Background of the invention Iron is an indispensable requirement for the activity of many essential metabolic processes. For example, Fe-containing proteins catalyse key reactions involved in energy metabolism, respiration, and DNA synthesis (eg. ribonucleotide reductase (RR), is the rate-limiting step in DNA synthesis). In fact, without Fe, cells are unable to proceed from the G, to the S phase of the cell cycle.

Neoplastic cells have a high Fe requirement relative to normal cells due to their greater ribonucleotide reductase activity and higher rates of proliferation. This is reflected by an increase in the expression in neoplastic cells of the transferrin receptor (TfR), which binds the serum Fe-transport protein, transferrin (Tf). Neoplastic cells are more sensitive to perturbations in cellular iron concentrations and the role of Fe in cellular proliferation and ribonucleotide reductase activity, means Fe is an important therapeutic target.

Iron is also an essential component for the functioning or regulation of a number of plasmoidial enzymes, such as ribonucleotide reductase, delta aminovulinate synthetase, and dihydroorotate dehydrogenase. In vitro studies have shown that iron chelating agents inhibit parasite growth and proliferation by depriving the intracellular parasites of this essential nutrient. Desferrioxamine (DFO) is an iron chelator that has been used for treating iron overload disease and as an anti-malarial agent. DFO has also been investigated as an anti-proliferative agent.

A key factor regarding the clinical use of Fe chelators for the treatment of cancer is their selectivity at inhibiting the growth of neoplastic compared to normal cells.

Neuroblastoma (NB) is an aggressive extracranial solid tumour. NB cells from patients with advanced disease contain increased amounts of the Fe storage protein,

ferritin, which is rich in Fe. The Fe (III) chelator, desferrioxamine (DFO), is capable of a cytotoxic effect on NB cells in vitro while having little effect on other cell types. DFO has also shown activity against leukemia cells both in vitro and in vivo. However, DFO suffers from a number of serious problems that limit its therapeutic use. In particular, DFO is extremely expensive, has poor membrane permeability, and is not orally available. In fact, DFO requires long subcutaneous infusions (12-24 hours/day, 5-7 day/week) to produce significant iron excretion, resulting in poor patient compliance.

Furthermore, the short plasma half-life of DFO and its low efficacy at permeating cell membranes limits its anti-proliferative activity.

There is a need for therapeutically acceptable iron chelators which are capable of permeating cell membranes and chelating intracellular Fe. In particular, there is a need for clinically useful iron chelators having anti-proliferative activity.

Summary of the Invention According to a first aspect of the invention there is provided a compound of the general Formula 1 a Formula la wherein E is 0 or S ; X is OH or SH ; R4 is selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, and substituted amino, wherein an R4 substituent can be at any available position on the naphthyl ring; and n is 1,2, 3,4, 5 or 6 ; R'is hydrogen or a group that does not prevent the compound chelating iron ions; R8 is hydrogen or C14 alkyl ; -G is where Rl is N, or CR5, wherein the bond between C and R5 may be a single, double or triple bond; and wherein when the bond between C and R5 is a double bond, one of R2

and R3 is absent, and wherein when the bond between C and R5 is a triple bond, R2 and R3 are both absent; R2 and R3 are the same or different and are individually selected from the following: hydrogen, halogen, substituted or unsubstituted Cl-8 alkyl group, substituted or unsubstituted C2 8 alkenyl group, substituted or unsubstituted C2 8 alkynyl group, substituted or unsubstituted amino group, substituted or unsubstituted heterocyclic group eg, 5-or 6-membered heterocyclic group, substituted or unsubstituted bicyclic group, and substituted or unsubstituted aromatic group; R5 is selected from the group consisting of hydrogen, halogen, substituted or unsubstituted Cl-8 alkyl group, substituted or unsubstituted C2 8 alkenyl group, substituted or unsubstituted C2 8 alkynyl group, substituted or unsubstituted amino group, substituted or unsubstituted heterocyclic group, eg 5-or 6-membered heterocyclic group, substituted or unsubstituted bicyclic group, and substituted or unsubstituted aromatic group; or-G is a substituted or unsubstituted heterocyclic group, eg 5-or 6-membered heterocyclic group, a substituted or unsubstituted bicyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted Cl-8 alkyl group; or a C3-8 substituted or unsubstituted cycloalkyl group; with the provisos that: (A) when E is 0, X is OH, R'is H, R8 is H, R4 is H and n is 6, G is not and (B) whenEisS, XisOH, R'isH, R8isH, R4isHandnis6, G is not-NH2,-CH3,-CH2CH3,-CH2CH2CH2CH3, In one embodiment, the compound according to the first aspect of the invention is a compound of Formula 2:

Formula 2 wherein E is 0 or S ; and R'and-G are as defined above, including the provisos.

In another embodiment of the invention, the compound according to the first aspect of the invention is a compound of Formula 3: Formula 3 wherein R', R2, R3, R4 and n are as defined above, including the provisos.

In a further embodiment of the invention, the compound of Formula 1 is 2-hydroxy- 1-naphthylaldehyde-4-methyl-3-thiosemicarbazone (abbreviated N4mT), 2-hydroxy-1- naphthylaldehyde-4-allyl-3-thiosemicarbazone (abbreviated N4aT), 2-hydroxy-1- naphthylaldehyde-4,4-dimethyl-3-thiosemicarbazone (abbreviated N44mT), 2-hydroxy-1- naphthylaldehyde-4,4-diphenyl-3-semicarbazone (abbreviated N44pH), 2-hydroxy-1- naphthylaldehyde-4-octoylhydrazone (abbreviated NoctH), as depicted in Figure 1.

The compound of Formula la, 2 or 3 may be suitable for use as an iron chelator.

The compound of Formula la, 2 or 3 may form a complex with Fe (II) or Fe (III). In particular, the compound of Formula la, 2 or 3 may be suitable for therapeutic use as an iron chelator in vivo.

According to a second aspect of the invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and at least one compound according to Formula lb Formula lb wherein E is 0 or S ; X is OH or SH ; R4 is selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, and substituted amino; and n is 1,2, 3,4, 5 or 6; R'is hydrogen or a group that does not prevent the compound chelating iron ions; R8 is hydrogen or Cl 4 alkyd ; -G is Rl is N, or CR5, wherein the bond between C and R5 may be a single, double or triple bond; and wherein when the bond between C and R5 is a double bond, one of R2 and R3 is absent, and wherein when the bond between C and R5 is a triple bond, R2 and R3 are both absent; R2 and R3 are the same or different and are individually selected from the following: hydrogen, halogen, substituted or unsubstituted Cl-8 alkyl group, substituted or unsubstituted C2 8 alkenyl group, substituted or unsubstituted C2 8 alkynyl group, substituted or unsubstituted amino group, substituted or unsubstituted heterocyclic group eg, 5-or 6-membered heterocyclic group, substituted or unsubstituted bicyclic group, and substituted or unsubstituted aromatic group;

R5 is selected from the group consisting of hydrogen, halogen, substituted or unsubstituted Cl-8 alkyl group, substituted or unsubstituted C2 8 alkenyl group, substituted or unsubstituted C2 8 alkynyl group, substituted or unsubstituted amino group, substituted or unsubstituted heterocyclic group, eg 5-or 6-membered heterocyclic group, substituted or unsubstituted bicyclic group, and substituted or unsubstituted aromatic group; or-G is a substituted or unsubstituted heterocyclic group, eg 5-or 6-membered heterocyclic group, a substituted or unsubstituted bicyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted Cl-8 alkyl group; or a C3-8 substituted or unsubstituted cycloalkyl group; with the proviso that G is not In another embodiment, the pharmaceutical composition according to the invention comprises a pharmaceutically acceptable diluent or carrier and at least one compound according to Formula 1 a as defined above.

In an alternative embodiment, the pharmaceutical composition according to the invention comprises a pharmaceutically acceptable diluent or carrier and at least one compound of Formula 2 or Formula 3: Formula 2 Formula 3 wherein E, G, R', R2, R3 and R4 are as defined above, with or without the provisos.

In related embodiments, the composition according to the invention may comprise one or more iron complex of a compound according to Formula la, lb, 2 or 3.

The pharmaceutical composition according to the invention may be suitable for iron chelation therapy, treating iron-overload disorders, inhibiting cellular proliferation, or treating proliferative disorders.

In alternate embodiments, the pharmaceutical composition according to the invention may be formulated for subcutaneous or intravenous injection, oral administration, inhalation, transdermal application, or rectal administration.

