an amino group. The fluorophore for reaction with the amino group may be, for instance, benzaldehyde, 2-furaldehyde, salicyladehyde, hydroxynaphthaldehyde, 4-(di-n-propylamino) benzaldehyde, butane-2-3-dione, phenyl glyoxal, terephthalaldehyde, glyoxal, isophthaldehyde, pyruric acid, propionaldehyde, 4- fluorobenzaldehyde, 2-pyridinecarboxaldehyde, or a suitable zinc bis(thiosemicarbazone) complex, which may be a zinc bis(thiosemicarbazone) complex of formula (I) or

Zn[ATSM/A-terephthalaldehyde], Zn[ATSM/A-glyoxal], Zn[ATSM/A-terephthaloyl chloride] and Zn[ATS WA-isophthalaldehyde]. Thus, fluorophore Z may be derived from any of the compounds in the preceding list.

When Z is a group containing a label, the label can be any moiety that permits the detection of the complex. The label may be a fluorophore, for instance as described above. Typically it is a radioisotope. Examples include 18 F, 11 C, 14 C 5 3 H, 99m Tc, 111 In, 123 I and 188 Re. In another embodiment it may be a stable isotope. Examples include 13 C, 2 H and 15 N. In one preferred embodiment it is a label that decays via positron emission, beta emission, electron

capture or Auger emissions. More typically, the label decays by positron emission. Most typically the label is 18 F. When the label is 18 F, because of the short half-life of 18 F (110 minutes), the fluorinated derivative must be prepared on the day of its clinical use and the reaction steps used to produce it should be optimised for speed, with yield as a secondary consideration. 18 F is typically prepared from a cyclotron in the form OfKH 18 F 2 and the 18 F in KH 18 F 2 replaces a suitable leaving group in the complex with 18 F. Examples of suitable leaving groups include imidazosulfonyl, triflate, mesylate and tosylate. Accordingly, in a preferred embodiment of the present invention, the group Z in the complexes of formula (I) as defined above may represent such a suitable leaving group. Images may be acquired from about 5 minutes after administration until about 8 hours after administration. The maximum period in which images may be acquired is determined by 3 factors: the physical half-life of 18 F (110 minutes); the sensitivity of the detectors and the size of the dose administered. Those of skill in the art can adjust these factors to permit the acquisition of images at an appropriate time. Details of imaging procedures are well known. Radionuclides which emit gamma radiation can be used for SPECT imaging. SPECT scanners are gamma cameras which detect the photons produced from this decay. Facilities for SPECT imaging are widely available in hospitals. Radionuclides suitable for PET imaging must undergo β + decay, in which the nucleus emits a positron and neutrino. When the positron encounters an electron the two annihilate and two gamma rays are emitted. These travel in opposite directions (in reality at an angle of 179.5° due to the residual momentum of the positron.) The encircling PET scanner simultaneously detects these photons. This technique has significantly reduced noise, higher sensitivity and has a superior resolution to SPECT, however PET scanners are less widely available than those used for SPECT. In one embodiment the present invention provide diagnostic agents comprising a complex of formula (I) as defined above, for use in SPECT imaging.

When Z is a cytotoxin it is, for instance, an agent which is cytotoxic to cancer cells. Examples of such cytotoxins include taxanes such as taxol and taxotere; Vinca alkaloids such as vincristine and vinblastine; anthracycline antibiotics such as daunorubicin, epirubicin and doxorubicin; epipodophyllotoxins such as etoposide and plicamycin; mitoxantrone; and actinomycin D.

Z may be a biologically active molecule ("BAM"), which shows specific in vivo targeting to a diseased tissue of interest. Many BAM' s, for instance monoclonal antibodies, antibody fragments and peptides, have been investigated in medical imaging. Indeed 111 In-

DTPA-B72.3(OnoScint) and 99m Tc-IMMU-4 (CEO-SCAN) both use antibody binding and are approved for clinical use in the US. Furthermore, octreotide labelled with 111 In using DTPA is a diagnostic imaging agent for neuroendocrine tumors. That agent uses binding of the cyclic peptide octreotide (shown below) to peptide based tumor receptors that interact with the hormone somatostatin. Ga and In complexes linked to folic acid via DTPA chelation have also shown promising results in tumor imaging. The structures of octreotide, DTPA and folic acid are shown below:

NH

Octreotide DTPA Folic acid Thus, Z may be a biologically active molecule, wherein the biologically active molecule is selected from a monoclonal antibody, an antibody fragment, a peptide, octreotide and folic acid.

Z may be an amino acid, a peptide, an oligopeptide or a polypeptide. When Z is an amino acid, a peptide, an oligopeptide or a polypeptide, any amino acid or combination of amino acids may be used. Z may be any amino acid, derivative thereof or combinations (in the form of a straight or branched chain) thereof. Examples include -Lys(Boc)-OH, - Orn(Boc)-OH, -Lys-OH and -octreotide. Z may be -ONSu, derived from N- hydroxysuccinimide.

When Z is a sugar moiety, it can be a monosaccharide, disaccharide or trisaccharide. It can, for example, be glucose, sucrose or lactose.

When Z is a bis(thiosemicarbazone) or thiosemicarbazone complex, typically it is a Cu or Zn complex. In one embodiment, Z of the complex of formula (I) is a Cu or Zn complex of a bis(thiosemicarbazone) or of a thiosemicarbazone, thereby creating a dimer wherein two complexes are linked by -Ll-Rl-Ll-, -Ll-alk-Rl-Ll-, -Ll-Rl-alk-Ll- or -Ll- alk-Rl-alk-Ll- wherein Ll and Rl are as defined above.

Typically the complex is a bis(thiosemicarbazone) Cu or Zn complex. More typically it is a Zn complex.

Typically in this embodiment the group Z is only present in one of Rl and Rl ' . In this embodiment, both of the metal atoms present are typically Zn, thus creating a bi-nuclear zinc complex. Linking two zinc centres in this way may have a significant effect on the

fluorescence properties as compared to the properties of a mono-nuclear zinc complex. Alternatively, the present invention provides complexes of formula (I) as defined above, wherein M is a copper radionuclide and Z represents a zinc bis(thiosemicarbazone) complex (for instance, a zinc bis(thiosemicarbazone) complex of formula (I)), thus providing a radiopharmaceutical copper complex tethered to a more fluorescent zinc complex.

In one embodiment, the invention provides a complex of the following formula (Ie):

(Ie) wherein: each M, which is the same as or different from the other M, is a transition metal; a, R2, R2' and Ll are as defined above for formula (I);

Q is -Ll- or -L1-R1-L3- wherein Ll and L3 are as defined above for formula (I); and

Rl is selected from hydrogen, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1 . 10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C3 -20 carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene.

In one embodiment, neither Rl nor Rl ' features a Z group.

Typically, Rl and Rl', which are the same or different, are each independently selected from hydrogen, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2 . 2 o alkenyl. C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino 5 C 3-2O carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, - alk-C 3-2 o heteroaryl, and -alk-aryl, wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene.

More typically, Rl and Rl', which are the same or different, are each independently selected from hydrogen, substituted or unsubstituted C 1-2O alkyl, substituted or unsubstituted aryl and substituted or unsubstituted C 2-20 alkenyl. Even more typically, Rl and Rl ' are each independently selected from hydrogen, unsubstituted C 1-20 alkyl, unsubstituted aryl and

unsubstituted C 2-20 alkenyl. Even more typically, Rl and Rl ' are each independently selected from H 5 unsubstituted C 1-6 alkyl, phenyl and allyl. Typically, Rl and Rl ' are the same. Typically, R2 and R2', which are the same or different, are each independently selected from hydrogen, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted aryl and substituted or unsubstituted C 2-20 alkenyl. More typically, R2 and R2' are independently selected from H 5 substituted or unsubstituted C 1-6 alkyl and substituted or unsubstituted phenyl. Usually, R2 and R2' are the same. Thus, usually R2 and R2' are both either H substituted or unsubstituted C 1-6 alkyl or substituted or unsubstituted phenyl. More typically, R2 and R2' are both H. Alternatively, Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group (typically a C 5-7 heterocyclyl group) or an unsubstituted or substituted C 3-20 heteroaryl group (typically a C 5-7 heteroaryl group); and Ll', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group (typically a C 5-7 heterocyclyl group) or an unsubstituted or substituted C 3-20 heteroaryl group (typically a C 5-7 heteroaryl group). Thus, Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, a group of any of the following formulae (Ilia), (HIc) and (IIIc):

Similarly, Ll', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached a group of any of formulae (Ilia), (IIIc) and (IIIc).

Typically, Ll is a covalent bond. Typically, Ll ' is a covalent bond. In one embodiment of the metal complexes employed in the present invention: Ll and Ll' are both covalent bonds; Rl and Rl', which are the same or different, are each independently selected from hydrogen, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2- 20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-2 O heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3 . 20 heteroaryl, and -alk-aryl, wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene; and

R2 and R2\ which are the same or different, are each independently selected from H, substituted or unsubstituted C 1-6 alkyl and substituted or unsubstituted phenyl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 5-7 heterocyclyl group or an unsubstituted or substituted C 5 . 7 heteroaryl group; and provided that Ll', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 5-7 heterocyclyl group or an unsubstituted or substituted C 5-7 heteroaryl group.

Typically, R7 is H or C 1-6 alkyl or phenyl. More typically R7 is H.

In one aspect, the present invention provides a metal complex of formula (I):

(I)

wherein: M is a transition metal; either (i) n is 0, b is a bond and c is not a bond; (ii) n is 1 , b is not a bond and c is a bond; or (iii) n is 1 , b is a bond and c is not a bond;

L is a ligand comprising an electron donor group; a is a C 5-I0 carbocyclic, C 5-I0 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, terra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O,

N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl and a group

comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7>, -alk-N(R7)-, -N(R7)C(0), -alk- N(R7)C(O>, -C(O)N(R7), -alk-C(0)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(O)O-, -alk-N(R7)C(0)0-, - 0C(0)N(R7)-, -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7)-, -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, ~alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3 _ 20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3 . 20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2 . 20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(R7), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl;

provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll', Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group.

The inventors have shown that the metal complexes of the invention are taken up into a range of human cancer cell lines, and exhibit significant cytotoxicity towards those cell lines. Thus, the complexes of the invention are candidates for use as therapeutic agents, in particular for use in treating cancers and other conditions characterised by undesirable cellular proliferation.

Certain complexes of the invention have the potential to be hypoxic selective, as described hereinbelow, and may therefore be suitable for therapy and/or imaging of hypoxic tumours. Additionally, or as an alternative to hypoxic selectivity, complexes of the invention may be conjugated to a biologically active molecule which serves to target the complex to the desired site in vivo.

Accordingly, the invention further provides:

- a pharmaceutical composition comprising a complex of the invention as defined above and a pharmaceutically acceptable carrier. - a complex of the invention as defined above for use in a method of medical treatment.

- use of a complex of the invention as defined above in the manufacture of a medicament for use in the treatment of a condition characterised by undesirable cellular proliferation. - a method of treating a condition characterised by undesirable cellular proliferation, which method comprises administering to a patient in need of such treatment an effective amount of a complex of the invention as defined above.

- a pharmaceutical composition for use in treating a condition characterised by undesirable cellular proliferation, comprising a pharmaceutically acceptable carrier or diluent and a complex of the invention as defined above.

- a complex of the invention as defined above for use in treating a condition characterised by undesirable cellular proliferation.

- an agent for the treatment of a condition characterised by undesirable cellular proliferation, comprising a complex of the invention as defined above.

The inventors have also found that complexes of the invention, including copper complexes, have enhanced fluorescence compared with known metal thiosemicarbazone compounds, and that the complexes possess intrinsic fluorescence. The uptake of the intrinsically fluorescent species, into a range of human cancer cell lines, was observed using fluorescence imaging. The fluorescent properties of the complexes allows the distribution of the complexes within cells to be monitored.

In addition, complexes of the invention may be radiolabelled with metastable metal radionuclides which are useful in medical imaging techniques such as PET (Positron Emission Tomography) and SPECT (Single Photon Emmission Computerised Tomography).

Thus, the complexes of the invention are candidates for use as imaging agents, using either a radioactive imaging technique such as PET and SPECT, or a non-radioactive optical technique, such as fluorescence. The radiolabelled complexes may be suitable for both non- radioactive and radioactive imaging, which may be used in combination. When combined with fluorescence imaging, radioactive imaging techniques such as PET and SPECT could provide an extremely powerful tool in the clinical diagnosis and treatment of disease.

Furthermore, the complexes of the invention are candidates for use as dual therapy and imaging agents, for example in the treatment and imaging of cancer tumours and other conditions characterised by undesirable cellular proliferation. Accordingly, the present invention further provides: a diagnostic agent or medical imaging agent which comprises a complex of the invention as defined above. use of a complex of the invention as defined above in the manufacture of a medicament for use as a diagnostic agent, an imaging agent and/or a therapeutic agent. a method of imaging a cell or in vitro biopsy sample, which method comprises: (a) contacting the cell or in vitro biopsy sample with a complex of the invention as defined above; and (b) imaging the cell or in vitro biopsy sample. a method of imaging a patient in need thereof, which method comprises: (a) administering to the patient a complex of the invention as defined above; and (b) imaging the patient.

Preparation of complexes employed in the present invention

The metal complexes of formula (Ia) can be prepared by a templating reaction in the presence of a salt of the metal M, in which the ligand is formed in situ around the metal centre M from ligand precursor compounds. The metal M acts as a templating agent.

Accordingly, the invention provides a process for producing a metal complex of formula (Ia):

wherein:

M is a transition metal or a p-block metal; a is a C 5-10 carbocyclic, C 5-10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a Cs -10 heteroaryl ring, wherein the ring system is unsubstituted or substituted; X and X', which are the same or different, are independently selected from N and P;

Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached; Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -O-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(0), -alk- N(RT)C(O)-, -C(O)N(RT), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(RT)-, - alk-C(S)N(R7)- 5 -N(RT)C(S)-, -alk-N(R7)C(S>, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7>, - N(RT)S(O) 2 -, -alk-N(R7)S(O) 2 - 5 -S(O) -, -alk-S(O)-, -N(RT)C(O)O-, -alk-N(R7)C(0)0-, - OC(O)N(RT)-, -alk-OC(O)N(R7)-, -N(RT)C(O)N(RT)-, -alk-N(R7)C(O)N(R7)- 5 - N(RT)C(S)N(RT)-, -alk-N(RT)C(S)N(RT)-, -N=C(RT>, -alk-N=C(RT)-, -C(RT)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted;

Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-I0 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3 . 20 heteroaryl, aryl, aryloxy, ~alk-C 3 . 20 carbocyclyl, -alk-C 3 _ 20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl 'are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule,, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2' s which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3 - 20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating, in the presence of a solvent, a salt of M with either: (i) a compound of following formula (X), a compound of the following formula (Y) and a compound of the following formula (Z):

, (Y) , (Z) ; or

(ii) a compound of the following formula (V) and a compound of the following formula (Z):

(V) , (Z) Any suitable solvent may be employed. Typically, the solvent is a polar solvent, more typically a polar protic solvent. Typically, the solvent is acetic acid, DMSO or an alcohol such as methanol or ethanol. More typically, the solvent is acetic acid. Typically, the reaction is carried out with heating. More typically the reaction is carried out with heating to the reflux temperature of the solvent used. For example, when the solvent is acetic acid the reaction may be carried out at a temperature of 118 °C or higher, e.g. at a temperature of 120 0 C. Typically, heating is carried out for about 2 hours or longer.

Any suitable salt of M may be employed. An example of a suitable salt of M is an acetate salt of M. Where M is Cu, Zn or Ni, the salt is typically M(O Ac) 2 .xH 2 0. Generally x is from 1 to 4. Typically, x is 1 for Cu, 2 for Zn and 4 for Ni. Typically, the salt of M is present in a molar excess with respect to the compound of formula (X) or the compound of formula (V), as the case may be. More typically, the salt of M is present in about a twofold or about a threefold molar excess with respect to (X) or (V). Where M is Zn, the salt of Zn is typically present in about a threefold molar excess with respect to (X) or (V). Where M is Ni, the salt of Ni is typically present in about a twofold molar excess with respect to (X) or (V).

In one embodiment, a catalytic amount of trifluoroacetic acid (TFA) is present in the reaction mixture. Typically, 0.1 to 1 ml TFA is present. More typically, 0.2 to 0.8 ml, for instance about 0.5 ml of TFA is present. The presence of TFA has been found in one case to result in a product (i.e. a metal complex of formula Ia) of increased purity. The presence of TFA has also been found to greatly reduce the reaction time. In the preparation of zinc bis(4-

allyl-3-thiosemicarbazone) acenaphthenequinone (18), the presence of TFA both reduced the reaction time and increased the purity of the product.

Typically, M is Ni or Zn.

Typically, the process for producing a metal complex of formula (Ia) comprises treating, in the presence of a solvent, a salt of M with a compound of following formula (X), a compound of the following formula (Y) and a compound of the following formula (Z):

(X) , (Y) (Z) wherein M, a, Y', Y 5 X', X, Ll ', Ll, Rl ', Rl, R2' and R2 are as defined above. Typically, the compounds of formula (Y) and (Z) are present in a molar excess with respect to the compound of formula (X). Generally, the compounds of formula (Y) and (Z) are each present in about a twofold molar excess with respect to the compound of formula (X).

In one embodiment of the process for producing a metal complex of formula (Ia), Y'=Y, X'=X, L1'=L1, Rl '=R1 and R2'=R2, in which case the compound of formula (Y) is the same as the compound of formula (Z). Generally, in this embodiment, that compound of formulae (Y) and (Z) is present in a molar excess, typically about a fourfold molar excess, with respect to the compound of formula (X).

Alternatively, the process for producing a metal complex of formula (Ia) comprises treating, in the presence of a solvent, a salt of M with a compound of the following formula (V) and a compound of the following formula (Z):

(V) , (Z) wherein M, a, Y', Y, X', X, Ll', Ll, Rl', Rl, R2' and R2 are as defined above. This embodiment is particularly useful for producing compounds of formula (Ia) in which Y'≠Y, X'≠X, Ll'≠Ll, Rl'≠Rl or R2'≠R2, i.e. asymmetric compounds of formula (Ia).

Compounds of formula (V) can be prepared by treating a compound of (X) with a compound of formula (Y). This reaction is shown in Scheme 1, below:

Scheme 1 : Preparation of compounds of formula (V)

Typically, the solvent is a polar solvent, more typically a polar protic solvent. Typically, the solvent is an alcohol, such as methanol or ethanol. More typically-, the solvent is ethanol. Typically, the reaction is carried out with heating, typically at the reflux temperature of the solvent used. For example, when the solvent is ethanol the reaction is suitably carried out at a temperature of 78 0 C or higher, e.g. at a temperature of 80 °C. Typically, heating is carried out for about 1 hour or longer. More typically, heating is carried out for about 2 hours or longer, for instance for about 2 hours. Comparative Examples 1, 3, 5 and 7 are examples of preparations of compounds of formula (V).

Compounds of the following formulae (X), (Y) and (Z) are either commercially available or readily prepared, without undue experimentation, using known procedures:

(Y) (Z) (X) .