A pharmaceutical composition of the invention may be made by mixing a compound of Formula la, lb, 2 or 3 with a pharmaceuticallyacceptable carrier and/or diluent and/or adjuvant.

In a third aspect, the present invention provides a method of iron chelation therapy in a mammal comprising administering to said mammal a therapeutically effective amount of at least one compound selected from the group consisting of a compound of Formula la, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, or a pharmaceutical composition according to the invention.

In a fourth aspect, the present invention provides a method of treating an iron- overload disorder in a mammal comprising administering to said mammal a therapeutically effective amount of at least one compound selected from the group consisting of a compound of Formula la, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, or a pharmaceutical composition according to the invention.

In a fifth aspect, the present invention provides a method of inhibiting cellular proliferation in a mammal comprising administering to said mammal a therapeutically effective amount of at least one compound selected from the group consisting of a compound of Formula la, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, or an iron complex of said compound, or a pharmaceutical composition according to the invention.

In a sixth aspect, the present invention provides a method of treating a proliferative disorder in a mammal comprising administering to said mammal a therapeutically effective amount of at least one compound selected from the group consisting of a compound of Formula la, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, or an iron complex of said compound, or a pharmaceutical composition according to the invention.

In a seventh aspect the present invention provides the use of at least one compound selected from the group consisting of a compound of Formula la, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, for the preparation of a medicament for iron chelation therapy.

In an eighth aspect, the present invention provides the use of at least one compound selected from the group consisting of a compound of Formula 1 a, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, for the manufacture of a medicament for treating an iron-overload disorder.

In a ninth aspect, the present invention provides the use of at least one compound selected from the group consisting of a compound of Formula 1 a, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, or an iron complex of said compound, for the manufacture of a medicament for inhibiting cellular proliferation.

In a tenth aspect, the present invention provides the use of at least one compound selected from the group consisting of a compound of Formula 1 a, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, or an iron complex of said compound, for the manufacture of a medicament for treating a proliferative disorder.

In an eleventh aspect, the present invention provides at least one compound selected from the group consisting of a compound of Formula la, a compound of Formula lb, a compound of Formula 2 and a compound of Formula 3, as defined above, or a pharmaceutical composition of the invention, when used for iron chelation therapy, treating an iron-overload disorder, inhibiting cellular proliferation, or treating a proliferative disorder in a mammal.

With reference to the above aspects of the invention, the mammal may be a human.

The proliferative disorder may be selected from the group consisting of angiogenesis-dependent diseases, cellular proliferative diseases, inflammatory disorders, and cancer. The proliferative disorder may be psoriasis or arthritis.

The cancer may be a metastatic tumour or benign tumour. The cancer may be a solid tumour.

In a twelfth aspect of the invention there is provided a process for identifying a compound having anti-proliferative activity, said process comprising: contacting a cell or cellular extract having DNA encoding one or more cell cycle inhibitors, with a compound, determining the level of expression of said DNA, and thereby determining whether said compound has anti-proliferative activity, wherein said anti-proliferative activity is determined by the ability of the compound to up-regulate expression of said DNA encoding one or more cell cycle inhibitors.

In a thirteenth aspect the present invention provides a process for screening a plurality of compounds to identify a compound having anti-proliferative activity, said process comprising:

contacting a cell or cellular extract having DNA encoding one or more cell cycle inhibitors, with a plurality of compounds, determining whether any of said compounds modify the level of expression of said DNA, and if so, separately determining the level of expression of a DNA for each compound, and thereby identifying a compound having anti-proliferative activity, wherein said anti-proliferative activity is determined by the ability of said compound to up-regulate expression of said DNA encoding one or more cell cycle inhibitors.

With reference to the twelfth or thirteenth aspect of the invention, the compound may be a compound of Formula la, lb, 2 or 3 as defined herein, or an iron complex thereof.

With reference to the twelfth or thirteenth aspect of the invention, the DNA expressing cell cycle inhibitor may be GADD45.

According to a fourteenth aspect of the invention there is provided a method of treating viral, bacterial, or fungal infection in a mammal, said method comprising administering to said mammal a therapeutically effective amount of at least one compound selected from the group consisting of a compound of Formula 1 a, a compound of Formula lb, a compound of Formula 2, and a compound of Formula 3, as defined above, or a metal ion complex of said compound, or a pharmaceutical composition according to the invention.

The compounds according to the present invention include substituted or unsubstituted naphthyl semicarbazone, naphthylhydrazone, naphthyl thiosemicarbazone, and naphthyl thiohydrazone derivatives. With reference to Formulae la, lb, 2, and 3 the compounds are sometimes referred to herein as"NT analogues"or the"NT series".

Definitions Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

The embodiments of the invention described herein with respect to any single embodiment and, in particular, with respect to an apparatus or a method of treatment shall

be taken to apply mutatis mutandis to any other embodiment of the invention described herein.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as"comprises"or"comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

All the references cited in this application are specifically incorporated by reference herein.

As used herein, the term"alkyl group"refers to a saturated aliphatic hydrocarbon radical including straight-chain, branched chain, cyclic groups, and combinations thereof.

The alkyl radical may be a Cl 8, Cl 7, Cz 6, Cl 5, Cl 4, Cl 3, Cl 2 or CH3 radical, or in the case of a cycloalkyl group, a C3 8, C3 7, C3 6, C3 5, or C3 4 radical. Typical alkyl groups include but are not limited to methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, tert- butyl, amyl, isoamyl, sec-amyl, 1, 2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1- dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1, 2-dimethylbutyl, 1,3- dimethylbutyl, 1,2, 2-trimethylpropyl, 1,1, 2-trimethylpropyl, heptyl, 5-methylhexyl, 1- methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2- dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2, 3-trimethylbutyl, 1, 1, 2- trimethybutyl, 1,1, 3-trimethylbutyl, octyl, cyclopropyl, methylcyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopentyl, di-methylcyclopentyl, trimethylcyclopentyl, ethylcyclopentyl, methylethylcyclopentyl, propylcyclopentyl, methylcyclohexyl, dimethylcyclohexyl, ethyl cyclohexyl, and methylcycloheptyl.

The term"alkenyl groupas used herein refers to an unsaturated aliphatic hydrocarbon radical including straight, branched chain, cyclic groups, and combinations thereof, having at least one double bond, of either E or Z stereochemistry where applicable. The alkenyl group may be a C2-8, C2-7, C2-6, C2-5, C2-4, C2-3, or C=C radical, or

in the case of a cycloalkenyl group, a C3 8, C3 7, C3 6, C3 5, or C3-4 radical. Examples of alkenyl groups include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-2- propenyl, 1-butenyl, 1,3-butadienyl, hexenyl, pentenyl, heptenyl and octenyl, cyclohexenyl, cyclopentenyl.

The term"alkynyl group"as used herein refers to an unsaturated aliphatic hydrocarbon group including straight, branched chain, cyclic groups and combinations thereof, having at least one triple bond. The alkynyl group may be a C2-8, C2-7, C2-6, C2-5, C2-4, C2-3 or C C radical. Examples of alkynyl groups include but are not limited to ethynyl, 1-propynyl, 1-and 2-butynyl, and 1-methyl-2-butynyl.

The term"amino"as used herein refers to the group-NR6R7 wherein R6 and R7 are individually selected from the group including but not limited to H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group.

The term"aryl"or"aromatic"as used herein refers to single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 12 carbon atoms. Examples of these groups include phenyl, biphenyl, naphthyl, tetrahydronaphthyl, fluorenyl, indenyl, indanyl, pyrenyl, and the like.

The term"bicyclic"as used herein refers to two fused cyclic rings, wherein the rings are individually selected from C3_8 aliphatic and C5-6 aromatic rings.

The term"halide"or"halo atom"as used herein refers to fluorine, chlorine, bromine or iodine.

The term"heteroatom"as used herein refers to any atom other than carbon or hydrogen and preferably means oxygen, nitrogen or sulfur.

The term"heterocyclic"as used herein refers to any saturated or unsaturated 3-12 membered, e. g 5-or 6-membered, aliphatic or aromatic ring systems containing from 1 to 3 heteroatoms selected from oxygen, nitrogen and sulfur. Examples include substituted and unsubstituted piperidinyl, morpholinyl, pyrrolyl, furanyl, imidazolyl, thienyl, pyridyl, pyradizinyl, 1H-indazolyl, quinoxalinyl, quinolinyl, indolinyl, oxazolidinyl, benzimidazolyl, benzisothiazolyl, tetrazolyl, benzofuranyl, purinyl and indolyl.

The term"substituted or unsubstituted alkyl","substituted or unsubstituted alkenyl", "substituted or unsubstituted alkynyl", "substituted or unsubstituted amino", "substituted or unsubstituted aromatic", "substituted or unsubstituted bicyclic", "substituted or unsubstituted heterocyclic", and"substituted or unsubstituted cycloalkyl"

means the respective alkyl, alkenyl, alkynyl, amino, aromatic, bicyclic, heterocyclic, or cycloalkyl group may be unsubstituted, or substituted with one or more groups selected from Cz-8 alkyl, C3-8 cycloalkyl, C3_8 heterocycloalkyl, C2-8 acyl, C5-10 aryl, &num 5-10 heteroaryl, amino, amide, nitro, cyano, halo, hydroxy, alkoxy, and thio group.

The language"therapeutically effective amount"is intended to include within its meaning a non-toxic but sufficient amount of a compound or composition of the invention to provide the desired therapeutic effect. The exact therapeutically effective amount of the analogue will vary according to factors such as the type of disease of the animal, the age, sex, and weight of the animal, mode of administration, and the ability of the analogue to permeate cell membranes and chelate iron in cells of the animal. Dosage regima can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

In the context of the present invention, a"mammal"includes a primate or non- human primate or other non-human mammal, a marsupial or a reptile. The mammal may be selected from the group consisting of human, non-human primate, equine, murine, bovine, leporine, ovine, caprine, feline and canine. For example, the mammal may be selected from a human, horse, cattle, sheep, goat, dog, cat, llama, rabbit and a camel.

Abbreviations "DFO" : desferrioxamine "311": 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone "NNH"or"31 lm": 2-hydroxy-1-naphthylaldehyde nicotinoyl hydrazone "ST" : salicylaldehyde thiosemicarbazone "NT": 2-hydroxy-1-naphthylaldehyde thiosemicarbazone <BR> <BR> <BR> "N2mT": 2-hydroxy-1-naphthylaldehyde-2-methyl-3-thiosemicarbazone< ;BR> <BR> <BR> <BR> <BR> <BR> "N4mT" : 2-hydroxy-1-naphthylaldehyde-4-methyl-3-thiosemicarbazone "N44mT: 2-hydroxy-1-naphthylaldehyde-4, 4-dimethyl-3-thiosemicarbazone <BR> <BR> <BR> <BR> "N4eT": 2-hydroxy-1-naphthylaldehyde-4-ethyl-3-thiosemicarbazone< BR> <BR> <BR> <BR> <BR> <BR> "N4aT" : 2-hydroxy-1-naphthylaldehyde-4-allyl-3-thiosemicarbazone< BR> <BR> <BR> <BR> <BR> <BR> "N4pT" : 2-hydroxy-1-naphthylaldehyde-4-phenyl-3-thiosemicarbazone "N44pH": 2-hydroxy-1-naphthylaldehyde-4, 4-diphenyl-3-semicarbazone "NoctH": 2-hydroxy-1-naphthylaldehyde-4-octoyl-3-hydrazone.

Brief description of the drawings Figure 1 Structures of representative NT Analogues.

Figure 2 X-Ray crystal structure of NT showing 30% thermal ellipsoids Figure 3 X-Ray crystal structure of N44mT showing 30% thermal ellipsoids (dimethyl sulfoxide molecule of crystallisation not shown).

Figure 4 X-Ray crystal structure of N4eT showing 30% thermal ellipsoids.

Figure 5 X-Ray crystal structure of N4pT showing 30% thermal ellipsoids.

Figure 6 The effect of NT chelators on 59Fe mobilisation from prelabelled SK-N- MC neuroepithelioma cells. Cells were labelled with 59Fe-Tf (0.75 uM) for 3 h at 37°C, washed, and then reincubated, (A) for 3 h at 37°C in the presence of medium alone (control) or medium containing DFO (25 uM) or the other chelators (25 M), or (B) reincubated as above for 3,6, 12 and 24 h. Results are expressed as the mean + SD of 3 replicates in a typical experiment of 2 experiments performed.

Figure 7 The effect of NT chelators on 59Fe uptake from 59Fe-transferrin (59Fe-Tf) by SK-N-MC neuroepithelioma cells. The cells were incubated for: (A) 3 h at 37°C in media containing 59Fe-Tf (0.75 uM) and either DFO (25 uM) or the other chelators (25 pM), washed, and then incubated with pronase (1 mg/ml) for 30 min at 4°C, or (B) incubated as described in (A) for 3,6, 12 and 24 h at 37°C and internalised 59Fe measured. Results are expressed as the mean SD of 3 replicates in a typical experiment of 2 experiments performed.

Figure 8 The effect of NT analogue concentration on the proliferation of SK-N- MC neuroepithelioma cells. Cells were incubated in the presence and absence of an NT analogue (0-12. 5 uM) for 72 h at 37°C. After this incubation period, cellular density was measured via the MTT assay. Each data point represents the mean of 2 replicates in a typical experiment of 3 experiments performed.

Figure 9 Antiproliferative effects of NT chelators in neoplastic and normal cells.

The influence of the cytotoxic NT analogues, NT, N4mT, N44mT and NNH, and 311 (internal standard) on the proliferation of SK-N-MC neuroepithelioma cells (S) compared to MRC-5 fibroblasts (F). Cells were incubated in the presence and absence of the chelators (0-25 uM) for 72 h at 37°C. After this incubation period, cellular density was measured via the MTT assay. Each data point represents the mean of 2 replicates in a typical experiment of 3 experiments performed.

Figure 10 Comparison of the effect of NNH, NT and N44mT and the clinically used anti-tumour agents cisplatin and doxorubicin on the inhibition of normal

granulocyte-macrophage (GM) stem cell colonies of normal human bone marrow. Normal bone marrow stem cells (1 x 105 cells/ml) were incubated for 14 days at 37°C with the agents (0.005-0. 4 I1M), and the colonies were then counted. Results are from a typical experiment of 3 experiments performed.

Figure 11 The relationship between the anti-proliferative activity of the chelators using SK-N-MC neuroepithelioma cells (ICSO values) and their lipophilicity (calculated log Pica) c values). Log Plate values were estimated by Brotos'method, Crippen's fragmentation procedure, and Viswanadhan's fragmentation procedure using the program Chem Draw (version 4.5 1997) and the results then averaged (see Table 2).

Figure 12 The effect of NT chelators on 3H-thymidine incorporation by SK-N-MC neuroepithelioma cells. Cells were seeded at 15,000/well and allowed to grow overnight and the chelators were then added in 0.1 mL of complete medium containing diferric transferrin (1.25 M). After a 20 h incubation at 37 °C, 3H-thymidine (1 pCi) was added and the cells re-incubated for 2 h at 37° C. Each data point represents the mean of 2 replicates in a typical experiment of 3 experiments performed.

Figure 13 The effect of NT chelators on the mRNA levels of TfR, GADD45, and ffi actin (loading control) in SK-N-MC neuroepithelioma cells. Total RNA was extracted from cells after a 20 h incubation with medium alone (control) or medium containing DFO (150 M) or the other chelators (25 uM). The isolated RNA underwent electrophoresis on a 1.2% agarose-formaldehyde gel, transferred to a hybridisation membrane, and probed under high stringency conditions as described in Example 8. The result illustrated is a typical experiment from 3 experiments performed.

Figure 14 Effect of NT analogues on the expression of cell-cycle control molecules. The effect in SK-N-MC neuroepithelioma cells of the cytotoxic NT analogues, NT and N4mT, compared to 311 on the protein levels of (A) cdk2, cdk4, and action (loading control), (B), cyclin A, E, D1, D2, D3 and action (loading control). Western analysis was performed as described in Example 8. The results shown are typical of 2 experiments performed.

Figure 15 The effect of EDTA, NNH, Triapine, and the cytotoxic NT analogues NT and N4mT on (A) the Fe (III) -induced oxidation of ascorbate, and (B) the Fe (II)- induced hydroxylation of benzoate. Ascorbate oxidation and benzoate hydroxylation were assessed as described in Example 9 (a) and 9 (b). The results are mean SEM of 3 experiments.