The structures of some examples of known compounds of formula (X) are given below:

C 78 O 6 C 1 OoO 6

Alternatively, metal complexes of formula (Ia) may be prepared by transmetallation. Accordingly, the invention further provides a process for producing a metal complex of the following formula (Ia) by transmetallation:

wherein:

M is a transition metal or a p-block metal; a is a C 5-10 carbocyclic, C 5-10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, terra- or polycyclic ring system, wherein the or each further ring of said fused M-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-1O heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O,

N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and LV, which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(O), -alk- N(R7)C(O)-, -C(O)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)- 5 -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, - N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(O)O-, -alk-N(R7)C(O)O-, - OC(0)N(R7)-, -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7>, -alk-N(R7)C(O)N(R7>, -

N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7>, -C(R7)=N-, -alk- C(RJ)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl ', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2- I 0 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di^i-i^alkylamino, C 3 . 20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3 _ 20 carbocyclyl, -alk-C 3 , 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by

L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2 -2 0 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating a salt of M, in the presence of a solvent, with a metal complex of the following formula (W):

wherein M' is a metal other than M and is either (i) a transition metal or (ii) a main group metal; and a, X', X, Y', Y 5 R2, R2\ Rl, Rl', Ll and Ll' are as defined above for the metal complex of formula (Ia). Any suitable solvent may be employed. Typically, the solvent is a polar solvent.

Typically, the solvent is DMSO or an alcohol such as methanol or ethanol. More typically, the solvent is methanol. Usually, no heating is required and the reaction is conducted at room temperature.

For mono- and bidentate ligands the thermodynamic stabilities of corresponding complexes of bivalent ions of the first transition series, irrespective of the particular ligand involved, usually vary in accordance with the Irving- Williams series: Mn(II) < Fe (II) < Co(II) < Ni(II) < Cu(II) > Zn(II). Chelating and macrocyclic ligands can form a cavity which can be used to select one ion over another based on size. In general, however, copper complexes are expected to be more stable than analogous zinc complexes or analogous nickel complexes. Accordingly, the reaction between a zinc or nickel έw(thiosemicarbazone) and a salt of copper, such as copper acetate, should result in the formation of the corresponding copper δw(thiosemicarbazone) complex and a salt of zinc or nickel, such as zinc or nickel acetate. Indeed, Examples 15 and 16 describe the synthesis of copper δzXthiosemicarbazone) complexes of the invention from the corresponding zinc ^^(thiosemicarbazone) complexes, and Example 27 describes the synthesis of a radiolabeled 64 Cu &w(thiosemicarbazone) complex of the invention by 64 Cu transmetallation of the corresponding zinc complex.

Thus, in the process of the invention for producing a metal complex of formula (Ia) by transmetallation, typically M is Cu, Ni or Zn. More typically, M is Cu. In one embodiment, M is Cu, which Cu is a radionuclide. More typically, it is a positron emitter such as 64 Cu. When M is Cu, typically M' is Ni or Zn.

In one embodiment, M' is a main group metal. Thus M' may be an alkali metal, an alkaline earth metal, or a p-block metal. For instance, M' may be selected from Li, Na, K, Be, Mg, Ca, Sr, Ba, Al, Ga, In, Tl, Ge, Sn, Pb, Sb, Bi, Po. Typically, M' is Sn, Li, Na, K, Be, Mg or Ca. More typically, M' is Sn.

Any suitable salt of M may be employed. An example of a suitable salt of M is an acetate salt of M. Where M is Cu 5 Zn or Ni, the salt is typically M(OAc) 2 .xH 2 O. Generally x is from 1 to 4. Typically, x is 1 for Cu, 2 for Zn and 4 for Ni.

Typically, the salt of M is present in a molar excess with respect to the compound of formula (W). More typically, the salt of M is present in about a twofold molar excess with respect to (W).

Metal complexes of formula (Ia) may be further derivatised to produce metal complexes of formula (Ib).

Accordingly, the invention provides a process for producing a metal complex of the following formula (Ib):

(Ib) wherein:

M is a transition metal or a p-block metal; either (i) b is a bond and c is not a bond, or (ii) c is a bond and b is not a bond;

L is a ligand comprising an electron donor group; a is a C 5-10 carbocyclic, C 5 .10 heterocyclic, C 5-10 aryl or C 5 ^o heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri- 5 tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-1O aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P;

Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl, phenyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(RT)C(O), -alk- N(RT)C(O)-, -C(O)N(RT), -alk-C(O)N(RT), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(RT)-, - alk-C(S)N(R7)-, -N(RT)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(RT)-, -HIk-S(O) 2 N(RT)-, -

N(RT)S(O) 2 -, -alk-N(R7)S(O) 2 - 5 -S(O) -, -alk-S(O)-, -N(RT)C(O)O-, -alk-N(RT)C(O)O-, - OC(O)N(RT)-, -alk-OC(O)N(RT>, -N(RT)C(O)N(RT)-, -alk-N(RT)C(O)N(RT>, - N(RT)C(S)N(R7)-, -alk-N(RT)C(S)N(RT)-, -N=C(RT)-, -alk-N=C(R7)-, -C(RT)=N-, -alk- C(RT)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-2O alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1 . 10 )alkylamino, C 3-20 carbocyclyl, C3. 20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-2 o carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2 _ 2 o alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-2O alkynyl, amino, C 1-10 alkylamino, di(C 1-1 o)alkylamino, C 3-20 carbocyclyl, C 3-2O heterocyclyl, C 3 . 2 o heteroaryl, aryl, aryloxy, -alk-C 3 . 20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(RT), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(RT), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group;

and provided that Ll ', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-2 o heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; the process comprising treating a compound of formula (Ia) as defined above, with either:

(i) L; or

(ii) a precursor compound comprising L; in the presence of a solvent, wherein L is said ligand comprising an electron donor group. When the electron donor group of L is a heteroatom which bears a lone pair of electrons, the compound of formula (Ia) is generally treated with L itself, rather than with a precursor compound that comprises L. Such heteroatoms are generally able to coordinate to metal centres as well as exist in a free, unbound state. For instance, where L is DABCO (in which the electron donor group is a nitrogen atom bearing a lone pair of electrons) the compound of formula (Ia) is treated with DABCO itself, rather than with another compound that comprises DABCO. Similarly, where L is a heterocyclic or heteroaryl ring, or a C 1-20 hydrocarbon molecule comprising one or more heteroatoms, the compound of formula (Ia) is generally treated with L itself, rather than with a precursor of L: in those cases, the electron donor group is typically a heteroatom of L, which heteroatom bears a lone electron pair and can interact with the metal centre as well as exist in an unbound state.

Thus, typically, said treatment of the compound of formula (Ia) is with L, wherein L is said ligand comprising an electron donor group. The solvent is any suitable solvent. Typically, the solvent is a polar aprotic solvent, for instance THF or diethyl ether, or a non- polar organic solvent, for instance pentane or hexane. Generally, L is present in a molar excess with respect to the compound of formula

(Ia). For instance, L may be present in a tenfold or greater molar excess with respect to the compound of formula (Ia).

Ususally the reaction does not require heating and is carried out at room temperature. Typically, said treatment of the compound of formula (Ia) with L is by co- crystallisation of (Ia) and L from a solution of (Ia) and L. Typically, the solvent is a polar aprotic solvent, such as THF or diethyl ether, layered with a non-polar organic solvent, such as pentane or hexane.

Where the electron donor group of L is an atom or group which bears an overall negative charge, or a radical electron, the compound of formula (Ia) is generally treated with a precursor compound comprising L. The precursor compound comprising L may be a salt of L with an appropriate cation, or an adduct of L with another group, such as a leaving group. For instance, if L is an organic radical, such as C 1-2O alkyl, C 1-10 alkoxy, C 1-10 alkylthio,

C 2-20 alkenyl, C 2-20 alkynyl, acyl, acylamido, acyloxy, ester, aryl heteroaryl, C 3-20 heterocyclyl or C 3-20 carbocyclyl group, then the compound of formula (Ia) may be treated with a salt of L. The salt of L may be an alkali metal salt, such as a lithium salt or sodium salt.

The metal complex employed in the present invention, of formulae (I), (Ia), (Ib) or (Ic), may incorporate a group Z, i.e. a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, or a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone.

As the skilled person will appreciate, such a group Z may be incorporated into a metal complex employed in the present invention by synthesising that complex according to the process of the present invention using a ligand precursor compound, of formula (Y), (Z) or (V) above, whose Rl or Rl ' group already contains that group Z.

Alternatively, the group Z may be incorporated by coupling a "precursor" metal complex of formula (I) to that group Z. For instance, a "precursor" metal complex of formula (I) may comprise a terminal carboxylic acid group (e.g. within a terminal benzoic acid group) which carboxylic acid group can be used to couple the precursor complex to a group Z. For example, that carboxylic acid group of the precursor complex can be readily converted into an activated ester, which is susceptible to nucleophilic attack by amines and hydrazines.

Thus, a metal complex of formula (I) can be formed which is different from the precursor complex of formula (I) and which comprises the group Z.

Generally, the group -Z, or the group -L3-Z- or the group -L3-alk-Z is coupled to a precursor metal complex of formula (I), or alternatively incorporated in a ligand precursor compound of formula (Y), (Z) or (V), using conventional techniques known to the person of skill in the art. For instance, Z may be coupled to a thiosemicarbazone derivative via one or more peptide bonds, typically one peptide bond. Typically, when Z is linked via a peptide

bond, this link is formed in a reaction between a -COO(R7) group and a -NH(R7) group in the thiosemicarbazone derivative and the group containing Z, respectively, or vice versa.

Alternatively, Z may be coupled to the terminal -NH 2 group of the thiosemicarbazone derivative (10a) via one or more irnine links, typically one imine link. An example of this embodiment is shown in the following scheme:

The dione (1 Ia) is typically symmetrical and nucleophilic attack on the dione takes place at both carbonyl groups, giving rise to a thiosemicarbazone derivative (13a) which is symmetrical about L". If thiosemicarbazone derivative (10a) is already complexed to a metal centre, and Z is also a thiosemicarbazone complex, the product (13a) is a binuclear metal thiosemicarbazone complex.

Accordingly, in one aspect, the present invention provides a metal complex of formula (I) as defined above, wherein Z is a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone.

Where Z is a complex of a transition metal with a bis(thiosemicarbazone), that complex may itself be a complex of formula (I). Certain of such binuclear bis(thiosemicarbazone) complexes may be prepared by reaction of (at least) two equivalents of the mørøo-keto-thiosemicarbazone (V) with a dithiosemicarbazide (U) in the presence of a salt of M according to the following scheme:

(V) (U)

M-salt

wherein Q is -Ll- or -L1-R1-L3- wherein Ll, L3 and Rl are as defined above for formula (I).

Alternatively, such binuclear thiosemicarbazone derivatives of formula (Ie) may be prepared by reaction of a dithiosemicarbazone and a thiosemicarbazide according to the following scheme:

wherein Q is as defined above in connection with formula (Ie).

In another embodiment, complexes employed in the invention wherein Ll is -N(R7)- may be prepared according to the following scheme:

wherein LG is a suitable leaving group such as Br or Cl.

In a further embodiment complexes employed in the present invention wherein Ll is • N=C(R7)- may be prepared according to the following scheme:

Typically, where it appears in the above schemes and in the complexes employed in the present invention as defined above, R7 is H.

In one aspect, the invention provides a process for producing a metal complex of the following formula (Ia):

wherein:

M is a transition metal; a is a C 5-10 carbocyclic, C 5-10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a Cs -10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O,

N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(0), -alk- N(RT)C(O)-, -C(O)NCRT), -alk-C(0)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(RT)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)- 5 - N(RT)SCO) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(0)0-, -alk-N(R7)C(0)0-, - OCCO)NCRT)-, -alk-0C(0)N(R7)-, -N(R7)C(O)N(R7)- 5 -alk-N(R7)C(O)N(R7)-, -

N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted;

Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2 . 20 alkynyl, amino, C^ 10 alkylamino, di(C 1-1 o)alkylamino, C 3-2O carbocyclyl, C 3-20 heterocyclyl, C 3 . 20 heteroaryl, aryl, aryloxy, -alk-C 3-2 o carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-1 o)alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(R7), O, S or arylene; and R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating, in the presence of a solvent, a salt of M with either:

(i) a compound of following formula (X), a compound of the following formula (Y) and a compound of the following formula (Z):

(X) . (Y) (Z) ; o

(ii) a compound of the following formula (V) and a compound of the following formula (Z):

(V) , (Z)

The invention further provides a process for producing a metal complex of the following formula (Ia) by transmetallation:

wherein:

M is a transition metal; a is a Cs -10 carbocyclic, C 5-10 heterocyclic, C 5-1O aryl or C 5-I0 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused bi-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted; X and X', which are the same or different, are independently selected from N and P;

Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk~ C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(O), -alk- N(R7)C(0)-, -C(0)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7>, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, -

N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 - 3 -S(O) -, -alk-S(O)-, -N(R7)C(0)0-, -alk-N(R7)C(0)0-, - OC(O)N(R7)- 5 -alk-OC(O)N(R7)-, -N(R7)C(O)N(R7)-, -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)- 5 -alk-N(R7)C(S)N(R7)-, -N=C(R7)-, -alk-N=C(R7)-, -C(R7)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-2O alkynyl, amino, C 1-10 alkylarnino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3- 2 o heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an amino acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(tbiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylarnino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-20 heterocyclyl, -alk-C 3-2 o heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

-alk- is unsubstituted or substituted C 1-2O alkylene which is optionally interrupted by N(R7), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3 . 20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group;

and provided that Ll', Rl' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; which process comprises treating a salt of M, in the presence of a solvent, with a metal complex of the following formula (W):

wherein M' is either (i) a transition metal other than M or (ii) a main group metal; and a, X', X, Y', Y 5 R2, R2', Rl, Rl', Ll and Ll' are as defined above for the metal complex of formula (Ia).

The invention further provides a process for producing a metal complex of the following formula (Ib):

(Ib) wherein:

M is a transition metal; either (i) b is a bond and c is not a bond, or (ii) c is a bond and b is not a bond;

L is a ligand comprising an electron donor group;

a is a C 5 - 10 carbocyclic, C 5-10 heterocyclic, C 5-10 aryl or C 5-10 heteroaryl ring, which ring is unsubstituted, substituted or part of a fused M-, tri-, tetra- or polycyclic ring system, wherein the or each further ring of said fused bi-, tri-, tetra- or polycyclic ring system is independently selected from a C 5-10 carbocyclic ring, a C 5-10 heterocyclic ring, a C 5-10 aryl ring and a C 5-10 heteroaryl ring, wherein the ring system is unsubstituted or substituted;

X and X', which are the same or different, are independently selected from N and P; Y and Y', which are the same or different, are independently selected from S, O, N(R5) and P(R5), wherein each R5 is independently selected from H, C 1 -C 6 alkyl and a group comprising an electron donor group, which donor group is separated by two carbon atoms from the N or P atom to which R5 is attached;

Ll and Ll', which are the same or different, are each independently selected from a covalent bond and a linker group selected from -alk-, -C(O)-, -alk-C(O)-, -C(O)O-,-alk- C(O)O-, -OC(O)-, -alk-OC(O)-, -0-, -alk-O-, -N(R7)-, -alk-N(R7)-, -N(R7)C(O), -alk- N(R7)C(0)-, -C(O)N(R7), -alk-C(O)N(R7), -C(S)-, -alk-C(S)-, -S-, -alk-S-, -C(S)N(R7)-, - alk-C(S)N(R7)-, -N(R7)C(S)-, -alk-N(R7)C(S)-, -S(O) 2 N(R7)-, -alk-S(O) 2 N(R7)-, -

N(R7)S(O) 2 -, -alk-N(R7)S(O) 2 -, -S(O) -, -alk-S(O)-, -N(R7)C(O)O-, -alk-N(R7)C(0)0-, - OC(O)N(RT)-. -alk-0C(0)N(R7)-, -N(RT)C(O)N(RT)-, -alk-N(R7)C(O)N(R7)-, - N(R7)C(S)N(R7)-, -alk-N(R7)C(S)N(R7)-, -N=C(RT)-, -alk-N=C(RT>, -C(RT)=N-, -alk- C(R7)=N-, arylene and arylene-alk-, wherein said arylene is unsubstituted or substituted; Rl and Rl ', which are the same or different, are each independently selected from hydrogen, Z, L3-Z, L3-alk-Z, and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-1O alkylamino, di(C 1-10 )alkylamino, C 3-20 carbocyclyl, C 3-20 heterocyclyl, C 3-20 heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3- 20 heterocyclyl, -alk-C3 -20 heteroaryl, and -alk-aryl, which group may be further substituted by L3-Z or L3-alk-Z, wherein L3 is as defined above for Ll and Ll ', and wherein said C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O, S or arylene;

Z is a moiety selected from a biologically active molecule, a fluorophore, a cytotoxin, an ammo acid, a peptide, an oligopeptide, a polypeptide, a sugar, a group containing a label, a leaving group which is replaceable by a group containing a label, and a complex of a transition metal with a bis(thiosemicarbazone) or a thiosemicarbazone;

R2 and R2', which are the same or different, are each independently selected from H and a substituted or unsubstituted group selected from C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, C 3 . 20 carbocyclyl, C 3-20 heterocyclyl, C 3-20

heteroaryl, aryl, aryloxy, -alk-C 3-20 carbocyclyl, -alk-C 3-2 o heterocyclyl, -alk-C 3-20 heteroaryl, and -alk-aryl, which C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl are optionally interrupted by N(R7), O 5 S or arylene;

-alk- is unsubstituted or substituted C 1-20 alkylene which is optionally interrupted by N(R7), O, S or arylene; and

R7 is H, C 1-6 alkyl, C 3-10 cycloalkyl or aryl; provided that Ll, Rl and R2 may form, together with the N atom to which Ll and R2 are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted C 3-20 heteroaryl group; and provided that Ll ', Rl ' and R2' may form, together with the N atom to which Ll ' and R2' are attached, an unsubstituted or substituted C 3-20 heterocyclyl group or an unsubstituted or substituted Cs -20 heteroaryl group; the process comprising treating a compound of formula (Ia) as defined above, with either: (i) L; or

(ii) a precursor compound comprising L; in the presence of a solvent, wherein L is said ligand comprising an electron donor group.

Uses of complexes employed in the present invention The metal complexes of formula (I) are taken up into a range of human cancerous cell lines, and exhibit cytotoxicity towards those cell lines. The cell lines used were: IGROV

(epithelial-like ovarian carcinoma), T24 (colon carcinoma), SW620 (bladder carcinoma), A431

(epidermic carcinoma) and HeLa (an immortal cell line derived from cervical cancer cells).

Cytotoxicity was found to be comparable to the known cytotoxic agent cis platin. Thus, the complexes of formula (I) are candidates for use as therapeutic agents, in particular for use in treating cancers and other conditions characterised by undesirable cellular proliferation.

Conditions which may be treated by the metal complexes and pharmaceutical compositions of formula (I) include conditions characterised by undesirable cellular proliferation, that is to say, conditions characterised by an unwanted or undesirable proliferation of normal or abnormal cells. Such conditions may involve neoplastic or hyperplastic growth of any type of cell.

Examples of conditions characterised by undesirable cellular proliferation include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocytoma, osteoma), hypoxic tumours, cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma) and leukemias.

Certain complexes of formula (I) have the potential to be hypoxic selective. Hypoxic cells have a lower than normal oxygen concentration. Typically, the partial pressure of oxygen falls topOj < 3 mmHg of the normal concentrations (20 - 80 mmHg), occurring usually as a result of insufficient blood supply to the affected tissue which in turn can lead to anaerobic respiration and the lowering of cellular pH from an accumulation of lactic acid. Since the complexes of formula (I) have been shown to be cytotoxic, the complexes of ' formula (I) that are hypoxic selective may be useful in therapy targeted specifically to hypoxic regions. Such hypoxic selective complexes of formula (I) are thus particularly suitable candidates for use in the therapy of hypoxic tumours. This is of particular importance, because conventional radiotherapeutic methods are poor at treating areas of hypoxia due to the absence of oxygen, a strong radiosensitizer. Thus, the cytotoxicity of complexes of formula (I) helps to overcome the problem of the radiation therapy resistance of hypoxic zones in tumours.

Hypoxic selective complexes also have the potential to be used as imaging agents to visualise hypoxic tissue in a wide range of oncological, neurological and cardiological applications.

Thus in one embodiment, the complex of formula (I) as defined herein is hypoxic selective. Typically, in this embodiment, M is Cu. The copper complexes of formula (I) show fast and reversible reduction couples at biologically compatible potentials, rendering them potentially useful hypoxic imaging and therapeutic agents.

The hypoxic selectivity of such complexes may be manipulated by suitable selection of X, X', Y and Y'. By varying these atoms and groups it is possible to exert control over factors which may influence hypoxic selectivity such as pK a , reduction potential and stability of a particular redox state of the central metal atom (for instance, the stability of the Cu(I) state when the metal is copper).

Complexes employed in the present invention, including copper complexes, have enhanced fluorescence compared with known thiosemicarbazone complexes of the same metal. The uptake of these intrinsically fluorescent species, into a range of human cancer cell lines, was observed using fluorescence imaging. The zinc(II) complexes of formula (I) showed particularly strong fluorescence emission with respect to known Zn thiosemicarbazone compounds (including known Zn bis- thiosemicarbazone compounds with aliphatic backbones), and showed promising uptake into a range of cancer cells.

Surprisingly, the copper(II) analogues also showed fluorescence. Indeed, it was possible for the first time to observe the uptake of a copper(II) complex into cells by fluorescence emission. It has not previously been possible to observe the uptake or cellular distribution of Cu(II) bis(thiosemicarbazone) complexes using fluorescence. Typically, thiosemicarbazone complexes of metals other than copper, such as zinc, were used to model the uptake mechanism that would be expected of the corresponding Cu complex. A disadvantage of that method, however, was that it did not indicate the distribution in cells that would be expected of corresponding Cu complexes. This was because, unlike the zinc complexes, the copper complexes are redox active (i.e. their tendency to dissociate increases with oxidation or reduction of the metal atom). The intrinsic fluorescence of the copper complexes of formula (I) gives rise to the possibility of optical imaging of hypoxia. The fluorescent properties of the complexes employed in the invention can be used to track their uptake in living cells and show where the complexes are being localised. They also allow the distribution of the complexes within cells to be monitored. These properties can be extremely useful for diagnostic and other clinical purposes. For example, fluorescence can be used to check whether a particular complex is taken up into a particular diseased cell line and where in the cells the complex becomes localised. This can be useful for determining whether that complex would be suitable for imaging that cell line or for therapy of such cells.

Thus, in one embodiment, the present invention provides complexes of formula (I) as defined above which are fluorescent. In this embodiment, M is typically Zn, Cu, or a further transition metal in an oxidation state that results in electronic properties that do not quench fluorescence, such as d 6 or d 10 . Examples of such further transition metals include Cd(II),

Re(I), Tc(I) and Ru(II). Typically in this embodiment, M is Zn, Cd or Cu. More typically, M is Zn.

In another embodiment, the invention provides fluorescent complexes that are taken up by a human cancer cell line. In this embodiment the fluorescence of the complexes allows the distribution of the complexes within the cells to be monitered. Typically, in this embodiment, M is Zn or Cu. More typically, M is Zn. Complexes employed in the invention may be radiolabeled with metastable metal radionuclides which are useful in medical imaging techniques such as PET (Positron Emission Tomography) and SPECT (Single Photon Emmission Computerised Tomography).

SPECT is a widely-used imaging technique which is capable of mapping the in vivo distribution of γ-emitting radionuclides in three dimensions. Radioisotopes suitable for SPECT include 99m Tc, 111 In, 67 Ga, 131 I and 123 I. 99m Tc has found widespread use as a result of its facile separation from 99 Mo on an ion exchange column, its suitable energy of 141 KeV and its ideal half life of 6 hours, making it the most widely used imaging agent for SPECT.

PET is the mapping of radiopharmaceutical distribution based on detection of annihilation photons resulting from the interaction of a positron with an electron. It is useful for oncological applications and has increased sensitivity with respect to SPECT (10-100 fold). However randomness, scattering and photons emitted with insufficient energy will reduce contrast. The short half lives of positron emitters mean that they must be produced close to where they are needed. They are made either by nuclear generators via parent/daughter radionuclide decay (though this is rare) or by use of a cyclotron (as for the isotopes of 11 C, 13 N, 15 0, 19 F and 64 Cu). The positron, β + , emitting isotopes 89 Zr, 60 Cu, 61 Cu 5 62 Cu and 64 Cu find use in PET imaging. It is widely recognised that PET will be one of the primary tools for the diagnosis of cancer and for the monitoring of the effects of therapy. 64 Cu is of particular interest, since its half-life (12.7 h) is sufficiently long to allow for the synthesis of many radiopharmaceutical compounds. The long-lived Cu isotope is also of interest in the context of PET (positron emission tomography) imaging of the biodistribution of drugs as part of the drug screening process.

Thus, the complexes employed in the invention are candidates for use as imaging agents, using either radioactive imaging techniques such as PET and SPECT, or a nonradioactive optical technique, such as fluorescence. The radiolabeled complexes may be suitable for both non-radioactive and radioactive imaging, which may be used in combination. Indeed, when combined with fluorescence imaging, radioactive imaging techniques such as PET and SPECT could provide an extremely powerful tool in the clinical diagnosis and treatment of disease. Given that the copper and gallium complexes employed in the invention

show fluorescence, if a radioisotope of copper is used (e.g. 64 Cu) there is the possibility of simultaneous optical imaging (due to the insrinsic fluorescence of the copper complex) and PET imaging (due to the radionuclide).

Accordingly, the present invention further provides a diagnostic agent or medical imaging agent which comprises a complex of formula (I) as defined above. Typically, in this embodiment, M is Zn 5 Ga, In, Tc or Cu. Thus, for instance, M may be 99m Tc, 111 Li, 67 Ga, 89 Zr 5 60 Cu, 61 Cu, 62 Cu or 64 Cu. More typically, M is Zn, Ga 5 In, Zr or Cu. Thus, for instance, M may be 111 In, 67 Ga, 89 Zr, 60 Cu, 61 Cu, 62 Cu or 64 Cu. Even more typically, M is Zn or Cu. In one embodiment M is 60 Cu, 61 Cu, 62 Cu and 64 Cu. In one embodiment, M is 64 Cu. In one embodiment, the diagnostic agent or medical imaging agent is suitable for both radioactive imaging and non-radioactive imaging, wherein the complex comprises a radionuclide. Typically, in this embodiment, the non-radioactive imaging is by fluorescence. Thus, M may be Cu or any transition metal in an oxidation state that results in electronic properties that do not quench fluorescence, such as d 6 or d 10 . Examples of such further metals include Zn (II), Cd(II), Re(I) 5 Tc(I) and Ru(II). Typically in this embodiment, M is Zn, Cd or Cu. Typically, the radioactive imaging is by PET or SPECT. Thus, in this embodiment, M may be a radionuclide of copper, such as 64 Cu. When M is a radionuclide of copper, the complex may comprise a group Z, wherein Z is a group containing a label, wherein the label is a fluorophore. Thus, the fluorescence of the Cu complexes employed in the invention may be further enhanced. Alternatively, in this embodiment, M may be Zn or any other transition metal in an oxidation state that results in electronic properties that do not quench fluorescence, provided that the complex comprises a group Z 5 wherein Z is a group containing a label, the label being a radioisotope suitable for use in radioactive imaging. Typically, the radioisotope is 18 F, 11 C, 14 C, 3 H, 99m Tc 5 111 In, 67 Ga, 89 Zr, 123 I or 188 Re. More typically, the label is 18 F.

Furthermore, the complexes employed in the invention are candidates for use as dual therapy and imaging agents, for example in the dual therapeutic treatment and imaging of cancer tumours and other conditions characterised by undesirable cellular proliferation.

Accordingly, the present invention further provides the use of a complex of formula (I) as defined above in the manufacture of a medicament for use as a diagnostic agent, an imaging agent and/or a therapeutic agent.

Typically, the medicament is for use as a combined imaging and therapeutic agent. Typically, in this embodiment, M is Cu or Zn. When M is Cu, M is typically a radionuclide of

copper. More typically, M is 64 Cu. Alternatively, M may be a stable (non-radioactive) isotope of Cu. When M is Cu, the complex may comprise a group Z 5 wherein Z is a group containing a label, the label being a fluorophore. Thus, the fluorescence of the Cu complex may be further enhanced, if desired. Alternatively, in this embodiment, M is Zn. When M is Zn 3 the complex may or may not comprise a group Z wherein Z is a group containing a label, the label being a radioisotope. This depends on whether the intrinsic fluorescence of the zinc complex is being used for the purpose of imaging the complex, or whether radioactive imaging (such as PET or SPECT) is desired. Typically, the radioisotope is 18 F, 11 C, 14 C, 3 H, 99m Tc, 111 In, 123 I or 188 Re. Most typically the label is 18 F. The use of complexes of formula (I) as defined above, in which the complex itself is conjugated to a functional substituent (for instance, a complex of formula (I) in which Rl or Rl ' is either Z or a group substituted by Z) might hinge on whether the complex is hypoxic selective or not.

If the complex is not hypoxic selective then attachment of an appropriate biologically active molecule as Z will permit the complex to be used as a targeted diagnostic imaging agent (for instance, when Z is a monoclonal antibody or a peptide). Biologically active molecules in this context may be those mentioned above, for instance therapeutic agents and agents which target the conjugated complex to the desired site in vivo. They include cytotoxins, monoclonal antibodies, folic acid and peptides. If the metal complex is hypoxic selective to start with, for instance due to the redox behaviour of the metal centre, then suitable manipulation of the linker group and functional substituent on the side-chain in the final conjugate of formula (I) allows that hypoxic selectivity to be retained. Cu is typically the metal M in complexes employed in the invention which are hypoxic selective. The complex can act as a hypoxic selective vector to deliver a range of functional molecules to the desired site in vivo, in particular to tumours, giving rise to a wide range of biomedical applications. Those applications include therapy, diagnosis and medical imaging, examples of which are as follows.

When Z in formula (I) as defined above is a metal based or organic fluorophore and M is Cu, there is the possibility of enhancing the intrinsic fluorescence of the copper complexes employed in the invention for the purpose of optical imaging of hypoxia. In particular, if the fluorophore exhibits two-photon fluorescence then the emission is at a sufficiently long wavelength to be detected externally to the body of the patient.

As noted above, in one embodiment the present invention provides complexes wherein M is zinc (Zn(II)) which are highly fluorescent. Manipulation of the substituents present may enable further enhancement of the quantum yield of the fluorescence. This brings advantages, such as the possibility to increase sensitivity in cellular measurements. When Z in formula (I) is or comprises another radionuclide and M is Cu, the complex can for instance be used in the SPECT imaging of tumours (using 99m Tc as the radionuclide) or in PET imaging (using 18 F as the radionuclide ). The latter option is particularly desirable

1 S since it combines the hypoxic selectivity of the Cu with the ready availability of F. This provides a convenient route to 18 F-based PET imaging agents for hypoxia and for the subsequent monitoring of therapy. If the radionuclide used in Z is a beta emitting radionuclide such as 188 Re 5 the complex can be used in targeted radiotherapy.

When Z in formula (I) is a therapeutic agent such as a cytotoxin and M is Cu, the resulting cytotoxin complex can be targeted specifically to tumours. In one embodiment Z is or comprises a reductively activated cytotoxin, and the copper complex releases the active agent in vivo once it is trapped inside the cells.

As is evident, the complexes employed in the present invention have a wide range of potential applications. Some of these applications may be modified, enhanced or realised by attachment of the appropriate group Z to the complex. Thus, in one embodiment, the present invention provides complexes as defined above, wherein LV, -Rl' and/or -Ll and -Rl, and R2 and R2', are such that the complex has one or more terminal functional group that provides a means for simple reaction with another compound to which the skilled person wishes to attach it. Thus, in one embodiment the present invention provides complexes which may serve as a building block for producing a diagnostic agent, which can be added to another compound as and when is convenient depending on the application. For example, terminal groups that may be suitable for such building blocks include Narylcarboxylate, - COOH, -CHO, -NH 2 , and -OH. For example, when the complex features a terminal -NNH 2 group, these can be readily reacted with another molecule with an activated ester or carboxyl chloride group to generate amide bonds, or with carbonyl groups to generate imides. By way of another example, when the complex features a Narylcarboxylate group, this can be readily reacted with another molecule with an -NH 2 group to generate an amide.

Complexes employed in the present invention may be also used in a method of imaging a cell, in vitro biopsy sample or patient. Accordingly, the invention provides a method of imaging a cell or in vitro biopsy sample, which method comprises: (a) contacting

the cell or in vitro biopsy sample with a complex employed in the invention as defined herein; and (b) imaging the cell or in vitro biopsy sample. When step (b) is imaging the cell, the imaging of the cell can be done using conventional techniques. When step (b) is imaging the in vitro biopsy sample, the imaging of the in vitro biopsy sample can be performed using conventional techniques. The invention further provides a method of imaging a patient in need thereof, which method comprises: (a) administering to the patient a complex employed in the invention as defined herein; and (b) imaging the patient. Step (b), of imaging the patient, can be done using conventional techniques.

Typically, in step (b), the imaging is fluorescence imaging. Thus, in one embodiment, the invention provides a method of imaging a cell or in vitro biopsy sample, which method comprises: (a) contacting the cell or in vitro biopsy sample with a complex employed in the invention as defined herein which complex is fluorescent; and (b) imaging the cell or in vitro biopsy sample using fluorescence imaging. The invention further provides a method of imaging a patient in need thereof, which method comprises: (a) administering to the patient a complex employed in the invention as defined herein which complex is fluorescent; and (b) imaging the patient using fluorescence imaging.

Alternatively, for imaging an in vitro biopsy sample or a patient, the imaging may be PET or SPECT. Thus, in one embodiment, the invention provides a method of imaging an in vitro biopsy sample, which method comprises: (a) contacting the in vitro biopsy sample with a complex employed in the invention as defined herein, which complex comprises a radionuclide suitable for PET imaging or SPECT imaging; and (b) imaging the in vitro biopsy sample using PET imaging or SPECT imaging. In another embodiment, the invention provides a method of imaging a patient in need thereof, which method comprises: (a) adrninistering to the patient a complex employed in the invention as defined herein, which complex comprises a radionuclide suitable for PET imaging or SPECT imaging; and (b) imaging the patient using PET imaging or SPECT imaging. Radionuclides suitable for PET and SPECT imaging respectively are discussed herein.

Alternatively, for imaging an in vitro biopsy sample or a patient, the imaging is both fluorescence imaging and eiher PET or SPECT imaging. The fluorescence imaging and eiher PET or SPECT imaging may be simultaneous. Thus, in one embodiment, the invention provides a method of imaging an in vitro biopsy sample, which method comprises: (a) contacting the in vitro biopsy sample with a complex employed in the invention as defined herein, which complex is fluorescent and comprises a radionuclide suitable for PET imaging

or SPECT imaging; and (b) imaging the in vitro biopsy sample using fluorescence imaging and PET imaging or SPECT imaging. In another embodiment, the invention provides a method of imaging a patient in need thereof, which method comprises: (a) administering to the patient a complex employed in the invention as defined herein, which complex is fluorescent and comprises a radionuclide suitable for PET imaging or SPECT imaging; and (b) imaging the patient using fluorescence imaging and PET imaging or SPECT imaging. Radionuclides suitable for PET and SPECT imaging respectively are discussed herein.

Some key design features of the metal complexes for use in the present invention as optical andTET imaging probes, with increased cytotoxicity include: (a) They are more soluble in biological media than known Zn(II) analogues.

(b) They incorporate flat, π-rich systems likely to bind to DNA in an intercalative manner.

(c) They show higher levels of fluorescence than known Zn(II) analogues.

(d) They have rapid and clean conversion to 64 Cu(II) or other radiolabeled analogues. (e) They incorporate functional groups suitable for further derivatisation with an

'address' for targeted delivery. The higher intrinsic fluorescence, coupled with solubility and stability in biologically compatible medium should facilitate the monitoring of cell delivery and biodistribution of imaging probes in cancer cells.

The present invention provides a pharmaceutical composition comprising a complex of the invention as defined above and a pharmaceutically acceptable carrier or diluent. A complex of the invention is formulated for use as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier or diluent. The compositions are typically prepared following conventional methods and are administered in a pharmaceutically suitable form. The complex may be administered in any conventional form, for instance as follows:

A) Orally, for example, as tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

Tablets contain the complex in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes. Formulations for oral use may also be presented as hard gelatin capsules wherein the complex is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is present as such, or mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Aqueous suspensions contain the complex in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides for example polyoxyethylene sorbitan monooleate.

The said aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, such as sucrose or saccharin.

Oily suspension may be formulated by suspending the complex in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occuring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, for example sorbitan mono-oleate, and condensation products of the said partial esters with ethylene oxide, for example poly oxy ethylene sorbitan monooleate. The emulsion may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose. Such formulations may also contain a demulcent, a preservative and flavouring and coloring agents.

B) Parenterally, either subcutaneously, or intravenously, or intramuscularly, or intrasternally, or by infusion techniques, in the form of sterile injectable aqueous or oleaginous suspensions. This suspension may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.

Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition fatty acids such as oleic acid find use in the preparation of injectables;

C) By inhalation, in the form of aerosols or solutions for nebulizers;

D) Rectally, in the form of suppositories prepared by mixing the drag with a suitable non-irritating excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and poly-ethylene glycols;

E) Topically, in the form of creams, ointments, jellies, collyriums, solutions or suspesions.

Abbreviations The abbreviations Cu(ATSM), Zn(ATSM), Zn(ATSEt) and Zn(ATSPh) are used herein. Cu(ATSM) refers to a compound of the following formula (X), in which M is Cu and each R is methyl. Zn(ATSM) refers to a compound of the following formula (X), in which M is Zn and each R is methyl. Zn(ATSM/ A) refers to a compound of the folio whig formula (X), in which M is Zn, one R is methyl and the other R is amino. Zn(ATSEt) refers to a compound of the following formula (X), in which M is Zn and each R is ethyl. Zn(ATSPh) refers to a compound of the following formula (X), in which M is Zn and R is phenyl.

(X)

The invention will be further described in the Examples which follow:

EXAMPLES

Mono(thiosemicarbazone) ligands, metal complexes of the mono(thiosemicarbazone) ligands, and Ni, Zn and Cu bis(thiosemicarbazone) complexes were synthesized and characterised as described in the following Comparative Examples 1 to 7 and Examples 8 to 16. NMR spectra were run on a Varian Mercury 300 MHz spectrometer at 298K in dβ -DMSO solution and referenced to residual solvent peak. IR spectra were run on a Perkin Elmer 1000 FT-IR spectrometer using KBr discs. UV spectra measurements were run in DMSO using a Perkin Elmer UV/Vis/NIR spectrometer Lambda 19. Fluorescence spectra were measured in DMSO using a Hitachi F-4500 Fluorescence Spectrophotometer. ES-MS was carried out by the Inorganic Chemistry Spectrometry Service using a MicroMass LC time of flight electrospray mass spectrometer. HPLC was done on a Gilson Unipoint instrument using a reverse phase column with a CH 3 CN/H 2 O mobile phase. Elemental analyses were performed by the University of Oxford Inorganic Chemistry Laboratory analytical department. X-ray crystal structural data was collected using an Enraf Nonius DIP2000 image plus plate diffractometer. Fluorescence Microscopy images were made using a Leica confocal microscope.