Figure 16 The effect of EDTA, NNH, Triapine, and the cytotoxic NT analogues NT and N4mT on integrity of the plasmid pGEM-7Zft+) when incubated in the presence of Fe (II) and hydrogen peroxide. Reagents were added in the following order: purified sterile water, chelator (1,10, and 30 uM), FeS04 (10 uM), H202 (1 mM), and plasmid (10 pg/ml), and incubated at room temperature for 30 min before loading onto a 1% agarose gel (Example 9 (c) ). Results are a typical experiment from at least 8 performed.

Detailed Description of the Invention The present invention is directed to novel substituted naphthyl semicarbazone, naphthylhydrazone, naphthyl thiosemicarbazone, and naphthyl thiohydrazone compounds of Formula la which are capable of chelating iron ions. The invention also encompasses pharmaceutical formulations comprising at least one compound of Formula la, Formula lb, Formula 2, or Formula 3 as defined herein, or an iron complex of said compound, and the therapeutic uses thereof.

With reference to Formulae la, lb, 2 and 3, the compounds are sometimes referred to herein as"NT analogues".

The NT analogues in accordance with the present invention have a substituted or unsubstituted naphthyl moiety and a thiosemicarbazone, semicarbazone, thiohydrazone or hydrazone moiety. Suitably, the naphthyl moiety may be substituted with one or more lipophilic and/or electron donating groups, such as substituted or unsubstituted Cl-8 alkyl, C2 8 alkenyl, C2 8 alkynyl, Cl-8 alkoxy, aromatic, heterocyclic, cycloalkyl, cycloaromatic groups, alkyl-substituted amines, and the like. Suitably, the thiosemicarbazone, semicarbazone, thiohydrazone or hydrazone moiety is substituted with one or more lipophilic groups, such as Cl-8 alkyl, C2 8 alkenyl, C2 8 alkynyl, C3-8 cycloalkyl, aromatic, cycloaromatic, heterocycloalkyl, heteroaromatic, alkyl substituted amines and the like.

The lipophilicity of the NT series of compounds according to the present invention is such that the NT compounds may be capable of permeating cell membranes. Inside cells, NT compounds are capable of chelating intracellular Fe.

Suitable methods for the synthesis of compounds of Formulae la, lb, 2 and 3 are well known to those skilled in the art and are described, for example, in J. March, Advanced Organic Chemistry, 4"'Edition (John Wiley & Sons, New York, 1992); and Vogel's Textbook of Practical Organic Chemistry, 5th Edition (John Wiley & Sons, New York, 1989).

Suitably, naphthyl semicarbazone, naphthylhydrazone, naphthyl thiosemicarbazone, and naphthyl thiohydrazone derivatives disclosed herein may be prepared by means of a Schiff base condensation reaction in which a substituted or unsubstituted 2- hydroxynaphthaldehyde is condensed with either an acid hydrazide or acid thiosemicarbazide of choice to produce the corresponding thiosemicarbazone, semicarbazone, hydrazone or thiohydrazone derivative having the desired substitution pattern.

A typical example of a general synthetic route for preparing NT analogues of Formula la, lb, 2 or 3 as defined above, is represented in the following general reaction scheme: GE H O ° INH2 Schiff base N OH OH I condensationI /+ ici E I E III (F) n where E, G and R4 are as defined above.

In particular, a synthetic route for preparing NT analogues of Formula 2 as defined above, is illustrated below for the compounds N44mT, N4pT and NoctH: Sq, NMe2 H O NH2 Schiff base HN, NOH OH condensation i i g N44mT H NS H O NH Schiff base I/HNN OH OH H condensation + HN y N NT s'- fj N4pT Oqw H O NH SChlff b2S2 HNN OH OH I condensation i i + I NoctH

The condensation reactions represented above may be carried out under conditions known to those skilled in the art. For example, suitable solvent systems include ethanol, methanol, ethanol/water, methanol/water, or other common organic solvents such as acetone, benzene, toluene, etc.

Preferred NT analogues suitable for use in accordance with the present invention include the following: 2-hydroxy-1-naphthylaldehyde thiosemicarbazone (NT) 2-hydroxy-1-naphthylaldehyde-4-methyl-3-thiosemicarbazone (N4mT) 2-hydroxy-1-naphthylaldehyde-4, 4-dimethyl-3-thiosemicarbazone (N44mT) 2-hydroxy-1-naphthylaldehyde-4-ethyl-3-thiosemicarbazone (N4eT) 2-hydroxy-1-naphthylaldehyde-4-allyl-3-thiosemicarbazone (N4aT) 2-hydroxy-1-naphthylaldehyde-4-phenyl-3-thiosemicarbazone (N4pT) 2-hydroxy-1-naphthylaldehyde-4, 4-diphenyl-3-semicarbazone (N44pH) 2-hydroxy-1-naphthylaldehyde-4-octoyl-3-hydrazone (NoctH).

The NT series of compounds in accordance with the present invention include compounds which have iron chelation and anti-proliferative properties. In particular, the NT series of compounds are effective Fe (III) and Fe (II) chelators.

Iron complexes in accordance with the present invention include: Fe [NT] 2, Fe [N4aT] 2, Fe [N4eT] 2, Fe [N4mT] 2, Fe [N44mT] 2, Fe [N4pT] 2, Fe [N44pH] 2 and Fe [N4octH] 2. Suitably, the iron ion is Fe (II) or Fe (III).

Iron complexes of NT analogues in accordance with the invention may be readily prepared using techniques and reagents well known to those skilled in the art. Suitable iron salts include, but are not limited to, halides, nitrates, sulfates, perchlorates, acetates, and triflates. Suitable iron salts for forming iron complexes include, but are not limited to, FeCl3, Fe (N03) 3, FeS04, Fe (OAc) 3, and Fe2 (C104) 3 (Richardson & Bernhardt, 1999).

NT derivatives in accordance with the present invention can function as tridentate ligands capable of forming iron complexes. A general example of an iron complex is represented below for an NT analogue of Formula 1 a or 1 b. where E is O or S, and G is as defined above.

By way of further example, an iron complex of the ligand N44mT, is illustrated below.

Suitably, the NT analogues disclosed herein have a range of activities, including iron chelation. The inventors have examined the effect of a range of NT analogues on cellular proliferation, iron chelation efficacy, the expression of cell cycle control molecules, iron-regulatory protein-RNA-binding activity, 3H-thymidine incorporation, and the effect of the NT analogues on iron-mediated free radical damage.

Suitably, NT analogues in accordance with the present invention may be capable of permeating cell membranes. Suitably, in accordance with the present invention, iron complexes of NT ligands in accordance with the invention may be neutral or charged.

In accordance with the present invention, NT analogues, especially for example, more lipophilic members of the NT series of compounds, may be potent Fe (III) and/or Fe (II) chelators and may exhibit anti-proliferative activity. Accordingly, the NT series of compounds are potentially useful as anti-tumour agents.

Suitably, Fe-NT complexes suitable for use in accordance with the present invention may exhibit anti-proliferative activity.

In accordance with the present invention, NT analogues disclosed herein may exhibit antimicrobial activity. Thus, NT analogues disclosed herein may be capable of treating bacterial, viral and fungal infection.

NT analogues in accordance with the invention may inhibit DNA synthesis, and preferred analogues may induce an increase of the mRNA levels of molecules vital for cell cycle arrest. In particular, expression of GADD45 mRNA may provide a useful indicator of the anti-proliferative potential of Fe chelators. Further, preferred NT analogues may exhibit anti-proliferative activity. Accordingly, NT analogues disclosed herein have potential therapeutic applications, including for example, the treatment of iron-overload disease, and the treatment of proliferative diseases, such as cancer.

Methods of measuring DNA synthesis are well known by persons skilled in the art. For instance, DNA synthesis can be measured indirectly by quantitating [3H]- hypoxanthine incorporation into the nucleic acids in the presence or absence of the agents to be tested.

As inhibition of the enzyme ribonucleotide reductase can interfere with the cellular synthesis of DNA, one useful anti-viral treatment strategy is the use of an Fe chelator to inhibit the replication of viral DNA. One method of assessing viral activity includes infecting human monocyte-derived macrophages and peripheral blood lymphocytes with HIV-lBa-L. The cells are then incubated in the absence or presence of the chelator for a suitable period. Virus replication is monitored by assessing the level of p24 core antigen in virus-inactivated supernatants by ELISA techniques (Georgiou et al. , 2000).

To evaluate the anti-bacterial properties of NT analogues, a microplate alamar blue assay (MABA) procedure can be used (Stevens et al. , 2001). For example, an appropriate strain of Mycobacterium tuberculosis (eg, H37Rv strain), can grown in Bactec 12 B medium, in the presence or absence of the test compounds. The reduction in fluorescence produced by the treated cultures is proportional to the inhibition of inoculum growth.