Comparative Example 1: Preparation of mono(thiosemicarbazone) acenaphthenequinone (1)

(D

Mono(thiosemicarbazone) acenaphthenequinone (1) was obtained from a 1 : 1 molar ratio of acenaphthenequinone and thiosemicarbazide in absolute ethanol as reported by Rodriguez- Arguelles, M. C. et al. Journal of Inorganic Biochemistry 1997, 66, (1), 7-17. Acenaphtenequinone (0.5 g, 2.74 mmol) and thiosemicarbazide (0.2502 g, 2.74 mmol) were suspended in absolute ethanol (15ml) and heated under reflux for 2 hours. The resulting solid was then isolated from the reaction mixture by filtration of the hot reaction mixture. The solid was re-suspended in hot methanol (10ml) and stirred for 15 minutes before filtering and washing with further methanol. The solid (1) was then dried under vacuum. Yield = 0.5642 g, 2.21 mmol, 81 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 12.50 (s, IH, H7') 5 9.12 (s, IH, NH 2 ), 8.83 (s, IH, NH 2 ), 8.34 (d, IH, H7), 8.11 (d,lH, H3), 8.09 (d, IH, H9), 7.99 (d, IH, Hl), 7.86(dd , IH, H8), 7.81(dd, IH, H2). 13 C NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 189.0(C=O) 3 179.3 (C-8'), 139.6(C-5'), 137.8(C-5), 133.2(C-3), 130.8(C-4), 130.4(C-IO), 130.3(C-8), 129.3(C-2), 129.0(C-6), 127.5(C-9), 122.8(C-7), 118.8(C-4). IR: 3404 m υ(NH 2 ), 3241 w υ(NH 2 ), 3147 m υ(N-H ), 1689 s υ(C=O), 1606 s υ(C=N), 1575 m υ(C=N), 1486 sb, 1451 s υ(ring), 1140 m υ(C-S). ES MS: m/z = 257.1 [M + H] + . HPLC: R f = 14.1 mins. X-ray: crystals of (1) suitable for X-ray diffraction analysis were grown, and the structure of (1) was confirmed by single crystal X-ray crystallography.

Comparative Example 2: Preparation of zinc bis [(mono-thiosemicarbazone) acenaphthenequinone] (2)

(2)

Mono(thiosemicarbazone) acenaphthenequinone (1) was prepared as described in Comparative Example 1. Mono(thiosemicarbazone) acenaphthenequinone (1) (0.350 g, 1.366 mmol), and zinc acetate bishydrate (0.150 g, 0.683 mmol) were then suspended in ethanol (50ml) with 10 drops of HCl (35 %) and heated under reflux for 24 hours. The resulting red- orange solid was then isolated from the reaction mixture by filtration of the hot reaction mixture. The solid was washed with diethyl ether (50 ml). The solid (2) was then dried under vacuum. Yield = 0.327 g, 0.572 mmol, 84 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 8.72 (d, 2H 5 NH 2 ), 8.63 (d, IH, H-7), 8.24 (d, IH, H-3), 8.05 (d, IH 5 H-9) 5 7.92 (d 5 IH 5 H-I) 5 7.79 (t, IH 5 H-8), 7.71 (t, IH, H-2). 13 C NMR (300 MHz 5 d 6 -DMSO 5 25 0 C ): δ 188. 3 (C-7") 5 186.8 (C-8'), 138.2 (C-5'), 138. 2 (C-5), 132.8 (C-3), 130.3 (C-4), 129.8 (C-10), 128.9 (C-8), 128.3 (C-2), 128.2 (C-6), 127.2 (C-9), 124.0 (C-7), 122.9 (C-I). ES MS: m/z = 573.0 [M + H] + . IR: 3446w 3355w υ(NH 2 ), 1685s υ(C=O), 1604sm υ(C=N), 1593s υ(C=N) 5 1486s υ(ring), 1176m υ(C-S) 5 1118mwυ(N-N). X- ray : crystals of (2) suitable for X-ray diffraction analysis were grown, and the structure of (2) was confirmed by single crystal X-ray crystallography.

Comparative Example 3: Preparation of mono(4-methyl-3-thiosemicarbazone) acenaphthenequinone (3)

9 1

(3)

Acenaphtenequinone (0.50 g, 2.74mmol) and 4-methyl-3-thiosemicarbazide (0.290 g, 2.80 mmol) were suspended in absolute ethanol (15ml) and heated under reflux for 2 hours. The resulting solid was then isolated from the reaction mixture by filtration of the hot reaction mixture. The solid was re-suspended in hot methanol (10ml) and stirred for 15 minutes before

filtering and washing with further methanol. The solid was then dried under vacuum: a yellow solid (3) was obtained. Yield = 0.604g, 2.245 mmol, 82.0 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C ): δ 12.62 (s, IH, N-N(H)), 9.38 (q, IH, N(H)Me) 5 8.36 (d, IH, H7), 8.12 (d, IH 5 H3) 5 8.07 (d, IH, H9), 7.95 (d, IH 5 Hl), 7.86 (t, 2H 5 H8) 5 7.82 (t, 2H 5 H2) 3.10 (t, 3H 5 (CH 3 )). 13 C NMR (300 MHz 5 d 6 -DMSO, 25 0 C): δ 188.8(CO) 5 178.3(C-8'), 139.3 (C-5'), 137.3(C-5), 133.1(C-3), 130.7(C-4) 5 130.4(C-IO), 130.2(08), 129.5(C-2), 129.2(C-6), 128.9(C-9), 122.8(C-7), 118.5(C-I) 5 31.9(C-Me). IR: 3230m υ(N-H), 3051wυ(N-H ) 5 1687s υ(C=O), 1610s υ(C=N), 1553s υ(C=N) 5 1486mb υ(ring) 5 1435 s υ(ring) 5 1142m υ(C-S). ES MS: m/z = 270.1 [M + H] + . HPLC: R f = 15.9mins. Elemental: Calculated N(15.61 %), C(62.45 %), H(4.59 %). Found N(15.40 %), C(62.37 %), H(4.36 %).

Comparative Example 4: Preparation of zinc bis(mono-4-ethyl-thiosemicarbazone) acenaphthenequinone] (4)

(4)

Mono(4-methyl-3-thiosemicarbazone)-acenaphthenequinone (3) was prepared as described in Comparative Example 3. Mono(4-methyl-3-thiosemicarbazone)-acenaphthenequinone (0.367 g, 1.366 mmol), and zinc acetate bishydrate (0.150 g, 0.683 mmol) were then suspended in ethanol (50ml) with 10 drops of HCl (35 %) and heated under reflux for 24 hours. The resulting red-orange solid was then isolated from the reaction mixture by filtration of the hot reaction mixture. The solid was washed with diethyl ether (50 ml). The solid (4) was then dried under vacuum. Yield = 0.307 g, 0.512 mmol, 75 %. 1 H NMR (300 MHz 5 d 6 -DMSO, 25 0 C ): δ 9.52 (m, IH 5 N(H)), 9.03 (m, IH, N(H)), 8.66 (d, IH 5 H7) 5 8.42 (d, IH, H7) 5 8.29 (d+d, 2H 5 H3) 5 8.10 (d 5 IH 5 H9) 5 8.08(d 5 IH, H9), 7.93(d+d,

2H, Hl) 3 7.85(t, IH, H8), 7.82(t, IH 5 H8), 7.72(t, IH, H2), 7.71(t, IH, H2). 13 C NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 188.2(C-7"), 187.0(C-7"), 183.3(C-8') 5 139.5(C-5'), 138.2(C- 5'), 137.2(C-5), 136.0(C-5), 132.5(C-3), 132.4(C-3), 129.9, 129.8, 129.64, 129.5, 129.0, 128.8, 128.5, 128.2, 128.0, 126.9, 123.4(C7), 123.3(C7), 122.8(Cl), 122.7(Cl), 31.5(CH 3 ) IR: 3299 w υ(N-H), 2910 m υ(N-H ), 1677 s υ(C=O), 1592 s υ(C=N), 1564 s υ(C=N), 1489 sb υ(ring), 1460 s υ(ring), 1169 m υ(C-S), 1141 s υ(NN). ES MS: m/z = 601.1 [M + H] + .

Comparative Example 5: Preparation of mono(4~ethyl-thiosemicarbazone) acenaphthenequinone (5)

(5)

Acenaphthenequinone (0.5 g, 2.74mmol) and 4-ethyl-3-thiosemicarbazide (0.358 g, 3.00 mmol) were suspended in absolute ethanol (15ml) and heated under reflux for 2 hours. The resulting solid was then isolated from the reaction mixture by filtration of the hot reaction mixture. The solid was re-suspended in hot methanol (10ml) and stirred for 15 minutes before filtering and washing with further methanol. The solid was then dried under vacuum: a yellow solid (5) was obtained. Yield = 0.659 g, 2.329 mmol, 85%. 1 H NMR (300 MHz, d 6 -DMSO, 25 0 C ): δ 12.45 (s, IH, N-N(H)), 9.45 (t, IH, N(H)Et), 8.35 (d, IH, H7), 8.10 (d, IH, H3), 8.05 (d, IH, H9), 7.95 (d, IH, Hl) 5 7.80 (t+t, 2H, H2 H8), 3.65 (m, 2H, (CH 2 )CH 3 ), 1.25 (t, 3H, CH 2 (CH 3 )). 13 C NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 188.9 (CO), 177.0 C-8'), 139.5(C-5'), 137.5 (C-5), 133.2(C-3), 130.9(C-4), 130.5(C-IO) 5 130.3(C-8), 129.4(C-2), 129.3(C-6), 129.0(C-9), 122.9(C-7), 118.7(C-I), 39.6(CH 2 ), 14.5(CH 3 ). IR: 3279 w υ(N-H), 3059 m υ(N-H ), 1686 s υ(C=O), 1607 s υ(C=N), 1536 s υ(C=N), 1475 sb υ(ring) 5 1452 s υ(ring), 1140 m υ(C-S). ES-MS: m/z = 284.1 [M + H] + . HPLC Rf= 17.3 mins. Elemental: Calculated N(14.84 %), C(63.60 %), H(4.59 %). Found N(14.52 %), C(63.12 %), H(4.52 %). X-ray: crystals of (5) suitable for X-ray diffraction

analysis were grown, and the structure of (5) was confirmed by single crystal X-ray crystallography.

Comparative Example 6: Preparation of zinc bis[mono(4-ethyI-3-thiosemicarbazone) acenaphthenequinone] (6)

(6)

Mono(4-ethyl-thiosemicarbazone)-acenaphthenequinone (5) was prepared as described in Comparative Example 5. Mono(4-ethyl-thiosemicarbazone)-acenaphthenequinone (0.387 g, 1.366 mmol) and zinc acetate bishydrate (1.50 g 0.683 mmol) were then suspended in ethanol (50ml) with 10 drops of HCl (35 %) and heated under reflux for 24 hours. The resulting red- orange solid was then isolated from the reaction mixture by filtration of the hot reaction mixture. The solid was washed with diethyl ether (50 ml). The solid (6) was then dried under vacuum. Yield = 0.65g, 1.0281 mmol, 75 %.

1 H NMR (300 MHz 5 d 6 -DMSO, 25 0 C): δ δ 9.52 (m, IH 5 N(H)), 9.03 (m, IH 5 N(H)), 8.64 (d, IH 5 H7), 8.43 (d, IH 5 H7), 8.28 (d+d, 2H 5 H3), 8.09 (d, IH 5 H9), 8.06 (d, IH 5 H9), 7.96(d+d 5 2H 5 Hl) 5 7.86(t, IH 5 H8) 5 7.82(t 5 IH 5 H8) 5 7.78(t 5 IH 5 H2) 5 7.74(t, IH 5 H2) 5 3.73 (m 5 2H 5 (CH 2 )CH 3 ), 3.64(m, 2H 5 (CH 2 )CH 3 ), 1.31(t, 3H 5 CH 2 (CH3)) 5 1.25 (t, 3H 5 CH 2 (OB)). 13 C NMR (300 MHz, d 6 -DMSO 5 25 0 C ): δ 188.3(C-7"), 187.4(C-7"), 182.6(C-8'), 139.7(C-5'), 138.5(C-5'), 137.4 (C-5), 136.6(C-5), 132.8(C-3) 5 132.6 (C-3), 130.3, 130.2, 129.8, 129.7, 129.1, 128.8, 128.6, 128.3, 128.2, 127.3, 123.6 (C7), 123.5(C7), 122.9(Cl), 122.8 (Cl), 39.2(CH 2 ), 14. 3(CH 3 ). IR: 3319 w υ(N-H), 2930 mυ(N-H ), 1683 s υ(C=O), 1606 s υ(C=N), 1577 s υ(C=N), 1496 s υ(ring) 5 1450 s υ(ring), 1171 m υ(C-S), 1139s υ(NN). ES MS: m/z = 629.1 [M + H] + X-ray: crystals of (6) suitable for X-ray diffraction analysis were grown, and the structure of (6) was confirmed by single crystal X-ray crystallography.

Comparative Example 7: Preparation of mono(4-phenyl-3-thiosemicarbazone) acenaphthenequinone (7)

(7)

Acenapthenequinone (0.50 g, 2.74 mmol) and 4-phenyl-3-thiosemicarbazide (0.468 g, 2.80 mmol) were suspended in absolute ethanol (15ml) and heated under reflux for 2 hours. The resulting solid was then isolated from the reaction mixture by filtration of the hot reaction mixture. The solid was re-suspended in hot methanol (10ml) and stirred for 15 minutes before filtering and washing with further methanol. The solid was then dried under vacuum: a yellow solid (7) was obtained. Yield = 0.62Og, 1.867 mmol, 68 %.

1 R NMR (300 MHz, d 6 -DMSO, 25 0 C ): δ 12.79 (s, IH 5 N-N(H)), 10.96 (t, IH 5 N(H)Ph) 5 8.38 (d, IH 5 H7), 8.14 (d, IH 5 H3) 5 8.12 (d, IH 5 H9) 5 8.01 (d, IH 5 Hl), 7.88(t, 2H 5 H8), 7.84(t 5 2H 5 H2), 7.62 (d, 4H 5 o-CH), 7.44 (t, 4H, m-CH) 5 7.29(t, 2H 5 p-CH). 13 C NMR (300 MHz 5 d 6 -DMSO 5 25 0 C): δ 188.9 (CO), 177.0(C-8'), 139.7 (C-5'), 138.9 (C-Ph) 5 138.0 (C-5), 133.2 (C-3), 130.7 (C-4), 130.3(C-18), 129.3(C-Ph) 5 129.1(C-8), 129.0(C-2), 128.9(C-6), 127.6 (C-9), 126.6(C-Ph) 5 126.2(C-Ph), 122.9(C-7), 119.3(C-I). IR: 3298 Wi)(N-H), 3054 w υ(N-H ), 1683 s υ(C=O), 1597 s υ(C=N) 5 1566 s υ(C=N), 1476 m υ(ring), 1445 s υ(ring) 5 1147 m υ(C-S). ES-MS: m/z = 333.0 [M + H] + . HPLC R f = 20.5 mins. X-ray: crystals of (7) suitable for X-ray diffraction analysis were grown, and the structure of (7) was confirmed by single crystal X-ray crystallography.

Example 8: Preparation of nickel bis(thiosemicarbazone) acenaphthenequinone (8)

(8)

Acenaphthenequinone (0.5 g, 2.74 mmol) and nickel acetate tetrahydrate (2.05 g, 8.25 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. Thiosemicarbazide (2.50 g, 27.44 mmol) was then added to the suspension and the mixture heated under reflux for 2 hours. The black-brown solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.934 g, 2.43 mmol, 89 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 8.01 (d, 2H, H3) 7.99 (s, 4H 5 NH 2 ), 7.77 (d, 2H, Hl), 7.68 (t, 2H, H2). 13 C NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 182.5 (C-8), 152.6 (C-5), 142.8 (C-3'), 131.4(C-2'), 129.1C-2), 128.1(C-I), 125.5(C-4), 122.7(C-3). IR: 3355 w I)(NH), 3132 wbr υ(NH), 1617 s υ(CN), 1583 s υ(CN), 1476 s υ(ring), 1436 s υ(ring), 1177 s υ(CS), 1140 s υ(NN). ES MS: m/z = 385.0 [M + H] + . HPLC R f = 21.8 mins. Elemental: Calculated: N(21.87 %), C(43.75 %), H(2.60 %), Ni(15.10 %). Found: N(21.87 %), C(42.96 %), H(2.52 %), Ni(15.04 %). X-ray: crystals of (8) suitable for X-ray diffraction analysis were grown, and the structure of (8) was confirmed by single crystal X-ray crystallography.

Example 9: Preparation of nickel bis(4-methyl-3-thiosemicarbazone) acenaphthenequinone (9)

(9)

Acenaphthenequinone (0.5 g, 2.74 mmol) and nickel acetate tetrahydrate (2.05 g, 8.25 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-methyl-3- thiosemicarbazide (2.50 g, 23.78 mmol) was then added to the suspension and the mixture heated under reflux for 2 hours. The black-brown solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.929 g, 2.254 mmol, 82 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 8.18 (d, 2H 5 N(H)Me) 5 7.97 (d, 2H, H3 ring), 7.94 (d, 2H 5 Hl ring), 7.64 (t 5 2H, H2 ring), 2.93 (d, 6H, Me). 13 C NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 179.5(C-8) 5 153.0(05), 142.5 (C-3'), 130.9(02'), 128.7 (O2), 127.8(C-I), 125.0 (C-4) ,123.9 (C-3), 32.4 (C-10). IR: 3353 mbr υ(NH), 1578 m υ(CN), 1532 s υ(CN), 1471 mbr υ(ring), 1393 s υ(ring), 1181 s υ(CS), 1149 s υ(NN). Elemental: Calculated N(20.34 %), C(46.51 %), H(3.42 %), Ni(14.21 %), Found N(19.72 %), C(46.01 %), H(3.42 %), Ni(14.09 %). ES MS: m/z = 413.0 [M + H] + . HPLC: R f = 17.8 minutes. X-ray: crystals of (9) suitable for X-ray diffraction analysis were grown, and the structure of (9) was confirmed by single crystal X-ray crystallography.

Example 10: Preparation of nickel bis(4-ethyl-3-thiosemicarbazone) acenaphthenequinone (10)

(10)

Acenaphthenequinone (0.5 g, 2.74 mmol) and nickel acetate tetrahydrate (2.05 g, 8.25 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-ethyl-3 -thiosemicarbazide

(1.32 g, 10.98 mmol) was then added to the suspension and the mixture heated under reflux for 2 hours. The black-brown solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.971 g, 2.21 mmol, 80 % 1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 8.32 (m, 2H, N(H)Et), 8.01 (d, 2H, H3), 7.89 (d, 2H, Hl), 7.68 (t, 2H, H2), 3.40 (m, 4H, H9), 1.14 (t, 6H 5 HlO). 13 C NMR (300 MHz, d 6 - DMSO, 25 0 C): δ 178.8(C-8), 152.8(C-5), 142.4 (C-3'), 130.9 (C-2'), 128.8(C-2), 127.7(C-I), 125.L(C-4), 122J(C-3), 40.8(C-9), 14.3(C-IO). IR: 3335 mbr υ(NH), 1576 s υ(CN), 1532 s D(CN), 1486 mbr υ(ring), 1343 s υ(ring), 1178 s υ(CS), 1142 s υ(NN). ES MS: m/z = 440.0 [M + H] + . HPLC R f = 22.1 mins. Elemental: Calculated N(19.09 %), C(49.09 %), H(4.12 %), Ni(13.17 %). Found N(19.03 %), C(48.56 %), H(4.01 %), Ni(12.52 %). X-ray: crystals of (10) suitable for X-ray diffraction analysis were grown, and the structure of (10) was confirmed by single crystal X-ray crystallography.

Example 11: Preparation of nickel bis(4-phenyl-3-thiosemicarbazone) acenaphthenequinone (11)

(H)

Acenaphthenequinone (0.5 g, 2.74 mmol) and nickel acetate tetrahydrate (2.05 g, 8.25 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-phenyl-3- thiosemicarbazide (1.32 g, 10.98 mmol) was then added to the suspension and the mixture heated under reflux for 2 hours. The black-brown solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield: 1.044 g, 1.948 mmol, 71 %.

1 H NMR (300 MHz, d 6 -DMSO 5 25 0 C): δ: 10.40 (s, 2H 5 H9), 8.09 (d, 2H, H3), 7.81 (d, 2H 5 Hl) 5 7.75 (t, 2H 5 Hl 3), 7.69 (d, 4H 5 Hl I) 5 7.32 (t, 4H 5 H12) 5 7.08 (t 5 2H 5 H2). 13 C NMR (300 MHz 5 d 6 -DMSO 5 25 0 C): δ 177.8(C-S) 5 155.8 (C-5) 5 143.3(C-3') 5 140.0(C-IO) 5 131.1(C-2') 5 129.1(C-2), 128.9(C-12), 128.7(C-I) 5 124.6(C-4), 123.7(C-13) 5 122.6(C-3) 5 120.3(C-Il). IR: 3313 m υ(NH), 1597 s υ(CN), 1541 s υ(CN) 5 1492 mbr υ(ring), 1435 s υ(ring), 1171 ms υ(CS), 1127 mbr υ(NN). ES MS: m/z = 537.0 [M + H] + . Elemental: Calculated N(15.67 %), C(58.2 %), H(3.36 %), Ni(10.82%). Found N(15.32 %), C(57.65 %), H(3.71 %), Ni(10.60 %). X-ray: crystals of (11) suitable for X-ray diffraction analysis were grown, and the structure of (11) was confirmed by single crystal X-ray crystallography.

Example 12: Preparation of zinc bis(4-methyl-3-thiosemicarbazone) acenaphthenequinone (12)

(12)

Acenaphthenequinone (0.5 g, 2.74 mmol) and zinc acetate bishydrate (1.807 g 5 8.23 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-methyl-3- thiosemicarbazide (1.20 g, 11.4 mmol) was then added to the suspension and the mixture heated under reflux for 2 hours. The orange-red solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.78 g, 1.87 mmol, 68%. 1 H NMR (300 MHz, d 6 -DMSO 5 25 0 C): δ 11.97 (s, IH 5 N-N(H)), 8.18 (d, 2H, H3 ), 7.98 (d, 2H 5 Hl), 7.88 (m, 2H, NH), 7.75 (t 5 2H 5 H2) 5 3.02 (d 5 6H, CH 3 ), 1.89 (s, 3H 5 CH 3 (acetic acid). 13 C NMR (300 MHz 5 d 6 -DMSO 5 25 0 C): δ 179.6 (C-8), 172.5 (acetic acid C=O) 140.4

(C-5), 138.5 (C-3'), 131.0 (C-2 5 ), 129.0 (C-2/4), 128.6 (C-2/4), 127.3 (C-I), 123.5 (C-3), 29.8 (C-IO), 21.8 (CH 3 acetic acid). ES MS: m/z = 419.0 [M + H] + . IR: 3200 wυ(NH), 305 Ow υ(NH), 1585 m υ(C=N), 1574 m υ(C=N), 1455 m υ(ring), 1174 s υ(C-S), 1120 mw υ(N-N). HPLC: R f = 16.9 mins. Elemental: Calculated (+CH 3 COOH): N(17.57 %), C(45.19 %), H(3.79 %), Ni(13.37 %). Found: N(16.73 %), C(44.57 %), H(3.58 %), Ni(13.47 %).

Example 13: Preparation of zinc bis(4-ethyl~3-thiosemicarbazone) acenaphthenequinone

(13)

(13)

Acenaphthenequinone (0.5 g, 2.74 mmol) and zinc acetate bishydrate (1.807 g, 8.23 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-ethyl-3-thiosemicarbazide (3.00 g, 25.16 mmol) was then added to the suspension and the mixture heated under reflux for 30 hours, under an atmosphere of nitrogen. The red solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.877 g, 1.97 mmol, 72 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ: 11.97 (slH, N-N(H)), 8.15 (d, 2H, H3 ring), 7.97 (d, 2H, Hl ring), 7.91 (m, 2H, N(H)Et), 7.70 (d, 2H, H2 ring), 3.35 (m, 4H, CH 2 (Et)), 1.21 (t, 6H, CH 3 (Et)), 1.87 (s, 3H 5 CH 3 acetic acid). 13 C NMR (300 MHz, d δ -DMSO, 25 0 C ): δ 178.9(C-8), 172.3 (acetic acid C-O), 140.1(C-5), 138.4(C-3'), 131.0(C-2'), 129.0(C-2/4), 128.7(C-2/4), 127.3(C-I), 123.2(C-3), 37.7(C-9) 5 21.9 (CH 3 acetic acid), 14.9(C-IO). IR: 3254 wbr υ(NH), 1591 mbr υ(CN), 1567 mbr υ(CN), 1384 m υ(ring), 1176 υ(CS), 1142m υ(NN). ES MS: m/z = 447.1 [M + H] + . HPLC: R f = 19.3 mins. Elemental:

Calculated(+CH 3 COOH) N(16.60%), C(47.42%) 5 H(4.38%), Zn(12.63%), Found; N(16.57%), C(46.51%) 5 H(4.27%), Zn(12.74%).

Example 14: Preparation of zinc bis(4-phenyl-3-thiosemicarbazone) acenaphthenequinone (14)

(14)

Acenaphthenequinone (0.182 g, 0.997 mmol) and zinc acetate bishydrate (0.656 g, 2.99 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-phenyl-3- thiosemicarbazide (1.50 g, 8.97 mmol) was then added to the suspension and the mixture heated under reflux for 24 hours, under an atmosphere of nitrogen. The dark red solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.397 g, 0.766 mmol, 77 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 10.05 (s, 2H, N(H)Ph), 8.25 (d, 2H, H3), 8.15 (d, 2H, Hl) 5 7.93 (d, 4H, Hl 1), 7.85 (t, H 3 H2), 7.40 (t, 4H, H12), 7.05 (t, 2H, H13). 13 C NMR (300 MHz, d 6 -DMSO 5 25 0 C): δ 177.2 (C-8), 142.3 (C-IO) 5 140.4 (C-5), 138.6 (C-3'), 130.7 (C-2') 5 128.4 (C-2), 128.3 (C-12), 128.0 (C-4), 127.5 (C-I) 5 123.5 (C-3), 122.7 (C-13), 120.6 (Cl 1). IR: 3402 m υ(NH) 5 1592 m υ(CN), 1568 m υ(CN) 5 1479 mbr υ(ring) 5 1392 ssh υ(ring) 5 1175 m υ(CS) 5 1142 m υ(NN). ES MS: m/z= 543.1 [M + H] + .

Example 15: Preparation of copper bis(4-methyl-3-thiosemicarbazone) acenaphthenequinone (15)

(15)

Zinc bis(4-methyl-3-thiosemicarbazide) acenaphthenequinone was prepared as described in Example 12. Copper acetate bishydrate (0.105 g, 0.478 mmol) was dissolved in methanol (100 ml) to give a methanol solution of copper acetate bishydrate. To this solution was added a suspension of zinc bis(4-methyl-3-thiosemicarbazide) acenaphthenequinone (0.100 g 0.239 mmol). The resulting suspension was stirred overnight for 20 hours. A dark red solid (15) was then isolated by filtration, washed with warm methanol (20ml) and dried under vacuum. Yield (0.076 g, 0.1830 mmol, 77 %).

ES MS: m/z = 418.0 [M + H] + . IR: 3315 ms υ(NH), 1577 sbr υ(CN), 1529 mbr υ(CN), 1455 s υ(ring), 1393 s υ(ring), 1179 s υ(CS), 1143 s υ(NN). HPLC R f = 20.3 mins. X-ray: crystals of 15 suitable for X-ray diffraction analysis were grown by slow evaporation of a DMSO solution of 15 over 3 weeks, and the structure of 15 was confirmed by single crystal X-ray crystallography.

Example 16: Preparation of copper bis(4-ethyl-3-thiosemicarbazone) acenaphthenequinone (16)

(16)

Zinc bis(4-ethyl-3-thiosemicarbazide) acenaphthenequinone (13) was prepared as described in Example 13. Copper acetate bishydrate (0.490 g, 0.224 mmol) was dissolved to give a methanol solution (100ml). To this was added a suspension of zinc bis(4-ethyl-3- thiosemicarbazide) acenaphthenequinone (13) (0.500 g 0.112 mmol). The resulting suspension was stirred overnight for 20 hours. A dark red solid (16) was then isolated by filtration, washed with warm methanol (20ml) and dried under vacuum. Yield (0.35 g. 0.798 mmol, 71%).

ES MS: m/z = 446.1 [M + H] + . IR: 3395 s υ(NH), 3315 ms υ(NH), 1511 sbrυ(CN), 1481 s υ(ring), 1418 s υ(ring), 1179 s υ(CS), 1143 s υ(NN). HPLC R f = 24.1 mins. X-ray: crystals of 16 suitable for X-ray diffraction analysis were grown by slow evaporation of a DMSO solution of 16 over 3 weeks, and the structure of 16 was confirmed by single crystal X-ray crystallography.

Example 17: Preparation of nickel bis(4-allyl-3-thiosemicarbazone) acenaphthenequinone (17)

(17)

Acenaphthenequinone (0.5 g, 2.74 mmol) and nickel acetate tetrahydrate (2.05 g, 8.25 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-allyl-3-thiosemicarbazide (1.44 g, 10.96 mmol) was then added to the suspension and the mixture heated under reflux for 2 hours. The black-brown solid was isolated by filtration whilst hot, then re-suspended in

warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.953 g, 2.053 mmol, 75 %. 1 H NMR (300 MHz, d 6 -DMSO, 25 0 C): δ 8.43 (m, 2H 5 N(H)Et) 3 8.06 (d, 2H, H3 ring), 7.80 (d, 2H, Hl ring), 7.66 (t, 2H, H2 ring), 5.81(m, 2H, H-Il), 5.23 (d, 2H 5 H-IO) 5 5.17 (d, 2H, H- 10), 3.99 (m, 4H, H-12)). ES MS: m/z = 465.1 [M + H] + . X-ray: crystals of 17 suitable for X-ray diffraction analysis were grown from a THF solution of 17 layered with pentane, and the structure of 17 was confirmed by single crystal X-ray crystallography.

Example 18: Preparation of zinc bis(4-allyl-3-thiosemicarbazone) acenaphthenequinone (18)

(18)

Acenaphthenequinone (0.5 g, 2.74 mmol) and zinc acetate bishydrate (1.807 g, 8.23 mmol) were suspended in glacial acetic acid (10 ml) and heated to 60 0 C. 4-allyl-3-thiosemicarbazide (3.00 g, 25.16 mmol) was then added to the suspension and the mixture heated under reflux for 30 hours, under an atmosphere of nitrogen. The red solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 ml) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 ml). The solid was recrystalised from a

THF/pentane solvent mix, filtered and then dried under vacuum. Yield = 0.877 g, 1.97 mmol, 72 %.

1 HNMR (SOO MHz, d 6 -DMSO 5 25 0 C): δ: 11.96 (s, IH 5 acetic acid), 8.15 (d, 2H, H3 ring), 8.09 (m, 2H 5 N(H)Et), 7.98 (d, 2H, Hl ring), 7.74 (t, 2H, H2 ring), 5.98(m, 2H 5 H-11), 5.24 (d, 2H 5 H-IO), 5.09(d, 2H, H-IO), 4.15 (m, 4H 5 H-12)), 1.89 (s, 3H 5 acetic acid). ES MS: m/z = 471.4 [M + H] + .

Example 19: Preparation of zinc[bis(4-methyl-3-thiosemicarbazone) acenaphthenequinone] [1,4-diaza-bicyclo [2,2,2] octane] (19)

(19)

Zinc compound 12 was co-crystallised with 1,4-diaza-bicyclo [2,2,2]octane ("DABCO"). Crystals of 19 were grown from a 1 :10 solution of 12 :D ABCO in THF layered with pentane at room temperature. The molecular structure of the 1 : 1 DABCO complex 19 was determined by x-ray crystallography.

Example 20: Preparation of zinc[bis(4-phenyl-3-thiosemicarbazone) acenaphthenequinone] [1,4-diaza-bicyclo [2,2,2] octane] (20)

(20)

Zinc compound 14 was co-crystallised with DABCO. Crystals of 20 were grown from a 1 : 10 solution of 14 :D ABCO in THF layered with pentane at room temperature. The molecular structure of the 1:1 DABCO complex 20 was determined by x-ray crystallography.

Example 21: Spectroscopic properties of zinc(II) bis(thiosemicarbazone) compounds 12, 13 and 14, and comparison with other compounds

UV visible and fluorescence studies were carried out on zinc compounds 12, 13 and 14 to identify: a) whether they are intrinsic fluorescent and suitable for cell imaging; b) how they compare to other compounds, including compounds 1 to 7, acenaphthenequinone and the known compounds Zn(ATSM), Zn(ATSEt) and Zn(ATSPh); and c) how their fluorescence is altered by changing the solvent for biocompatible media, hi addition, DFT calculations were performed in order to investigate the nature of the frontier orbitals of compounds 12, 13 and 14, in order to appreciate the number and intensities of the absorption bands of those compounds.

Example 21a: Fluorescence studies

Fluorescence measurements were carried out in DMSO and in a 5:95 DMSO:water solvent mix to mimic biological conditions.

Fluorescence of the acenaphthenequinone starting material was observed only at a concentration of 2.0 mM in 100 % DMSO 9 with emission observed at 590 nm for excitation at 480 nm.

The mono substituted ligands I 5 3, 5 and 7 showed fluorescence when measured in 1.0 mM and 0.1 mM concentrations. A shift in their emission to shorter wavelengths was observed as a result of the increased conjugation, with emission at 561, 554, 546 and 562nm respectively for excitation at 480 nm.

For the zinc complexes 2, 4, and 6, fluorescence of the mono-substituted ligands (1, 3 and 5 respectively) was nearly or completely quenched over all concentration ranges when tested over all excitation wavelengths. As a result of this it was not possible to calculate quantum yields for the zinc(II) complexes 2, 4, and 6. Without wishing to be bound by theory, one reason for the quenching may be found from the 1 H NMR spectra of 2, 4, and 6, which suggest that, as a result of the long labile Zn-O bond, the ligand is exchanging rapidly between being bidentate (S and N) and tridentate (S, N and O), with the vacant coordination site on the metal being occupied by solvent molecules. This exchange with the solvent and the rearrangement of the ligand may well account for a dynamic fluorescence quenching mechanism. That mechanism may provide a rapid energy transfer route by which the excited state can relax with respect to the timescale of fluorescence. Other possible reasons for quenching might include the interactions with the solvent of intermolecular packing arrays. The zinc(II) bis(thiosemicarbazone) species showed fluorescence at concentrations of

0.ImM 5 O.OlmM and O.OOlmM with emission at 547, 551and 564nm for 12, 13 and 14 respectively. Maximum fluorescence emission was observed for excitation at wavelengths around 480 nm. These emissions occur in very similar ranges to the mono substituted ligands, suggesting that the fluorescence is strongly ligand based. The fluorescence was much more intense then that found for complexes with aliphatic backbones. Without wishing to be bound by theory, this increased fluorescence may be a result of increased conjugation, high symmetry and enforced planarity on binding with zinc. The bis-substituted compounds 12, 13 and 14 do not suffer from the degree of quenching observed for the mono substituted complexes 2, 4 and 6, possibly in part due to the rigid non-labile nature of the ligand set. It was possible to calculate the quantum yields for 12, 13 and 14 in DMSO and draw comparison with the corresponding bis(thiosemicarbazone) complexes which are derived from 2,3-butadione as the ligand backbone, namely Zn(ATSM), Zn(ATSEt) and Zn(ATSPh).

The quantum yield (the ratio of photons emitted to photons absorbed) is calculated by the following expression:

φ = quantum yield D = integrated area under emission peak s = sample A = absorbance of solution at excitation wavelength r = reference n - refractive index of (pure) solvent

/= maximum intensity of emission peak

[Ru(bipy) 3 ] [PF 6 ] 2 in water was used as the reference material, where φ r = 0.042.

Absorbance was calculated according to the Beer Lambert Law with A=εcl where ε= molar absorptivity of sample (LmOr 1 Cm '1 ), c= concentration (molL '1 ) and 1= pathlength (cm).

Table 1 below lists the quantum yields for the zinc species 12, 13 and 14 (in which Ll-Rl and Ll '-Rl' of formula (I) are Me, Et and Ph respectively) and the comparative data for Zn(ATSM) and Zn(ATSPh) (i.e. the corresponding Zn(ATSR) equivalents in which R= Me and Ph). Figs. Ia and Ib provide comparative fluorescence spectra. Fig. Ia is a fluorescence profile comparing the fluorescence intensities of the acenaphthenequinone derived Zn complex 14 (solid line) and the corresponding butadiene-derived zinc species Zn(ATSPh) (dashed line) in DMSO at concentrations of 0.0 ImM. Fig. Ib compares the fluorescence intensities of the acenaphthenequinone-derived Zn complex 12 (solid line) and the corresponding butadiene-derived zinc species Zn(ATSM) (dashed line) in DMSO at concentrations of 0.0ImM. The comparative data indicates that incorporation of the naphthalene group into the backbone has led to a substantial increase in fluorescence intensity.

Table 1 : Comparison of quantum yields for new and known zinc compounds

It was also possible to observe fluorescence from the zinc species 12 and 13 in the DMSO:water mix at emission wavelengths of 533 and 538nm: Fig. 2 compares the fluorescence intensities of 13 in (a) DMSO (solid line), and (b) 5:95 DMSO:water solvent mix (dashed line), at concentrations of 0. ImM.

Example 21b: UV visible absorption spectra

UV visible measurements were carried out in DMSO and in a 5:95 DMSO:water solvent mix to mimic biological conditions. UV visible data for selected compounds is listed in Table 2 below. For all complexes the absorbance was measured at 0.0ImM concentration and λι shows the strongest intensity for all complexes, with 3 to 4 times the absorbance of that observed for λ 2.