Anti-fungal activity of the NT analogues can be assessed by methods known to those skilled in the art, for example, the recommendations of the National Committee for Clinical Laboratory Standards (U. S. A).'6 Briefly, the standardised test compares the turbidity of treated vs untreated control cultures. An inoculum of test yeast is grown at 35°C and adjusted to a concentration of approximately 1 x 103 CFU/mL, and then re- incubated (35°C) for a suitable time period before turbidity is measured.

Pharmaceutical and/or Therapeutic Formulations In accordance with the present invention, when used for the treatment of disease or infection, NT analogues may be administered alone. However, it is generally preferable that the analogues be administered as a pharmaceutical formulation which comprises at least one NT analogue, or a metal ion complex thereof. The NT analogues may also be present as suitable pharmaceutically acceptable salts.

By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.

For instance, suitable pharmaceutically acceptable salts of NT analogues in accordance with the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention. Suitable pharmaceutically acceptable salts of the compounds of the present invention therefore include acid addition salts.

For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66 : 1-19. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, asparate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like.

Representative alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

Convenient modes of administration include injection (subcutaneous, intravenous, etc. ), oral administration, inhalation, transdermal application, or rectal administration.

Depending on the route of administration, the analogue may be coated with a material to protect the analogue from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the analogue. The analogue may also be administered parenterally or intraperitoneally.

Dispersions of NT analogues can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use,

pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.

In a preferred embodiment, the analogue is administered orally, for example, with an inert diluent or an assimilable edible carrier. The analogue and other ingredients can also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the analogue can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

Suitably, such compositions and preparations may contain at least 1% by weight of active compound. The percentage of the NT analogue in pharmaceutical compositions and preparations can, of course, be varied and, for example, can conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit. The amount of analogue in therapeutically useful compositions is such that a suitable dosage will be obtained.

The language"pharmaceutically acceptable carrier"is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the analogue, use thereof in the therapeutic compositions and methods of treatment is contemplated. Supplementary active compounds can also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of analogue is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly

dependent on (a) the unique characteristics of the analogue and the particular therapeutic effect to be achieve, and (b) the limitations inherent in the art of compounding such an analogue for the treatment of iron-related or iron overload diseases in individuals. The principal analogue is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit.

In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

In a preferred embodiment, the carrier is an orally administrable carrier.

A particularly suitable form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration.

In the case of injectable solutions, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents. Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose,

lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the analogue can be incorporated into sustained-release preparations and formulations.

Preferably, the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.

Single or multiple administrations of the pharmaceutical compositions according to the invention can be carried out. One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the disorders or diseases to which the compounds and compositions are applicable.

Further, it will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the compound or composition of the invention given per day for a defined number of days, can be ascertained using convention course of treatment determination tests.

Generally, an effective dosage of NT analogue per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; suitably, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight. More suitably, an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight.

Alternatively, an effective dosage may be up to about 500 mg/m2. Generally, an effective dosage may be in the range of about 25 to about 500 mg/m2, about 25 to about 350 mg/m2, about 25 to about 300 mg/m2, about 25 to about 250 mg/m2, about 50 to about 250 mg/m2, or about 75 to about 150 mg/m2.

By way of example, suitable dosage forms in accordance with the present invention include the following: Tablet NT Analogue 0. 01 to 20 mg, generally 0.1 to 10 mg Starch 10 to 20 mg Lactose 100 to 250 mg Gelatin 0 to 5 mg Magnesium Stearate 0 to 5 mg Iniectable Solution NT Analogue 0.01 to 20 mg, generally 0.1 to 10 mg Sodium Chloride 8.5 mg Potassium Chloride 3 mg Calcium Chloride 4.8 mg Water for injection, q. s. to 10 ml It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The invention will now be described in greater detail by reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

EXAMPLE 1-Synthesis of Compounds DFO was purchased from Ciba-Geigy Pharmaceutical Co. , Summit, NJ, USA.

Representative structures of the NT series of compounds, DFO and"311"are provided in Figure 1.

Example l (a) -General Synthesis of NT Analogues Representative NT analogues were synthesised by Schiff base condensation using standard procedures. Equimolar amounts of 2-hydroxy-1-naphthylaldehyde was heated with an appropriate acid hydrazide or thiosemicarbazide in refluxing ethanol. The product was collected by filtration. Other suitable solvents for the Schiff base condensation include methanol, ethanol/water, methanol/water, acetone, benzene, and toluene. The NT compounds were characterised by a combination of elemental analysis,'H-NMR spectroscopy, infrared spectroscopy, and X-ray crystallography using.

Representative structures of NT analogues are provided in Figure 1.

Example l (b)-Synthesis of 2-hydroxy-1-naphthylaldehyde thiosemicarbazone (NT) Equimolar amounts of 2-hydroxy-1-naphthaldehyde and thiosemicarbazide were refluxed in ethanol. The compound was collected by filtration and crystals suitable for X-ray crystallography were obtained by slow evaporation of an ethanolic solution of the compound. The crystal structure and data of NT is provided in Figure 2.

Microanalytical data: values expected C=58. 78%, H=4. 52%, N=17.13% values observed C=58.79%, H=4.48%, N=16.99% Example l (c)-Synthesis of 2-hydroxy-1-naphthylaldehyde-4, 4-dimethyl-3- thiosemicarbazone (N44mT) Equimolar amounts of 2-hydroxy-1-naphthaldehyde and 4,4- dimethylthiosemicarbazide were refluxed in ethanol. The title compound was collected by filtration and crystals suitable for X-ray crystallography were obtained by slow evaporation of an ethanolic solution of the compound. The crystal structure and data of N44mT is provided in Figure 3.

Microanalytical data: values expected C=61.52%, H=5.53%, N=15.37% values observed C=61.47%, H=5.60%, N=15.23% Example l (d)-Synthesis of 2-hydroxy-1-naphthylaldehyde-4-ethyl-3- thiosemicarbazone (N4eT)

Equimolar amounts of 2-hydroxy-1-naphthaldehyde and 4-ethylthiosemicarbazide were refluxed in ethanol. The title compound was collected by filtration and crystals suitable for X-ray crystallography were obtained by slow evaporation of an ethanolic solution of the compound. The crystal structure and data of N4eT is provided in Figure 4.

Microanalytical data: values expected C=61.52%, H=5.53%, N=15.37% values observed C=61.58%, H=5.45%, N=15.09% Example l (e)-Svnthesis of2-hvdroxv-l-naphthyladehyde-4-phenyl-3- thiosemicarbazone (N4pT) Equimolar amounts of 2-hydroxy-1-naphthaldehyde and 4-phenylthiosemicarbazide were refluxed in ethanol. The title compound was collected by filtration and crystals suitable for X-ray crystallography were obtained by slow evaporation of an ethanolic solution of the compound. The crystal structure and data of N4pT is provided in Figure 5.

Microanalytical data: values expected C=67.27%, H=4.70%, N=13.07% values observed C=67.19%, H=4.68%, N=12.79% Example 1 (fl-Preparation of Iron complexes Iron complexes of NT analogues were prepared using techniques described by Richardson & Bernhardt, 1999. Generally, 1 mole equivalent of an Fe (III) salt is refluxed in hot ethanol with 2 mole equivalents of NT analogue for 1 hour. A dark green/black complex solid forms and is isolated from the mother liquor by filtration under reduced pressure. The complex is then washed with ethanol and air dried.

In Vitro STUDIES General Methodology for Examples 2-8 Presentation of Chelator Compounds NT chelators were dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock solutions immediately prior to an experiment and then diluted in 10% fetal calf serum (FCS; Commonwealth Serum Laboratories, Melbourne, Australia) so that the final DMSO concentration was equal to or less than 0.5 % (v/v).

In studies examining hydroxyl radical generation by the chelator-Fe complexes, DMSO was not used as a solvent due to its ability to scavenge hydroxyl radicals. In these experiments, suitable solutions of the NT chelators were prepared using acetonitrile at

concentrations < 0.45% v/v. Control experiments demonstrated that under these conditions, acetonitrile had no effect on the hydroxyl radical assays.

Cell Culture Preparation Human K562 erythroleukemia, SK-Mel-28 melanoma, SK-N-MC neuroepithelioma cells, MCF-7 breast cancer cells and the normal cell lines MRC-5 and L8 rat skeletal muscle were from the American Type Culture Collection (ATCC), Rockville, MD, USA.

The cell lines were grown in Eagle's modified minimum essential medium (MEM; Gibco BRL, Sydney, Australia) containing 10 % FCS, 1 % (v/v) non-essential amino acids (Gibco), 2 mM L-glutamine (Sigma Chemical Co. , St. Louis, MO, USA), 100 Fg/ml of streptomycin (Gibco), 100 U/ml penicillin (Gibco), and 0.28 pg/ml of fungizone (Squibb Pharmaceuticals, Montreal, Canada). This growth medium will be subsequently referred to as complete medium. Cells were grown in an incubator (Forma Scientific, OH, USA) at 37°C in a humidified atmosphere of 5% C02/95% air and subcultured as described previously (Richardson et al. , 1995). Primary cultures of human umbilical vein endothelial cells (HUVECs) and human monocyte derived macrophages (HMDM) were prepared using standard techniques (Brown et al. , 2000; McCrohon et al. , 1999). Bone marrow stem cell cultures were prepared using established methods (Eaves, 1995) to study the effect of the chelators on the growth of granulocyte-macrophage (GM) colonies.

Cellular growth and viability were monitored using phase-contrast microscopy and trypan blue staining.

Statistics Experimental data were compared using Student's paired t-test. Results were considered statistically significant when p < 0.05. Results are presented as the mean or mean standard deviation (SD) of 3-4 separate experiments.

EXAMPLE 2-Iron Mobilisation Experiments Labelling of Transferrin with 59Fe Apotransferrin (Sigma Chemical Co. , St. Louis, USA) was prepared and labelled with 59Fe (as ferric chloride in 0.1 M HCI, Dupont NEN, MA, USA) to produce 59Fe- transferrin (59Fe-Tf) using standard procedures described by Richardson & Baker, 1992.

Example 2 (a)-Comparison of the Effect of NT Chelators on Iron Mobilisation from Prelabelled SK-N-MC Neuroepithelioma Cells Methodology : Iron Mobilisation Assay The ability of NT chelators to mobilise 59Fe from SK-N-MC neuroepithelioma cells was studied using standard procedures (Richardson et al. , 1995). Briefly, cells were prelabelled with 59Fe-transferrin (0.75 M) for 3 h at 37°C. The cells were then placed on ice and washed 4 times with ice-cold BSS, and then (A) reincubated for 3 h at 37°C in the presence and absence of the internal standard DFO (100 uM) or the NT chelators (50 M) ; or (B) reincubated for 3,6, 12 and 24 h. DFO was used at a concentration of 100 uM due to the low efficacy of this chelator at mobilising 59Fe from cells and preventing 59Fe uptake from 59Fe-Tf. The overlying medium was then removed and placed into y- counting tubes. The cells were removed from the petri dishes in 1 ml of BSS using a plastic spatula and transferred to a separate set of y-counting tubes.

Broadly, the ligands can be grouped into two classes depending upon their ability to mobilise 59Fe. The first class exhibit activity similar to DFO, and include the chelators ST, NT, and N2mT, which mobilise 3-13% of total cellular 59Fe (Fig. 6A). The second class of chelators are the remaining NT analogues which are significantly (p < 0.0001) more effective than DFO, resulting in the release of 28-43% of cellular 59Fe (Fig. 6A). Of this latter group, N44mT is the most efficient analogue having activity that is comparable to 311 and NNH, resulting in the release of 43% of cellular 59Fe (Fig. 6A). Of this latter group, N44mT shows significant antiproliferative activity, resulting in 43% release of cellular 59Fe (Fig 6A). The parent analogue, NT, only mobilised 13% of cellular 59Fe and is markedly less efficient than the other NT analogues (Fig. 6A).

As NT has high anti-proliferative efficacy (Fig. 8 and Table 1), but relatively low activity at mobilising 59Fe from cells (Fig. 6A), time-course experiments were performed in an attempt to understand its mechanism of action (Fig. 6B). In these experiments, N44mT was also assessed as it has high activity at both inhibiting proliferation and mobilising cellular 59Fe. These results with NT and N44mT were compared to 311 and DFO. The experiments show that DFO and NT behave similarly, both increasing 59Fe efflux from the SK-N-MC cells as a function of time (Fig. 6B). These data suggest that NT and/or its Fe complex may be subject to similar membrane permeability limitations as DFO, which forms a relatively hydrophilic Fe complex which does not exit cells easily.

Example 2 (b)-Effect of the NT Chelators on Iron Uptake from Transferrin bv SK-N-MC Neuroepithelioma Cells Methodology : Iron Uptake Assay The ability of NT analogues to inhibit cellular uptake of 59Fe from 59Fe-Tf by SK- N-MC Neuroepithelioma Cells was examined using standard procedures (Richardson et al. , 1995). SK-N-MC Cells were incubated for 3 h at 37°C in media containing 59Fe-Tf (0. 75 uM) in the presence or absence of an NT chelator (25 J. M) or in the presence of DFO (100, uM) (Fig 7A-B). The cells were then placed on ice and washed four times with ice-cold Hank's balanced salt solution (BSS) to remove non-specifically bound 59Fe-Tf.

The amount of 59Fe internalised by cells was measured by incubation with the general protease, pronase (1 mg/ml), for 30 min at 4°C to remove membrane-bound 59Fe and Tf.

In agreement with their lower ability to mobilise 59Fe, ST, NT, and N2mT, had little effect at preventing 59Fe uptake (Fig. 7A). The most efficient chelators, namely, 311, 311m and N44mT limited 59Fe uptake to 5-9% of the control, their activity being significantly greater (p < 0.0001) than DFO (Fig. 7A). These results agree with the ability of these ligands to mobilise cellular 59Fe (Fig. 6A). It is significant that all analogues (except ST, NT and N2mT) show much greater efficacy than DFO in preventing 59Fe uptake from 59Fe-Tf by SK-N-MC cells (Fig. 7A).

In view of the limited permeability of NT and/or its Fe complex in kinetic studies assessing 59Fe mobilisation (Fig. 6B), time-course experiments were undertaken to examine the effects of NT on 59Fe uptake from 59Fe-Tf. These results were compared to the most effective NT analogue, N44mT, and the standards 311 and DFO (Fig. 7B).

These experiments suggest that NT and/or its Fe complex may be subject to similar permeability limitations as DFO, as both chelators only moderately reduce 59Fe uptake from 59Fe-Tf even for incubation periods of 24 h (Fig. 7B). For example, after a 6 h incubation, NT and DFO reduced 59Fe uptake by SK-N-MC cells to 82% and 85% of the control, respectively. In contrast, after 24 h, NT and DFO reduce 59Fe uptake to 61% and 48% of the control, respectively (Fig. 7B).

Example 2 (c)-NT Chelators Inhibit 59Fe Uptake Only After Intracellular 59Fe Delivery From Tf The ability of the NT chelators to prevent cellular 59Fe uptake from 59Fe-Tf (Fig.

7A) was examined to determine whether it was due to direct removal of 59Fe from

transferrin. The most effective chelators at inhibiting proliferation namely 311,311m, NT, N4mT and N44mT were ineffective at removing 59Fe from Tf over the duration of an uptake experiment (3 h), resulting in the release of 0.1-0. 3% of the bound 59Fe, while DFO removed 0.7% (data not shown). These results demonstrate that the chelators have very little effect on direct 59Fe release from 59Fe-Tf. Further, these data suggest that the ligands inhibit 59Fe uptake only after intracellular delivery from Tf.

EXAMPLE 3-Effect of NT Compounds on Cellular Proliferation Example 3 (a) -Effect of NT Analogues on the Proliferation of Neoplastic Cells Methodology : Cellular Proliferation Assay The effect of NT compounds on the proliferation of the SK-N-MC neuroepithelioma cell line (Table 1) was examined using a MTT (3- (4, 5-dimethylthiazol- 2-yl) -2,5-diphenyl tetrazolium) assay following substantially the same method as that described by Richardson et al. , 1995. MTT colour formation was directly proportional to the number of viable cells measured by Trypan blue staining. In all MTT proliferation experiments, the known iron chelators 311 and DFO were used as relevant internal controls (Table 2). Results of the MTT assays are expressed as a percentage of the control volume. The effect of potent NT analogues on bone marrow stem cell growth was determined by microscopy, only colonies containing over 40 cells were counted. In these studies the clinically used drugs cisplatin and doxorubicin were used as internal controls.