Table 2: Selected UV absorption data

Compound λ 1 (nm) λ 2 (nm)

1 341 420

2 360 453

3 336 430

4 341 445

5 336 436

6 364 458

7 339 418

12 359 481

13 361 488

14 372 510

Table 3 : Calculated energies δE(eV) and molar absorbancies ε for the Zn(II) compounds of interest, 12, 13 and 14

Compound λ^nm) E(eV) ε (Lmol cm ) λ 2 (nm) E(eV) ε (Lmol " cm " )

12 359 3.46 28 .76 481 2.58 7.68 13 361 3.45 33 .74 488 2.54 11.76 14 372 3.34 32 .74 510 2.43 16.58

Although the compounds 12, 13 and 14 all show their strongest absorbance in the UV- visible spectra at around 350 nm, the fluorescence emission as a result of excitation at this wavelength is small compared to that from excitation at wavelengths around 480 nm.

Similarly for the mono-substituted ligands 1, 3, 5 and 7, the maximum fluorescence intensity is observed for excitation at the weaker absorbing wavelengths. This suggests that although the zinc(II) bis(thiosemicarbazones) absorb strongly at around 350 nm, the excited state does not fluoresce either for symmetry reasons or as a result of the energy being dissipated rapidly via non radiative processes. Excitation at the weaker absorbing, higher wavelength gives rise to population of a state that fluoresces strongly and on a fast timescale with respect to non radiative processes.

Example 21c: DFT calculations

In order to appreciate the number and intensities of the absorption bands, the nature of the frontier orbitals was investigated by use of DFT calculations. ADF calculations were performed using Vosko, Wilke and Nusair's local functional (Can. J. Phys: 58 (1990) 1200), with the Becke 88 (Phys. Rev. A38 (1988) 2398) and the Perdew 86 (Phys. Rev. B33 (1986) 8822) non local exchange and correlation gradient corrections, on ADF version 2000.02 (E. J. Baerends et al., Chem. Phys. 2 (1973) 41; C. Fonseca Guerra, Theor. Chem. Ace. 99 (1998) 391; B. te Velde et al., J. Comput. Phys. 99 (1992) 84; L. Verluis et at., J. Chem. Phys. 88 (1988) 322; S. ADF2002.01, Theoretical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands, http://www.scm.com). The basis sets used were uncontracted triple-ξ Slater- type orbitals (STOs). The cores of atoms were frozen, C and N up to the Is level, S and Zn up to the 2p level.

Table 4 shows the respective relative energies for the HOMO(-l), HOMO and LUMO orbitals for each of the zinc(II) complexes, 12, 13 and 14. The accompanying figure, Fig. 3, illustrates, for the case of 12, the nature of the electron density on the frontier orbitals. A similar diagram for the frontier orbitals was obtained for compounds 13 and 14. The shapes and energies of the frontier orbitals are similar for 12, 13 and 14.

Table 4: Relative energies of frontier orbitals for 12, 13 and 14 and the relevant energy gaps

50

115

Compound HOMO(-l) HOMO LUMO HOMO(-l)- HOMO- (eV) energy energy LUMO LUMO (eV) (eV) Energy Energy Gap Gap (eV) (eV)

12 -5.332 -4.781 -3.212 2.120 1.569

13 -5.269 -4.845 -3.231 2.038 1.613

14 -5.455 -5.001 -3.516 1.939 1.485

The DFT calculations show similarities with results on Zn(ATSM). In general, the DFT calculated frontier orbitals show similar electron distribution with calculations done on Zn(ATSM) although here contribution from the naphthyl group is observed. Fig. 4 compares the UV spectra of Zn(ATSM) (dashed line) and compound 12 (solid line). The absorption bands of 12 appear at wavelengths different from those of Zn(ATSM). Zn(ATSM) shows two strong absorption bands whereas compound 12 shows one strong and several weaker lower energy bands. The absorption band at 435 nm for Zn(ATSM) gives rise to the most intense fluorescence. This was previously assigned to a HOMO-LUMO transition, whilst the absorption at 314 nm (which gives rise to negligible fluorescence) is assigned to a HOMO(- I)-LUMO transition. Since the frontier orbital sets for both classes of compounds showed strong similarities, similar transitions were assigned to the observed absorption bands in the spectra of 12, the strong observed absorption at 359 nm being associated with a transfer of electron density from metal based orbitals to those of the diimine naphthyl backbone (H0M0(- I)-LUMO). The much weaker lower energy absorption bands which are associated with the intense fluorescence are likely to be associated with (HOMO-LUMO(type)) transitions with transfer of electron density from sulphur lone pair based orbitals to those of the diimine and naphthyl backbone type.

As a result of relaxation to various vibrational and rotational states the LUMO calculated does not necessarily represent the final state from which fluorescence occurs. It is therefore difficult to assign any explanation to the variation in intensity of the fluorescence from the DFT calculations. The large increase in fluorescence with respect to Zn(ATSM) is likely to be due to the naphthyl group as a result of the enforced rigidity and the distribution of electron density on the backbone associated with the HOMO-LUMO absorption transition. The DFT calculations are however in vacuo calculations and as such do not take into account

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116 solvent interactions and the closer intramolecular contacts. It would be of interest to look at solution phase DFT calculations to ascertain what contribution the individual orbital transitions make to the observed absorbance bands.

The work in Example 21 indicates that the naphthalene backbone unit leads to enhanced fluorescence of zinc bis(thiosemicarbazone) units with respect to the known aliphatic backbone compounds.

Example 22: In vitro fluorescence imaging study using zinc(II) bis(thiosemicarbazone) compounds 12, 13 and 14

With the fluorescent studies complete it was possible to carry out in vitro fluorescent tests on a range of cancer cells.

Cells were cultured and fluorescence cell plates prepared according to the following methods: Cells were cultured at 37 0 C in a humidified atmosphere of 5% CO 2 in air and diluted once confluence had been reached. Cells were cultured in DMEM medium with 10% foetal calf serum (FCS) and 100U/ml penicillin. The medium contained no fluorescent indicator dyes such as phenol red and was therefore suitable for use in fluorescentstudies. Samples for fluorescence were prepared in the following way: surplus supernatant containing dead cell matter and excess protein was discarded; the live adherent cells were then washed with two 5 ml aliquots of Phosphate Buffer Saline solution to remove any remaining medium containing FCS. FCS inhibits resuspension of the cells as it contains protease inhibitors which inactivate trypsin. To resuspend the cells in solution, they were incubated in 3 ml of trypsin/EDTA (500 mg/L Trypsin, 200 mg/L EDTA) solution for five minutes at 37 0 C. After trypsinising, fresh DMEM was added to the suspended cells to give a sufficient concentration of cells. The concentration of cells required varies between cell lines and is chosen to be optimal for achieving sufficient coverage and optimal imaging. The cells were plated in a Petri dish containing a glass cover slip and left for 24 hours to adhere before fluorescence imaging measurements were made. The following data details the use of the dyes that were used in conjuction with cell uptake studied:

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LysoTracker Red DND-99 is a red-fluorescent dye that stains acidic compartments in live cells such as lysosomes. A lysotracker solution was made up and the cells loaded to give a final concentration of dye of 100 nM. The sample was then irradiated with a 568 nm red laser and all emitted fluorescence at above 590 nm collected with images taken for 15 minutes. Fluorescence and bright field image of lysotracker and compound of interest are overlaid to see concomitant areas of uptake.

MitoTracker Green FM is green-fluorescent mitochondrial stain which localizes in mitochondria regardless of mitochondrial membrane potential. A 1 μM solution of Mitotracker was loaded into the cells to give a final concentration of 10 nM and left for 20 minutes, bright field and fluorescent images were taken at regular intervals. For these preliminary tests we note that the Mitotracker solution available excited and emitted in a similar region to the compounds of interest. Therefore, it was not possible to observe a direct overlay comparison of Mitotracker and compound uptake in the same cell.

The studies were carried out on a confocal microscope. The fluorescent uptake was imaged by laser-scanning confocal microscopy (TCS NT, Leica), using the 488 nm line of an argon ion laser for excitation and the emission was long pass filtered (515 nm) and detected with a photomultiplier tube. The intensity of the laser was modified to reduce the possibility of photobleaching of the cell over time and the PMT voltage adjusted to be just above the autofluorescence limit of the starting conditions, effectively recording a background image before addition of compound, whilst maximising sensitivity and ensuring the correct focal plane is in focus.

The compounds of interest were made up to 100 μM solutions in a 1 :99 DMSO:DMEM cell medium solvent mix. All complexes gave highly stable solutions in this solvent mix, showing no signs of precipitation over several hours. A solution of complex (1 ml) was added to the cell plate containing cell medium (1 ml) to give an overall complex concentration of 50 μM with overall DMSO concentration at 0.5 %. Fluorescent images were then recorded every 30 seconds and the increase in fluorescence over time observed. To interpret the intracellular distribution of the complex it was of interest to compare the fluorescence of the complexes with that of a dye with a known uptake in specific organelles of the cell. Two dyes; Lysotracker Red and Mitotracker Green were used with 12 for some very preliminary qualitative work on subcellular distribution.

In vitro uptake studies were carried out on a range of adherent human cells to gain an interpretation of the effects different cell lines have on the intracellular distribution. The cell

lines used were: IGROV - an epithelial-like ovarian carcinoma, plated at 5,000 cells/ml, T24 - colon carcinoma, plated at 2,000 cells/ml, SW620 - bladder carcinoma, plated at 6,000 cells/ml and A431 - epidermic carcinoma, plated at 3000 cells/ml.

This work was primarily driven by the question of whether or not uptake of the zinc(II) compounds could be observed. It was also of interest to know the effects of the nature of the fluorescent compound and the type of cell line on the rate of uptake of the fluorescent compound. In order to compare directly with the work done by Shore et al., Chem. Commun. (Cambridge, United Kingdom) 2005, 845-847, it was of interest to investigate the nature of uptake more thoroughly with the IGROV cell line. As such, a comparison between the cellular distribution of the zinc compounds, and that of the established Mitotracker and Lysotracker were carried out. The confocal images of cellular uptake, with their corresponding bright field images of the cell in focus are shown in Figs. 5, 1, 9, 11 and 13. Each confocal image is a real time 16 shot average of fluorescence uptake.

Uptake in IGROV cells was followed for compounds 12, 13 and 14, with an incubation period of 30 minutes. With 13 a direct comparison of lysotracker uptake was also performed. Significant uptake was observed with all three compounds, and the fluorescence profiles for 12 and 13 are given in Figs. 6 and 8 respectively. They show that uptake is rapid, in particular for 13 with maximum uptake being observed after only 10 minutes. Once maximum uptake was reached the fluorescence intensity remained constant over time, with some photobleaching observed for 12. Compounds 12, 13 and 14 are well distributed within the cytoplasm with 12 and 13 also showing areas of heightened fluorescence intensity. Work on compound 13 in the presence of lysotracker highlights areas of concomitant fluorescence emission which seems to be in line with the studies on Zn(ATSM) uptake carried out by epi- fluorescence microscopy (Shore et al., Chem. Commun. (Cambridge, United Kingdom) 2005, 845-847). Compound 12 shows promising results towards nuclear uptake.

The cell uptake of 12, 13 and 14 in the S W620 cell line was monitored, with an incubation period of 30 minutes for 13 and 60 minutes for 12 and 14. Strong uptake was observed for all three compounds. Compound 12 showed the strongest nuclear uptake, as was also the case for IGROV cells. Compound 14 showed strong uptake in large specific areas of the cells adjacent to the nucleus.

The increase in fluorescence signal for compounds 13 and 14 is shown in Fig. 10a and Fig. 10b respectively. This gives a direct indication of active uptake, which is indicative of all three species (12, 13 and 14) in this cell line. The uptake of 13 in S W620 appears comparable

with the IGROV study, with similar fluorescence uptake observed over the same incubation period. The uptake of 14 indicates that a longer incubation is required to observe similar levels of fluorescence intensity.

Studies on A431 cell uptake with 12, 13 and 14 were carried out. AU three samples were incubated for an hour with the cell line. Again, strong cellular uptake is observed by all three compounds. There is little or no obvious nuclear uptake by any of the three compounds and uptake of 12 and 13 appears to occur at a faster rate then is observed for 14.

Fluorescence uptake plots over the 60 minute Incubation are shown in Figs. 12a and 12b, for 13 and 14 respectively, revealing active cellular uptake over time. The most intense fluorescence of all the cell lines tested is observed with uptake, suggesting that the A431 cells take up more compound then the other cell lines. Both 12 and 13 show rapid uptake with intense fluorescence apparent after only 10 minutes incubation. Compound 14 shows an abnormally long delay before any fluorescence is observed but then shows an equally rapid fluorescence uptake. Without wishing to be bound by theory, this perhaps infers some form of aggregation of molecules as key to emission.

Uptake of compounds 13 and 14 in T24 cells with was observed only with incubation over 60 minutes. Figs. 14a and 14b show the increase of fluorescence with time for compounds 13 and 14 respectively. Complex 13 shows comparable uptake in this cell line. Complex 14 shows localised uptake and some partial nuclear uptake. As with the A431 cell line, there was negligible uptake in T24 cells for the first 20 minutes followed by a rapid increase in fluorescence intensity.

Thus it has been possible to observe the uptake of the intrinsically fluorescent new zinc compounds 12, 13 and 14 into a range of cell lines, with uptake being evenly distributed in the cytoplasm. Studies with localised dyes on IGROV cell uptake suggests that some localisation in the acidic organelles of the cells takes place.

Example 23: Cytotoxicity studies of zinc(II) bis(thiosemicarbazone) compounds 12, 13 and 14

It was noted during fluorescence microscopy studies that cells showed considerable reduction in size, reduced binding to the coverslip and cell vesicularisation, upon exposure to compounds 12, 13 and 14. It was therefore of interest to investigate further the cytotoxic effect of these zinc complexes. In addition, it was believed that studies on the Zn(II)

complexes could model the behaviour of the corresponding Cu(I) complexes; the studies might therefore give insight into the possible cytotoxic behaviour of Cu(I) complexes in hypoxic tissue, i.e. once reduced and trapped inside cells.

Work was initially carried out on a non-adherent U937 (human myeloid) cell line, and subsequently on SW620, T24 and A431 cell lines. Experimental details pertaining to cell culturing are as follows:

The following method details how U937 cells were cultured and the setup of the cytotoxicity experiment with this cell line:

Cells were cultured at 37 0 C in a humidified atmosphere of 5% CO 2 in air. The cells were grown in suspension in RPMI 1640 medium (Invitrogen) with added 2mM glutamine and antibiotics (100 units/ml penicillin and lOOμg/ml streptomycin). Cells were diluted when they reached a density of about 5xlO 5 /ml.

Cells were seeded onto a 96 well plate at cell concentrations of approximately 2xlO 5 /ml in 200 μl of cell medium. To this 50 μl of the complex solution was added. The following methods detail how the adherant cell lines were cultured and the setup of the cytotoxicity experiment with these cell lines.

As for the U937 cell line the adherent cells were cultured in RPMI 1640 medium as opposed to the DMEM used for fluorescence imaging as the former is the preferred medium for optimal growth. In testing the cytotoxicity it was of interest to find the LD 50 of a compound, which is the concentration of the compound that gives rise to a 50% cell death over an incubation period with respect to what is observed in medium alone.

Preliminary work involved looking for the best technique by which to measure the numbers of live and dead cells. A selection of methods was attempted before deciding to use a trypan blue stain and haemocytometer grid to count a sample of the cells. The trypan blue stain is taken up by dead cells but cannot permeate the cell membrane of live cells.

Initial experiments were run with cell counts taken at 0, 6, 12, 24, 48 and 96 hours, using an arbitrarily set final concentration of compound of 100 μM in a 1:99 DMSO.cell medium solution. Details of experimental setup are as follows: SW620 cells were seeded in 6- well plates at a suspended cell concentration of approximately 5x10 5 cells/ml in RPMI 1640 medium (4ml per well). The cells were left to adhere for 6 hours. The supernatant was then discarded and replaced with 4 ml fresh medium containing a solution of the compound of interest (100, 50, 25 and 12.5 μM)in DMSO to give a final overall DMSO concentration of

1%. After 48 hours, the supernatant was collected and the adhered cells washed carefully with 2xlml PBS and trypsinised with lOOμL of trypsin/EDTA solution for 5 minutes at room temperature.

The cell suspension was then made up to 1 ml with fresh medium and cell counts performed. Counts were done on both the supernatant collected and for the newly resuspended cells i.e. those still adhered after 48 hours.

A similar setup was followed for the T24 cell line. These cells were seeded at a suspended concentration of 2x10 5 cells/ml, for compound concentrations of 50, 25, 12.5 and 6.25μM. A similar setup was followed for the A431 cell line. These cells were seeded at a suspended concentration of 4x10 5 cells/ml, for compound concentrations of 100, 50, 25 and 12.5μM.

Counts were made for a sample of cells in a) medium alone, b) in medium containing 1%DMSO and in c) medium containing compound of interest and 1% DMSO. Results indicated that a 48 hour time lapse was sufficient for a significant cytotoxic effect to be observed. Both lack of proliferation and cell death were observed.

Studies progressed to 48 hour runs with the Zn compounds 12, 13 and 14 at 100 μM concentrations. Results of the cell count (Fig. 15) show that each of the zinc compounds had a significant cytotoxic effect. The number of live cells after 48 hours was greatly reduced compared to the number in the medium and medium/DMSO standards, with fewer live cells present than had been initially seeded. The results represent an average of four separate counts with error bars included to indicate the range in numbers recorded.

Work proceeded to the cell lines of interest from the fluorescent imaging studies. Three cell lines of the four studied, SW620, T24 and A431, were cultured as described above. Cell counts for 100, 50, 25 and 12.5 μM concentrations of compound 13 were looked at over a 48 hour period. Figs. 16, 17 and 18 show the results of these tests. All results are averages from four separate counts with error bars indicating the range in numbers recorded.

Cell counts for the SW620 cell line showed that compound 13 has a strong cytotoxic effect over a broad range of concentrations. The LD 50 cytotoxic limit over 48 hours was approximately 15 μM. A similar cytotoxic effect is observed for compounds 12 and 14 on the same cell line for a concentration of 100 μM.

A similar set up was followed for the A431 and T24 cell lines. Cell counts show that the cytotoxic effect of compound 13 is less for the A431 and T24 cell lines than for the SW620, with the LD 50 over 48 hours being at concentrations of approximately 25 μM.

In summary, the fluorescent zinc complexes 12, 13 and 14 all show a similar cytotoxic effect towards a range of human cancerous cell lines, which renders them potentially useful in the dual therapy and imaging of cancers and other proliferative diseases.

Example 24: Measurement of redox potentials of copper complexes 15 and 16

It was of interest to study the copper (II) bis(thiosemicarbazone) acenaphthenequinones 15 and 16 because such complexes may be hypoxic selective by virtue of their redox chemistry. Thus, it was of interest to measure the cyclic voltammetry of the copper(II) compounds 15 and 16. AU measurements were made in dried and degassed DMF containing 0.1M [NBu 4 ][BF 4 ] as a support electrolyte. A three electrode system consisting of a platinum disc working electrode, a platinum counter electrode and a Ag/Ag + reference electrode was used. Ferrocene (Ei /2 (DMF) = +0.53V) was used as an internal reference and all redox potentials were corrected according to this. Both 15 and 16 had similar voltammagrams with essentially reversible reduction processes, corresponding to the Cu(II)/Cu(I) couple. The reduction potentials, E^ 2 , for the two copper compounds were -0.517Vfor 15 and -0.536V for 16. The corresponding reduction potential for Cu(ATSM) is -0.581V. Thus the reduction potentials of 15 and 16 are comparable to that of the hypoxic selective complex Cu(ATSM). This would suggest that complexes 15 and 16 are also likely to be hypoxic selective.

Example 25: Studies on copper complex 16 using coupled EPR/CV

The EPR effect of electrochemical oxidation/reduction for complex 16 was measured. This allowed the nature of the copper species in solution to be followed by EPR whilst scanning over a range of electrode potentials.