The ability of the ligands to inhibit cellular proliferation was assessed in SK-N-MC neuroepithelioma cells (Fig. 8 and Table 1). These studies identified NNH, NT and N4mT as chelators with very high anti-proliferative activity (ICso= 0.3-0. 5 M ; Table 1). These ligands were significantly (p < 0.0001) more active than DFO (IC$o = 22 M), and have comparable efficacy to chelator 311 (IC50= 0.3 M ; Table 1). Like the Fe complexes of DFO and 311, the Fe complex of NT was not very active in retarding the proliferation of SK-N-MC cells (ICso > 12.5 LM ; Fig. 8 and Table 1). Other chelators with appreciable activity against SK-N-MC cells included N44mT and N4eT that had IC50 values of 1.5 and 1.6 pM respectively (Table 1).

To investigate the spectrum of anti-neoplastic activity, the most active analogues against SK-N-MC cells, namely, NNH, NT, N4mT and N44mT, were also assessed against K562 erythroleukemia, SK-Mel-28 melanoma and MCF-7 breast cancer cell lines (Table 1). The NNH, NT, N4mT and N44mT ligands were far more active than DFO at

inhibiting the growth of K562 and MCF-7 cells (Table 1). Against SK-N-MC cells, NT and N4mT are more active (IC50 = 0.5 RM) than N44mT (ICso = 1.5 µM ; Table 1).

However, of these chelators, N44mT is the most active against K562 (ICso = 1.8 µM), SK-Mel-28 (IC50= 2.9 pM) and MCF-7 (IC50 = 5. 9 µM) cells (Table 1). Significantly, chelator NNH demonstrated significant broad spectrum activity against all neoplastic cells examined (Table 1).

Table 1. The effect of the chelators on cellular proliferation of neoplastic and normal cells<BR> IC50 ( M) Neoplastic Cells Normal Cells human umbilical vein human monocyte SK-N-MC K562 SK-Mel-28 MCF-7 MRC-5 L8 rat skeletal endothelial cells derived macrophages neuroepithelioma eythroleukemia melanoma breast cancer fibroblasts muscle (HUVEC) (HMDM) DFO 22 ~ 8 >25 7.5 ~ 1.3 >25 >25 >25 >25 >25 311 0.3 ~ 0.1 0.5 ~ 0.1 0.8 ~ 0.4 0.9 ~ 0.3 >25 7.2 ~ 1.8 5.4 ~ 0.7 20.1 ST >12.5 * * * * * * * NT 0.5 ~ 0.1 6.6 ~ 1.9 9.6 ~ 2.0 11.6 ~ 0.8 >25 >25 4.5 ~ 0.7 24.0 N2mT >12.5 * >25 >25 8.6 ~ 4.6 >25 N4mT 0.5 ~ 0.1 5.2 ~ 1.5 11.6 ~ 0.8 14.4 ~ 3.1 >25 >25 >25 >25 N44mT 1.5 ~ 0.2 1.8 ~ 0.3 2.9 ~ 1.6 5.9 ~ 0.1 >25 9.8 ~ 1.8 9.4 ~ 0.4 >25 N4eT 1.6 ~ 0.9 * * * * * * * N4aT 2.3 ~ 1.4 * * * * * * * N4pT 3.3 ~ 0.4 * * * * * * * N44pT 5.2 ~ 0.6 * * * * * * * NoctT 10.5 ~ 1.4 * * * * * * * NNH 0.3 ~ 0.1 0.6 ~ 0.2 2.1 ~ 0.1 1.2 ~ 0.2 >25 9.9 ~ 1.4 4.6 ~ 0.2 21.6 FeNT >12.5 * * * * * * * *not determined<BR> The chelators and the iron complex of NT were incubated with cells for 72 h. At the end of this incubation period, cell density was determined by the MTT assay,<BR> as described in Material and Methods. Results are mean ~ Sd (3 experiments) except HMDM (mean of 2 experiments).

Example 3 (b)-Comparison of the Effect of NT Analogues on the Proliferation of Normal Cells and Neoplastic Cells The anti-proliferative effect of cytotoxic NT analogues (311, NNH, NT, N4mT, N44mT) was assessed for a range of neoplastic and normal cells (Fig. 9 and Table 1). In these experiments, N2mT was included as a negative control since the methyl group at the 2-position hinders electron delocalisation and metal ion binding. These studies showed that in contrast to SK-N-MC cells where 311, NNH, NT, N4mT and N44mT exhibited in a potent anti-proliferative effect (ICso = 0.3-1. 5 M), these chelators had relatively little effect on fibroblast proliferation (ICso values >25 M) (Fig. 9 and Table 1). The difference in the anti-tumour effect may in part be due to the lower rate of proliferation of the MRC 5 fibroblasts (doubling time = 22 h) compared to the SK-N-MC neuroepithelioma cells (doubling time = 16 h).

For the most active chelators examined (311, NNH, NT, N4mT and N44mT), their ability to inhibit growth was most pronounced for the neoplastic cells compared to the normal cell type MRC-5 (Table 1). Of the normal cell types examined, MRC-5 fibroblasts were the least sensitive to the action of the five NT chelators tested, while the more rapidly proliferating HUVEC cultures were the most sensitive (Table 1). Despite the similar replicative rates of HUVEC and SK-N-MC cells, the cell types assessed, SK-N- MC neuroepithelioma cells were the most sensitive, while MCF-7 breast cancer cells were the least affected.

Further studies examined the effect of the potent anti-proliferative agents NNH, NT and N44mT on the proliferation of GM colonies of normal human bone marrow over a period of 14 days. As relevant controls, the effect of the cytotoxic drugs cisplatin and doxorubicin were compared to the chelators (Fig. 10). The ability of NNH, NT and N44mT to inhibit proliferation of GM colonies was far less marked than doxorubicin and less marked or similar to cisplatin (Fig. 10). It is important to note that clinically, cisplatin is not a significant myelosuppressive agent, whereas doxorubicin is. Since the inhibition of colony growth by the chelators was relatively similar to cisplatin, it can be suggested that myelosuppression with NNH, NT or N44mT may not be marked.

EXAMPLE 4-The Relationship Between Lipophilicity and Anti-proliferative Activity of the NT Analogues Lipophilicity of the NT chelators was measured as the average log P values (n- octanol-water partition coefficients). Log P values were estimated (Table 2) according to

Brotos'method (Broto et al., 1984), Crippen's fragmentation procedure (Ghose & Crippen, 1987), or Viswanadhan's fragmentation procedure (Viswanadhan et al., 1987) using Chem Draw Pro. (v. 4.5, Cambridge Software, 1997). The means of these values (average calculated log P values; Table 2) show a strong linear correlation with anti- proliferative activity (r = 0.95 ; Figure 11), with the exception of NoctH.

These investigations identify a significant chelator structure-activity relationship dependent on lipophilicity. This is demonstrated by N4mT and N44mT which have one and two methyl groups at N4, and ICso values of 0.5 and 1.0 pM, respectively, in SK-N- MC cells. In addition, N4pT and N44pH (both more lipophilic than N4mT and N44mT; Table 2) have one and two phenyl groups at N4 and IC50 values of 3. 3 uM and 5.2 mM, respectively. Thus, for the NT series, anti-proliferative activity against SK-N-MC cells decreased when substituents on the terminal nitrogen atom became increasingly lipophilic.

Table 2: Calculated n-octanol partition coefficients (log Pâte) tour NT chelators.

Crippens'Viswanadhans'Brotos'Method Average Fragmentation Fragmentation log Pcalc 311 2.63 2.83 2.17 2.54 ST-0. 57-0. 12 0.74 0.02 NT 2.31 2.87 1.76 2.31 N2Mt 2.54 3.11 2.72 2.79 N4mT 2.83 3.27 2.19 2.76 N44mT 3.2 3.63 2.3 3.04 N4eT 3.16 3.62 2.4 3.06 N4aT 3.55 3.98 2.85 3.46 N4pT 4.49 5.1 3.75 4.45 N44pH 6.69 7.02 * 6. 86 NoctH 4.81 4.84 4.73 4.79 NNH 2.63 2.83 2.17 2.54 * Unable to be calculated by Brotos'method Log Peak values were calculated using the program Chem Draw Pro V 4.5.