The experimental setup involved a tailor made EPR tube equipped with electrodes, permitting the investigation of oxidised and reduced forms in situ. Solution CV-coupled EPR X- BAND spectra were recorded at room temperature, at 9.43 GHz, with a microwave power of 2 mW and a modulation amplitude of 6 G. The electrochemical setup was identical to the initial

CV work, with a platinum working electrode, a Ag/Ag + reference electrode and run in a DMF solution with NH 4 BF 4 support electrolyte.

The EPR plots in Fig. 22 show how the characteristic four-peak plot of Cu(II) equilibrates and then collapses as the potential changes from 0 V to -1.2 V and then builds back up as the potential scans back up to OV and remains constant as the potential rises to IV. For monoisotopic nuclei of Cu(II) 5 1 =3/2. Coupling with the spin of an electron S=I /2 gives 21+1 signals; 2(3/2)+l = 4. For Cu(I), on the other hand, there is no unpaired electron so no signal is expected. The fine structure observed is a result of coupling with two 15 N (1=1/2) atoms. Thus, it was possible using this technique to show that the reduction of Cu(II) in DMF is reversible within experimental error at room temperature. This is a key result supporting the CV work in Example 24. If the copper species is to be selectively trapped in hypoxic tissue then the reduction couple must be reversible. It also needs to have fast kinetics to ensure that the complex is washed out of normoxic cells as Cu(II) rapidly, avoiding the possibility of acid catalysed dissociation of the ligand.

The nature of the copper species under oxidizing conditions was investigated further. The four peak signal remained constant suggesting that at oxidizing potentials comparable to the oxidation wave observed in the CV 5 there is no further change in the oxidation state of the copper centre. This suggests that there is no oxidation of Cu(II), since no collapse of the four point signal of Cu(II) to a single signal for diamagnetic Cu(III) was observed.

A similar result was obtained when complex 15 was analysed using coupled EPR/CV. The spectra obtained for both 15 and 16 demonstrated that the Cu(II) centre in complexes 15 and 16 undergoes a fully reversible reduction-oxidation reaction at biologically favourable potentials. The EPR data also confirm that the redox process at 0.9 V (see Fig. 20 (b) and Fig. 21 (b)) does not invole the copper centre thus backing up the hypothesis of a 1 -electron process involving the naphthyl group.

Electrochemical in-situ X-band (9.435 GHz) EPR spectra of cancer cells containing complex 15 or 16 in [NH 4 ][BF 4 J Based on a generally accepted mechanism, bis(tbiosemicarbazone) complexes of

Cu(II) can be reduced to Cu(I) in all cells. However, in hypoxic cells this is not reoxidised and as a result it can be trapped within the cell. In normoxic (normal level of oxygen) cells the complex is reoxidised and subsequently washed out. A crucial condition for this proposed

7 002950

124 mechanism is a biologically favourable reduction potential of Cu(II) complex. Redox switching from Cu(II) to Cu(I) inside hypoxic cells would produce a fluorescent agent, which could be used to monitor its cell uptake and distribution. Oxidation - reduction effects of Cu(II) complexes inside tumour cells were therefore probed in an attempt to test whether the weak fluorescence observed is due to the intrinsic fluorescence of the compound or due to redox switching inside the cells.

IGROV cells were incubated with a solution of compound 15 or 16 as described in Example 22. Cell material was washed several times with non-fluorescent medium, centrifuged, separated from the liquid phase and resuspended in the electrolyte [NH 4 ][BF 4 ]. Electrochemical in-situ EPR spectra of 15 and 16 incorporated inside the tumour cells

(Fig. 34) resembled the shape of the powder spectra of the pure Cu(II) complexes (Fig. 35) and are centred at g 2.060. This observation suggests that molecular rotation is restricted inside the cells. EPR spectra recorded upon applying a positive potential (+1 V), after reduction at -1.2 V, shows slightly enhanced resolution which can be due to change in the EPR "active" complex concentration. Spectra with increased resolution have resonances centred at g 2.060 and g = 2.123 (Fig. 34).

Example 26: Fluorescence studies on copper complexes 15 and 16

It was possible to observe fluorescence for the copper(II) compounds 15 (see Fig. 23) and 16 in DMSO, at a concentration of 0. ImM. The observed fluorescence intensities of the copper compounds 15 and 16 were comparable to that of the known zinc compound Zn(ATSM) inDMSO.

Surprisingly, it was also possible to observe by fluorescence the uptake of the copper(II) complexes 15 and 16 in the IGROV cell line, with the complexes showing enhanced solubility in cell medium. This is believed to be the first result of fluorescence imaging in cells using copper-based species. Fig. 24 shows the fluorescence image for compound 16. Without wishing to be bound by theory, there are two possibilities for this observed fluorescence. Either fluorescence of the ligand has not been entirely quenched by the Cu(II) centre or the Cu(II) has been reduced to Cu(I) by the reducing environment of the cancer cells.

Uptake by the copper bis(thiosemicarbazone) complexes shows similar distribution to that observed for the zinc(II) analogues, supporting the use of zinc(II) as a model for copper.

Example 27: Radiolabelling by 64 Cu transmetallation of zinc bis(thiosemicarbazones)

HPLC showed that it is possible to make 64 Cu bis(thiosemicarbazone) acenaphthenequinones complexes cleanly by transmetallation from the corresponding zinc species. The concentrations Of 64 Cu which are required for detection and imaging are approximately 10 "6 M, making the detection of radiolabeled species in the presence of the non-radioactive starting material difficult. It was however possible to follow the radiolabelling by HPLC, with gamma detection, which allowed determination of the extent and efficiency of transmetallation.

64 Cu was produced from cyclotron proton irradiation Of 64 Ni. The 64 Cu 2+ (aq) was then purified from 64 Ni 2+ (aq) using an ion exchange column, and obtained as an aqueous 64 CuCl 2 solution in 0.1 mol dm "3 HCl. 64 Cu(CH 3 CO 2 )2 was prepared and used for the radiolabelling experiments by diluting 0.2 mL 64 CuCl 2 in 0.1 mol dm "3 HCl with 0.1 mol dm '3 sodium acetate (1.8 mL, pH 5.5).

Radiolabelling was achieved by reacting 64 Cu(CH 3 CO 2 ) 2 (200 μL, < 10 MBq), with 100 μL of the zinc complex 13 in DMSO (1.0 mg 13 in 1 ml DMSO) and water (400 μL). The reactions were stirred at room temperature for 30 minutes, and then 25 μL of each reaction solution was removed for analysis by reverse phase radio-HPLC. A 25 min gradient elution method was employed using a water/acetonitrile mobile phase solvent system. It was possible to show, by comparison with the characterised non-radioactive equivalent (using UV detection), that 13 radiolabels cleanly. As shown in Fig. 25, the 64 Cu-radiolabelled species was found to have a retention time (R f ) of 19.96 mins. The difference between this figure for the R f value and that presented in Example 16 for the non-radiolabelled copper complex 16 is a result of the use of different columns.

Thus it has been shown that the zinc(II) bis(thiosemicarbazone) acenaphthenequinone complexes in this family may be labelled cleanly with radio copper by transmetallation. The copper complexes show fast and reversible reduction couples at biologically compatible potentials, which potentially renders them useful as hypoxic imaging agents and/or hypoxic therapeutic agents. The copper complexes also show significant fluorescence emission when taken up in IGROV cells.

Example 28: Cytotoxicity studies of zinc(II) bis(thiosemicarbazone) compound 13, copper(II) bis(thiosemicarbazone) compound 16 and direct comparison with the known cytotoxic agent cis platin

Further cytotoxicity studies were carried out on the IGROV cell line. The IGROV cell line was prepared as described in Example 23 for the other adherant cell lines, with the cells seeded at 2.5x10 5 cells/ml. A comparitive study over 48 hours was carried out. Once adherant, the IGROV cells were loaded with 100, 50, 25 and 12.5μM concentrations of the zinc complex 13, the copper complex 16 and cis platin. Cell counts were made as described in Example 23. The data, shown in Fig. 19, suggest that the Zn complex is more cytotoxic than the analogous Cu complex. The Zn compound shows comparable cytotoxicity to cis platin.

Comparative Example 29: Cytotoxicity of Zn[ATSM] in IGROV cancer cells

An attempt was made to calculate the cytotoxicity of [Zn(ATSM)] in the IGROV cancer cell line by way of a lethal dose (LD 50 ) measurement. To calculate the LD 50 value, approximately 1500 IGROV cells were seeded in each well of a 96-well plate and grown in an incubator for 24 hours. Then, a 0.1 mM solution of [Zn(ATSM)] in 5% DMSO/95% cell culture medium was added to the end well. Half of this solution was then added to the medium in the next well. The rest of the wells were then serially diluted to half the concentration of the previous well along the whole row of wells. The plate was incubated for a further 24 hours. The medium was then removed from the wells and the cells washed with phosphate buffered saline before more medium was added to the wells. After incubation for another 24 hours, a cell proliferation assay was performed on the plate. The assay used was the CellTiter 96 aqueous one solution cell proliferation assay (Promega). This is a colorimetric method for determining the number of viable cells in a sample. After 2 hours incubation with the assay, the absorbance of each well in the plate was read automatically by a plate reader. The absorbance values for each well were then plotted against the concentration of compound in that well. This was then compared with the absorbance of a well of control cells which contained no added compound. The concentration of [Zn(ATSM)] which gave an absorbance value half that of the control could then be calculated from this graph to give the LDs 0 value. All measurements were performed in triplicate for reproducibility.

The LD 50 for [Zn(ATSM)] in IGROV cells could not be calculated in this experiment because of the relatively low cytotoxicity of [Zn(ATSM)] in this cell line. Indeed, the absorbance of the treated wells never got below the halfway value of absorbance for the control wells, although the presence of [Zn(ATSM)] at high concentrations does cause the number of cells to diminish to approximately 87% of that of the control wells. The LD 5 O of [Zn(ATSM)] could theoretically be calculated by using a higher [Zn(ATSM)] concentration, but there are problems with its solubility in the cell growth medium at concentrations higher than 0.1 mM. Fluorescence imaging experiments have shown that [Zn(ATSM)] is taken up by IGROV cells, so an inability of [Zn(ATSM)] to cross the cell membrane would not appear to be a reason for its lack of cytotoxicity.

Example 30: Alternative synthesis of Zinc bis(4-allyl-3-thiosemicarbazone) acenaphthenequinone (18)

(18)

Acenaphthenequinone (0.5 g, 2.74 mMol) was suspended in glacial acetic acid (10 mL) and heated to 120 0 C. Zn(OAc) 2 » 2H 2 O (1.81 g, 8.24 mMol), 4-allyl-3-thiosemicarbazide (3.24 g, 24.66 mMol) and trifluoroacetic acid (0.5 mL) were then added to the suspension and the mixture heated under reflux for 4 hours under an atmosphere of nitrogen. The dark orange solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (10 mL) and stirred for 15 minutes. The suspension was filtered and washed with diethyl ether (100 mL) and then dried under reduced pressure to give compound 18 as an orange macrocrystalline solid. Yield = 0.631 g, 1.41 mMol, 52 %.

50

128

1 H NMR (300 MHz 5 d 6 -DMSO, 25 0 C ): δ 8.16 (d, 2H, HS), 8.10(t, 2H, NH), 7.98 (d, 2H 5 Hl), 7.75 (t, 2H 5 H2\ 5.98 (m, 2H 5 HIl), 5.25 (dd 5 2H 5 H12), 5.10 (dd, 2H 5 H13), 4.16 (b m 5 4H 5 #70) . 13 C NMR (300 MHz, d 6 -DMSO 5 25 0 C ): δ 138.0 (Cu) 5 132.8 (C2), 128.2 (Ci) 5 126.5 (C3) 5 118.4 (Ci2), 48.2 (CiO). Quaternary aromatic carbons: δ 184.2, 178.4, 176.2, 161.0. ES MS: M/z = 471.1 (100%) [M + H] + . LCMS: 3 peaks R f =4.22 mins: M/z = 471.1 [M + H] + ;4.81 mins: M/z = 471.1 [M + H] + ; 5.15 mins: M/z = 471.1 [M + H] + . HPLC: R f =19.7 mins. IR: 3414 sbr υ(NH) 5 1560 m υ(CN) 5 1531 m υ(CN), 1407 m υ(ring), 1177 υ(CS) Elemental analysis: % Found (Calculated): N 17.79 (17.81), C 50.91 (50.90), H 3.84 (3.84), S 13.58 (13.59), Zn 13.78 (13.86).

It was found that addition of the catalytic amount of trifluoroacetic acid (TFA) and careful temperature control enabled a successful synthesis. One equivalent of acenaphthenequinone and three equivalents of zinc acetate were suspended in acetic acid and TFA and heated to 115 C . It was important to ensure that this temperature was reached before the excess (9 equivalents) of 4-allyl-3-thiosemicarbazide was added in order to avoid the formation of the kinetic product of the mono substituted acenaphthenequinone. Care was taken to avoid the temperature exceeding 12O 0 C.

Example 31: Co-crystallisation of compound 18 with DABCO

Crystals of 18 suitable for X-ray diffraction analysis were grown according to the following method:

Zinc bis(4-allyl-3-thiosemicarbazone) acenaphthenequinone (18) and 1 molar equivalent of DABCO were dissolved in the minimum of THF and mixed together in a vial. Pentane was layered on top of the resulting THF solution (THF: pentane ratio 1:2). Crystals suitable for X-ray diffraction were allowed to grow slowly over several weeks. X-ray diffraction analysis showed that a 1 : 1 ratio complex with DABCO was formed.

The solid state structure of the complex 18 with DABCO 5 as confirmed by X-ray crystallography, is shown schematically below:

(18-DABCO)

Example 32: Preparation of bis(4-allyl-3-thiosemicarbazonato) acenaphthenequinone Copper (II) (21)

(21)

Bis(4-allyl-3-thiosemicarbazonato) acenaphthenequinone Zinc (II) (18) was prepared as described in Example 30. Cu(OAc) 2 2H 2 O (0.104g, 0.478 mMol) was dissolved in methanol (100 mL). To this solution a suspension of zinc bis(4-allyl-3-thiosemicarbazonato) acenaphthenequinone (0.112 g, 0.239 mMol) was added. The resulting suspension was stirred overnight for 20 hours. A dark red solid (21) was isolated by filtration, washed with warm methanol (20 mL) and dried under vacuum.

Yield 38 mg, 0.081 mMol, 34%. ES MS: M/z = 470.1 (40%) [M + H] + . LCMS: 2 peaks R f =6.81 mins: M/z = 470.1 [M + H] + , 7.41 mins: M/z = 470.1 [M + H] + . HPLC: 23.7 mins. IR: 3430 br υ(NH), 1504 s υ(CN), 1415 s υ(ring), 1465 s υ(ring), 1181 s υ(CS) Elemental analysis: % Found (Calculated): N 17.53 (17.88), C 51.07 (51.10), H 4.02 (3.86). Single

crystals suitable for X-ray diffraction analysis, were grown from a THF solution of 3 layered with pentane. The molecular structure was confirmed by synchrotron X-ray crystallography.

Example 33: Preparation of bis(4-ethyl-3-thiosemicarbazone) aceanthrenequinone (22)

(22)

Aceanthrenequinone (0.25 g, 1.08 mMol) and Zn(OAc) 2 ^H 2 O was suspended in glacial acetic acid (4.5 mL) and heated to 110 0 C. To this 4-ethyl-3-thiosemicarbazide (1.13 g, 9.4 mMol) and a catalytic amount of TFA (0.25 mL) was added and the mixture was subsequently heated under reflux for overnight. The solid was isolated by filtration whilst hot, then re-suspended in warm acetic acid (5 mL) and stirred for 15 minutes. The suspension was filtered again and washed with diethyl ether (50 mL) and then dried under vacuum.

1 H NMR (500 MHz 5 d 6 -DMSO, 25 0 C): δ 7.72 (s, IH, HS), 7.20 (d, IH, H4), 8.30 (s, IH, HS), 8.91 (d, IH, H3), 7.40 (d, IH, HT), 8.20 (d, IH, Hi), 7.10 (dd, 1η, η2), 7.00 (dd, IH, H5), 6.90 (dd, IH, H6), 2.70 (b, HiO), 0.50 (b HIl). ES-MS: M/z = 500.096 (20%) [M + H] +

The desired product (22) was impurified by the mono compound (22B) (M/z = 725.420 [M + H] + (60%)). Compound (22) can be purified (in order to remove the mono compound 22B) by column chromatography, using a silica gel solid phase and a pentane/THF solvent mixture (e.g. 1:1 pentane:THF).

(22B)

Example 34: Preparation of Zinc bis(4-ethyl-3-thiosemicarbazide) 9,10- phenanthrenequinone (23)

9,10-phenanthrenequinone (0.25g, 1.2 mMol), 4-ethyl-3-thiosemicarbazide (0.3Ig 5 2.64 mMol) and Zn(OAc) 2 '2H 2 O (0.4g, 1.82 mMol) were added to methanol (5 mL). One drop of sulphuric acid was then added and the mixture was heated under reflux for 24 hours under an atmosphere of argon. The reaction mixture was then filtered under gravity and washed with ethanol and diethyl ether and 23 was subsequently recrystalised from THF.

1 HNMR (300 MHz, d8 - THF 5 25 0 C ): δ: 8.5 (dd, 2H 5 H3) 5 8.2 (d, 2H 5 H4), 7.9 (b, 2H, HlO) 5 7.6 (dd 5 2H 5 H2), 7.3 (d, 2H 5 Hl) 5 1.24 (m, HIl), 0.91 (t, Hl 2). ES MS: 491.1 (65%) [M + H+ H 2 O] + , HPLC: R f =18.4 mins.

The desired product (23) was impurified by Zn(II) bis[mono(4-ethyl-3-thiosemicarbazide)

9,10-phenanthrenequinone] (23A) (m/z = 681; 5%). Compound (23) can be purified (in order to remove the mono compound 23A) by column chromatography, using a silica gel solid phase and a pentane/THF solvent mixture (e.g. 1:1 pentane:THF).

Example 35: Preparation of Nickel bis(4-ethyl-3-thiosemicarbazide) 9,10- phenanthrenequinone (24)

9,10-Phenatithrenequinone (0.25 g, 1.2 mMol) and Ni(O Ac) 2 '4H 2 O (0.90 g, 3.6 mMol) were suspended in Acetic acid (5 mL) and heated to 115 0 C. To this was added 4-ethyl-3- thiosemicarbazide (1.43 g, 12.0 mMol) and the mixture was heated under reflux for 2 hours. The solid formed was isolated by filtration whilst hot and stirred in warm acetic acid for 15 minutes followed by filtration, washing with diethyl ether and drying under vacuum.

Example 36: Spectroscopic properties of bis(4-aIlyl-3-thiosemicarbazonato) acenaphthenequinone nickel(II), zinc(II) and copper(II) complexes (17, 18 and 21)

The UV/vis and fluorescence spectra of 17, 18 and 21 were recorded in DMSO solutions (cone. 100 μM) (Figure 26). Although the compounds 17, 18 and 21 all show their strongest absorbance in the UV/vis spectra in the region of 350 run, for 18 the fluorescence emission as a result of excitation at this wavelength is small compared to that from excitation at wavelengths above 400 nm. This suggests that although the Zn(II) bis(thiosemicarbazonato) complexes absorb strongly at around 350 nm, the excited state does not fluoresce either for symmetry reasons or as a result of the energy being dissipated rapidly via non radiative processes. Excitation at the weaker absorbing, higher wavelength gives rise to population of a state that fluoresces strongly and on a fast timescale with respect to non radiative processes. This is consistent with earlier observations in the fluorescence studies of Zn(ATSM). For 18, fluorescence intensities decayed significantly within 90 minutes therefore the quantum yield was not estimated (Figure 27; fluorescence spectrum of 18 in DMSO monitored over 90 minutes; λex=480 nm, 0.10 mM). It is difficult to assign the precise reasons for the

significant loss of intensity for the fluorescent emission; however it is likely that this is due to protonation in wet DMSO or to the change in the ligand coordination mode mediated by the solvent binding to the fifth coordination site of Zn(II) (Scheme 2). However, the fluorescence intensity was still significantly greater then that reported for Zn(ATSM).

L = DMSO, THF, DABCO

Scheme 2

To confirm the increased intrinsic fluorescence of 18 with respect to that of related compounds with aliphatic ligand backbones a fluorescence comparison study was devised. A compound believed to show the highest fluorescence in the aliphatic bis(thiosemicarbazonato) series (Compound D) was selected:

A concentration of 100 μM was chosen for both probes in order to enable the fluorescence of Compound D to be resolved and the peak maximum was shown to be 2708 A.U. (Fig. 33 (a)). However this resulted in the spectrum of 1 going off-scale. Fig. 33 (b) shows the same measurements made at 1 μM. Whilst the fluorescence intensity of 1 is a maximum of 2227 a.u., at the same concentration, the fluorescence of D is barely detectable with a maximum of just 480a.u. Without wishing to be bound by theory, the increased intrinsic fluorescence of 18 may be assigned to the presence of the extended aromatic backbone coupled with the presence of two allyl groups.

Example 37: In vitro fluorescence imaging study of bis(4-allyl-3-thiosemicarbazonato) acenaphthenequinone zinc(II) (18) using fluorescence microscopy

The behaviour of compound 18 was observed in HeLa cells using a Nikon TE2000-E microscope. Illumination was provided by a IOOW mercury arc lamp which was filtered for excitation at 330-380 nm and signal was collected at 523-643 nm. The cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing phenol red indicator. In order to achieve the best possible signal to noise the cells were transferred to CPB mediumf for at least one hour prior to addition of compound 18 in order to allow any phenol red to be washed out of the cells. It was discovered that CPB offered much lower background fluorescence than DMEM medium (even without phenol red) and maintained the cells in a healthy condition (addition of Zn(ATSM) in 1% DMSO-PBS (Phosphate Buffered Saline) resulted in cell death). Compound 18 was initially loaded at 50 μM by adding a 10 mM solution of 18 in DMSO to CPB to obtain a final DMSO concentration of 1%. The camera gain was adjusted so as to almost totally exclude the auto-fluorescence of the cells before the compound was added and the cells observed. The difference between the cell signal and the background (assessed well away from the cell) was observed as a function of time. The cell fluorescence increased rapidly during the first 20 minutes after which the signal became constant. Figure 28a shows a representative pattern of uptake; sub-cellular structures are picked-out by the fluorescence, but the precise organelles involved have not been determined yet.

The uptake of 18 in two other types of cancer cell lines (MCF-7, a breast carcinoma and IGROV, an ovarian carcinoma) was also imaged by laser-scanning confocal microscopy (TCS NT 5 Leica), using the 488 nm line of an argon ion laser for excitation. The emission was long- pass filtered (515 nm) and detected with a photomultiplier. The intensity of the laser was modified to reduce the possibility of photobleaching over time and the photomultiplier (PMT) voltage adjusted to be just above the autofluorescence limit of the starting conditions, effectively recording a background image before addition of the compound, whilst maximising sensitivity and ensuring the correct focal plane was achieved. Compound 18 was brought to 100 μM concentration in a 1 :99 DMSO-.DMEM solvent mix. The medium contained no fluorescent indicator dyes such as phenol red. Complex 18 gave stable solutions in this mixture, showing no significant precipitation over the period when the uptake was

monitored (90 minutes). A solution of complex 2 (1 mL) was added to the cell plate containing cell medium (1 mL) to give an overall complex concentration of 50 μM with an overall DMSO concentration of 0.5 %. Confocal fluorescence images were recorded every minute and the increase in fluorescence intensity over time was observed. The imaging showed that uptake in both MCF-7 and IGROV cells was rapid for 18 with maximum uptake being observed at 40 minutes. Once maximum uptake has been reached the fluorescence intensity remained constant over time with some photobleaching observed after 60 min. In both cases compound 18 seems well distributed within the cytoplasm but the nature of the localisation has not yet been established. There appears to be no significant uptake in the cell nucleus in these cell lines (Figures 29 and 30).

Temperature-dependent studies on the cell uptake in MCF-7 were also carried out to investigate the uptake mechanism. To test whether compound 18 was taken up through an endocytosis pathway, cells were incubated with 100 μM compound in 2 ml DMEM for 3 h at either 37°C or 4°C. In each case, images were recorded at 3 h after loading. There was a clear distinction in fluorescence between cells incubated with compound 18 at 37°C and those incubated at 4 0 C 5 suggesting the mechanism of uptake of this compound might be through endocytosis, follwing a method described by Silverstein et α/.and Veldhoen et al. In the images corresponding to incubation at 4°C, the cells appeared perfectly healthy when compared to the images taken for the cells incubated at 37°C, which showed a reduction of the coverslip binding suggesting early stages of cell death. Co-localisation with the endocytic marker Dextran was investigated. Cells were incubated for 30 min at 37 0 C with 100 μM of compound 2 in 1 ml DMEM. Subsequently, 0.5 ml was removed, mixed with 100 μl of a 10 mg/ml Alexa Red-conjugated Dextran solution and re-loaded. The uptake of 18 was imaged on the confocal microscope every 5 min over a period of 65 min. The solution was then removed and the cells rinsed 3 times using a 10 mg/ml Dextran (Sigma- Aldrich) solution in DMEM. Only partial co-localisation of 18 with Dextran was observed. This may suggest endocytosis as the main uptake pathway of 18 in MCF-7, though we cannot rule out passive diffusion as a potential uptake mechanism.

Thus, fluorescence microscopies showed clear uptake of 18 in HeLa 5 MCF-7 and IGROV cells. The uptake profile shown is reproducible and gives an idea of the kinetics of this compound's uptake in cancer cells.

7 002950

136

IGROVand MCF-7 Cells culturing and fluorescence cell plates preparation

Cells were cultured at 37 0 C in a humidified atmosphere of 5% CO 2 in air and diluted once confluence had been reached. Cells were cultured in DMEM medium with 10% foetal calf serum (FCS) and 100 U/mL penicillin. The medium contained no fluorescent indicator dyes such as phenol red and was therefore suitable for use in confocal fluorescence imaging studies. Samples for fluorescence were prepared in the following way: surplus supernatant containing dead cell matter and excess protein was discarded; the live adherent cells were then washed with two 5 mL aliquots of phosphate buffer saline solution to remove any remaining medium containing FCS. FCS inhibits resuspension of the cells as it contains protease inhibitors which inactivate trypsin. To resuspend the cells in solution, they were incubated in 3 mL of trypsin/EDTA (500 mg/L Trypsin, 200 mg/L EDTA) solution for five minutes at 37 0 C. After trypsinising, fresh DMEM was added to the suspended cells to give a sufficient concentration of cells. The concentration of cells required varies between cell lines and is chosen to be optimal for achieving sufficient coverage and optimal imaging. The cells were plated in a Petri dish containing a glass cover slip and left for 24 hours to adhere before fluorescence imaging measurements were made.

HeLa Cells culturing and fluorescence cell plates preparation Cells were cultured at 37 0 C in a humidified atmosphere in air and diluted once confluence had been reached. Cells were cultured in DMEM medium with 10% foetal calf serum (FCS) and 100 U/mL penicillin. Samples for fluorescence were prepared in the following way: surplus supernatant containing dead cell matter and excess protein was discarded; the live adherent cells were then washed with two 7 mL aliquots of Phosphate Buffer Saline solution to remove any remaining medium containing FCS. FCS inhibits resuspension of the cells as it contains protease inhibitors which inactivate trypsin. To resuspend the cells in solution, they were incubated in 2 mL of trypsin/EDTA solution (500 mg/L Trypsin, 200 mg/L EDTA) for three minutes at 37 0 C. After trypsinising, 5 mL of DMEM was added to inactivate the trypsin and the solution was centrifuged for five minutes to remove any remaining dead cell matter. The supernatant liquid was poured off and DMEM added to the cell matter left behind to give a sufficient concentration of cells. The cells were plated in a Petri dish containing a glass cover slip and left for 24 hours to adhere. 1 hour before fluorescence imaging measurements were made, the

DMEM was replaced with DMEM containing no fluorescent indicator dyes such as phenol red, therefore making it suitable for fluorescent studies.

Example 38: Cytotoxicity studies of 13, 16, 18 and 21 in MCF-7 cells

To date there is no evidence of a strong cytotoxic effect in Zn(ATSM). It was noted by the fluorescence microscopy of Example 37 that the IGROV, MCF-7 and HeLa cells all showed considerable reduction in size, followed by reduced binding to the coverslip within the period 60-90 minutes. The vesicularisation of the cells was particularly evident upon exposure to 18 over extended periods of time.

The cytotoxicties of 18 and 21 in the cancer cell line MCF-7 were therefore evaluated. Cell counts for 100, 50, 25 and 12.5 μM probe concentrations were taken over a 48 hour period. AU results are averages from four separate counts with error bars indicating the range in numbers recorded. Cell counts for this cell line showed that these compounds have a strong cytotoxic effect over a broad range of concentrations; the LC 50 cytotoxic limit over 48 hours is approximately 12.5 μM. Once adherant, the MCF-7 cells were loaded with solutions of 100, 50, 25 and 12.5 μM concentrations of the zinc complex 18, the copper complex 21 and cis-platin. Cell counts were made as described below. The data, shown in Fig. 31, suggest that the Zn complex 18 has a comparable cytotoxicity to cis-platin in this cell line, under the conditions described.

The cytotoxicties of 13 and 16 in the cancer cell line MCF-7 were also evaluated. Cell counts for 100, 50, 25 and 12.5 μM probe concentrations were taken over a 48 hour period. All results are averages from four separate counts with error bars indicating the range in numbers recorded. Cell counts for this cell line showed that these compounds have a strong cytotoxic effect over a broad range of concentrations. Once adherant, the MCF-7 cells were loaded with solutions of 100, 50, 25 and 12.5 μM concentrations of the zinc complex 13, the copper complex 16 and cis-platin. Cell counts were made as described below. The data, shown in Fig. 37, suggest that the Zn complex 13 has a comparable cytotoxicity to cis-platin in this cell line, under the conditions described.

Experimental details:

MCF-7 cells; an adherent cell line, were cultured at 37 0 C in a humidified atmosphere of 5% CO 2 in air. The cells were cultured in DMEM (Sigma) with added 10% Foetal Calf serum, 2mM glutamine and antibiotics (100 units/ml penicillin and lOOμg/ml streptomycin). Cells were diluted when they reached a density of about lxlO 6 /ml. Cells were seeded in 6-well plates at a suspended cell concentration of approximately 2x10 5 cells/ml in DMEM (4 mL per well). The cells were left to adhere for 6 hours. The supernatant was then discarded and replaced with 4 ml fresh medium containing a solution of the compound of interest (100, 50, 25 and 12.5 μM) in DMSO to give a final overall DMSO concentration of 1%. After 48 hours, the supernatant was collected and the adhered cells washed carefully with 2xlml PBS and trypsinised with lOOμL of trypsin/EDTA solution for 5 minutes at room temperature.

The cell suspension was then made up to 1 ml with fresh medium and cell counts performed using a trypan blue stain and haemocytometer grid to count a sample of the cells (The trypan blue stain is taken up by dead cells but cannot permeate the cell membrane of live cells). Repeat counts of live and dead cells for each experiment were made on four separate wells. The average number of viable cells is recorded with error bars indicating the range in counts made.

Example 39; Radiolabelling of bis(4-allyl-3-thiosemicarbazonato) acenaphthenequinone zinc(II) (18) with 64 Cu

The synthesis of the 64 Cu bis(thiosemicarbazonato) acenaphthenequinone complex by transmetallation from the zinc species 18 was investigated. The concentrations of Cu required for detection and imaging are of approximately 10 "9 M, making the detection of radiolabeled species in the presence of the non-radioactive starting material difficult. It was however possible to follow the radiolabelling by HPLC, with gamma detection, which allowed determination of the extent and efficiency of the transmetallation reaction. Cu was produced from cyclotron irradiation Of 64 Ni. The 64 Cu 2+ (aq) was subsequently purified from 64 Ni 2+ (aq) using an ion exchange column, and obtained as an aqueous 64 CuCl 2 solution in 0.1 mol dm "3 HCl. 64 Cu(OAc) 2 was prepared and used for the radiolabelling experiments by diluting 0.2 mL 64 CuCl 2 in 0.1 mol dm '3 HCl with 0.1 mol dm "3 sodium acetate (1.8 mL, pH 5.5). Radiolabelling was achieved by reacting 64 Cu(OAc) 2 (200 μL, < 10 MBq), with 100 μL of 18 in DMSO (1.0 mg 18 in 1 mL DMSO) and water (400 μL). The reaction mixtures were stirred at room temperature for 30 minutes, and then 25 μL of the sample was removed for

analysis by reverse phase radio-HPLC. A 25 min gradient elution method was employed using a water/acetonitrile mobile phase solvent system. The 64 Cu-radiolabelled species was found to have a retention time R f = 19.9 minutes under the conditions used. This analysis required 0.1% TFA in the acetonitrile and water mobile phase. Radiolabelling of 18 proceeded cleanly, giving a single species. The difference between this figure for the R f value and those found by HPLC for the non-radiolabelled copper complex 21 are a result of the use of different columns. However, these results clearly indicate that the zinc(II) bis(thiosemicarbazonato) acenaphthenequinone complexes in this family may be labelled rapidly and cleanly with 64 Cu by transmetallation (Figure 32).

This result is of relevance to PET imaging with 64 Cu since aliphatic bis(thiosemicarbazonato) copper(II) complexes are hypoxic selective. Additionally, or as an alternative to hypoxic selectivity, the 4 Cu-radiolabelled species may be conjugated to a biologically active molecule using standard coupling chemistry of the terminal allyl groups, which could serve to target the complex to the desired site in vivo.

Example 40: Fluorescence of copper complexes 15, 16 and 21

An advantage of the copper complexes used in the present invention is that they show fluorescence despite the presence of a d 9 paramagnetic Cu(II) centre. Coupled with the successful radiolabelling of the analogous Zn complexes to form the 64 Cu comlexes, these complexes may be suitable for both fluorescence and PET imaging. Figure 35 shows the fluorescence emission spectrum of the three copper complexes 15, 16 and 21 at 100 μM in DMSO (λ ex 480nm). It is worth noting that the concentration required for these measurements (100 μM) is ten times higher than that normally used for the Zinc analogues.

Example 41; Investigation into the mechanisms of action of complexes 13 and 18 in MCF-7 cells

There is currently no single model to explain Cu[ATSM] loading, distribution and effect on cells. To test the hypothesis that the compounds 13 and 18 could be taken up through an endocytosis pathway, cells were incubated with 100 μM ZnEt (13) or Zn allyl (18) compounds in 2 ml DMEM for 3 h at either 37°C or 4°C (Veldhoen S et al. Nucleic Acids Res

2006;34(22):6561-73; reviewed in Silverstein SC et al Annu Rev Biochem 1977;46:669-722), with images acquired at 3 h after loading. Co-localisation with the endocytic marker Dextran was also investigated in both cases. Cells were incubated for 30 min at 37 0 C with 100 μM of each compound in 1 ml DMEM. Subsequently, 0.5 ml was removed, mixed with 100 μl of 10 mg/ml Alexa Red-conjugated Dextran solution and re-loaded. Both compounds were imaged on the confocal microscope every 5 min over a period of 65 min. The solution was then removed and the cells rinsed 3 times using a 10 mg/ml Dextran (Sigma- Aldrich) solution in DMEM.

There was a clear distinction in fluorescence between cells incubated with Zn-allyl compound 18 at 37 0 C and those incubated at 4°C for all time points, suggesting the mechanism of uptake of this compound is endocytosis (Veldhoen S. et al, 2006; reviewed in Silverstein S.C. et al, 1977). In the images representing incubation at 4 0 C, the cells which were not fluorescing appeared perfectly healthy when compared to the 37°C images which slowed a reduction of the coverslip binding suggesting cell death. Cells were incubated with each compound for 10 min prior to Dextran addition, to allow adequate fluorescence visualisation.

Co-loading of 18 with Dextran was only partial, giving rise to partial co-localisation. Although this suggests endocytosis as the main uptake mechanism of 18 in MCF-7, we cannot rule out the fact that the uptake by passive diffusion may take place simultaneously for this compound.

For compound 13, no temperature effect was observed and no co-localisation with Dextran, under the conditions tested. This indicated that the principal mechanism of uptake for this compound in MCF-7 is through passive diffusion.

The experiments described above for 13 and 18 on MCF-7 (breast cancer carcinoma) suggest that uptake is highly dependent on compound modifications and the possibility of endocytosis cannot be ruled out in addition to the passive diffusion mechanism. The subsequent rapid effects on mitochondria, which seems to be associated with apoptosis suggests this compound localises primarily to these organelles, however it has been observed to locate in the nucleus after 12 h incubation.

Variable temperature uptake of Compounds 13 and 18 indicate that different uptake mechanisms may be induced by minor structural modifications

Example 42: Preparation of gallium(ll) chloride bis(4-allyl-3-thiosemicarbazonato) acenaphthenequinone (25) and fluorescence study thereof

(25)

Gallium chloride (1.864 g, 10.59 mmol) was dissolved in ethanol (50 ml). To this, a suspension of zinc bis(4-allyl-3-thiosemicarbazide) acenaphthenequinone (0.500 g 1.059 mmol) in EtOH (50 mL) was added, and the mixture was heated under reflux overnight for 20 hours. The solution was allowed to cool and the dark red solid was isolated by filtration, washed with warm ethanol (20 mL) and ether (50 mL) and dried under reduced pressure. Yield: 0.412 g, 0.805 mmol, 76 %.

1 H NMR (300 MHz, d 6 -DMSO, 25 0 C ): δ 9.76 (m, IH, N(H)), 9.05 (m, IH, N(H)'), 8.50 (d, IH, H3), 8.11 (d, IH, H3'), 8.02 (d, IH, Hl), 7.91 (d, IH, Hl'), 7.77(t, IH, H2), 7.75(t, IH 5 H2'), 5.96(m, IH, H5), 5.92(m, IH, H5'), 5.21 (q, 2H, H4), 5.20 (q, 2H H4'), 4.18 (m, 2H, H6), 4.12 (m, 2H, H6'). ES MS: m/z = 507.06 (10%) [M-H] + , 967.03 (100%) [2M + OH - 2Cl] + . X-ray: crystals of 25 suitable for X-ray diffraction analysis were grown, and the molecular structure of 25 was confirmed by single crystal X-ray crystallography.

The fluorescence of the Ga complex 25 was measured; Fig. 44 shows the resulting graph of fluorescence intensity (y axis) versus wavelength in units of nm (x axis) for the Ga complex 25 in 100% DMSO at a concentration of 20 μM, at room temperature (λ ex = 400 nm).




 
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