EXAMPLE 5-Relationship Between Iron Chelation and Anti-proliferative Activity of NT Analogues in SK-N-MC Neuroepithelioma Cells The relationship between Fe chelation efficacy (Figs. 6 and 7) and anti-proliferative activity (Table 1 and Fig. 8) was examined. N4mT and N44mT exhibit high chelation activity (Figs. 6and 7) and anti-proliferative activity (IC50 = 0.5 and 1.5 M, respectively; Table 1). However, N44pH and NoctH, also demonstrate high Fe chelation efficacy (Fig.

6 and 7), but have much lower anti-proliferative activity (IC50 = 5. 2 and 10. 5 uM, respectively; Table 1). Additionally, NT has marked anti-proliferative effects (Table 1) but has relatively low Fe chelation efficacy (Figs. 6 and 7). The lack of correlation between anti-proliferative activity and Fe chelation efficacy may reflect differences in the Fe pools targeted by these chelators and/or the relative efficacies of the ligand or its Fe complex to permeate cellular membranes.

EXAMPLE 6-Effect of NT Analogues on 3H-Thymidine Incorporation by SK-N-MC Neuroepithelioma Cells Methodology : 3H-Thymidine Incorporation Assay The effect of the chelators on 3H-thymidine incorporation in SK-N-MC cells was assessed using standard procedures (Richardson & Milnes, 1997). Briefly, cells were seeded in 96-well microtitre plates at 15,000 cells/well in 0.1 ml of complete medium containing human diferric Tf (1. 25 lit). This seeding density resulted in exponential growth of the cells during the duration of the assay. The cells were allowed to grow overnight and the chelators were then added in 0.1 ml of complete medium containing diferric Tf (1.25 pM). After a 20 h incubation at 37 °C, 3H-thymidine (1 pCi) was added and the cells reincubated for 2 h at 37° C. Subsequently, the cells were harvested onto glass fibre mats and the radioactivity directly measured on a p-scintillation counter (Wallac 1205 Betaplate Reader, Pharmacia, Turku, Finland).

The effect of the NT analogues on DNA synthesis was examined (Fig. 12; Table 3).

Of the NT analogues, the most active inhibitor of DNA synthesis was N44mT (IC50 = 1. 5 pM) which had activity that was significantly higher (p < 0.0001) than DFO (IC50 = 23 µM) and slightly less than chelator 311 (IC50= 0. 6 I1M) (Fig. 12; Table 3). It is relevant to note that N44mT also showed the highest Fe chelation efficacy of the NT series in terms of its ability to increase 59Fe mobilisation from cells (Fig. 6A, B) and inhibiting 59Fe uptake from 59Fe-Tf (Fig. 7A, B). On the other hand, ST, NT, and N2mT were among the

least effective inhibitors of DNA synthesis (Fig. 12; Table 3) and have relatively low Fe chelation efficacy (Figs. 6 and 7). However, overall, their was a weak relationship between inhibition of DNA synthesis and either preventing 59Fe uptake from 59Fe-Tf (r = 0.48) or increasing 59Fe mobilisation from prelabelled cells (r = 0.32). For example, in terms of inhibiting DNA synthesis, NoctH was much less active (IC50 = 17.4 µM ; Table 3) than N4pT (IC50 = 3. 8 µM ; Table 3), despite both ligands having comparable Fe chelation efficacy (Figs 6 and 7). When the relationship between anti-proliferative activity (Table 1) and 3H-thymidine incorporation (Table 3) was examined, a weak linear relationship was found (r = 0.54) (data not shown).

Table 3: The effect of the NT ligands on [3H] thymidine incorporation into SK-N-MC neuroepithelioma cells.

Chelator IC50 (µM) DFO 23i4. 6 311 0. 6 0. 1 311m 0. 6 ~ 0. 1 ST >25 NT 9 2. 3 N2mT 15. 5+3. 4 N4mT 6. 3 2. 0 N44mT 1.5 0. 4 N4eT 5.7 ~ 1. 8 N4aT 7.9 ~ 1. 4 N4pT 3. 8 2. 4 N44pH 4.3 ~ 1. 6 NoctH 17.4 ~ 6. 2 NNH 0.6 ~ 0. 1 FeNT >25 The chelators and the iron complex of NT were incubated with cells for 20 hours. At the end of this incubation period, [3H] thymidine was added and the cells were reincubated for 2

hours. Incorporation of ['H] thymidine was determined as described above. Results are the mean SD (3 experiments).

IC50 indicates 50% inhibitory concentration EXAMPLE 7-Effect of NT Analogues on Expression of TfR and Cell Cycle Control Molecules Methodology : (A) Northern Blot Analysis Northern blot analysis was performed by isolating total RNA using the Total RNA Isolation Reagent (Advanced Biotechnologies Ltd, Surrey, United Kingdom). The RNA was then cross-linked to the membrane using a UV-crosslinker (UV Stratalinker 1800, Stratagen Ltd, USA). The membranes were hybridised with probes specific for human GADD45, and ß-actin. The GADD45 probe consisted of a 760 bp fragment from human GADD45 cDNA cloned into pHu145B2 (kindly supplied by Dr. Albert Fornace, National Cancer Institute, NIH, Maryland, USA). The p-actin probe consisted of a 1.4 kb fragment from human p-actin cDNA cloned into pBluescript SK- (ATCC; Cat. No. 37997).

Hybridisation of probes to the membranes and their subsequent washing were performed as described previously. 5 The membranes were then exposed to Kodak XAR films at- 70°C with an intensifying screen. Densitometric data were collected with a Laser Densitometer and analysed by Kodak Biomax I Software (Kodak Ltd, USA).

(B) Western Blot Analysis Western blot analysis was performed by standard techniques. Briefly, lysates were prepared as previously described (Darnell & Richardson, 1994; Gao & Richardson, 2001) and after electrophoresis, the proteins were electroblotted for 24 h at 4°C onto a PVDF membrane (NEN, Boston, USA). The membranes were stained with Ponceau S (Sigma) to ensure all lanes contained equal amounts of protein. To also ensure uniformity of protein loading, membranes were probed for ß-actin. The films were scanned and montages assembled with Adobe Photoshop.

Antibodies Monoclonal antibody against p-actin (clone AC-15; used at a dilution 1: 5000) was obtained from Sigma. The remaining antibodies were from Santa Cruz (CA, USA). These are listed below in two sections: mouse anti-human MoAbs or rabbit anti-human polyclonal antibodies. The catalogue number and working concentrations (ug/ml) are listed next to each antibody. (1) MoAbs: cyclin A (BF683/SC239; 2 g/ml) ; cyclin D1

(R124/SC6281 : 2 llg/ml) ; cyclin E (HE12/SC247; 1 tg/ml) ; cdk2 (D12/SC6248; 2 g/ml) ; Rb (IF8/SC102; 2 pg/ml). Polyclonal antibodies: cyclin D2 (H289/SC754; 2 pg/ml) ; cyclin D3 (C16/SC182 ; 0.2 g/ml) ; cdk4 (C22/SC260; 1 µg/ml).

TfR plays a crucial role in Fe uptake while GADD45 plays a key role in cell-cycle arrest. NT analogues were assessed in order to determine whether they up-regulated these genes. DFO (150 uM) and 311 (25 uM) both up-regulated TfR and GADD45 mRNA levels (Fig. 13). Of the NT series, N44mT (25 pM) markedly up-regulated the levels of TfR and GADD45 mRNA, while NT, N4mT, and particularly N2mT, showed considerably less activity. The Fe complex of NT did not increase the level of TfR or GADD45 mRNA (Fig. 13) As Fe deprivation causes GI/S arrest, changes in the expression of key cell cycle control molecules that play roles in mediating Gl/S progression were investigated. The effect of NT and N4mT on the protein levels of several key cyclins (cyclin A, E, D1, D2 and D3), and cyclin-dependant kinases (cdk2 and cdk4) was examined. 311 was used as a control. Hyperphosphorylation of pRb is essential for GI/S progression, and further D- type cyclins bind cdk4 and/or cdk2 to phosphorylate pRb. In this investigation 311 (25 pM) caused a marked decrease in the expression of cyclins D1, D2, D3 and also cdk2 in SK-N-MC cells (Fig. 14A, B). Both NT (25 µM) and N4mT (25 pM) also caused a marked decrease in the expression of these cell cycle control molecules, but were slightly less effective than 311 (Fig. 14A, B). Relative to the control, all of the chelators had little effect on cdk4 or cyclin A protein levels, while 311 and NT (25 pLM) increased cyclin E levels (Fig. 14A, B).

EXAMPLE 8-The Effect of NT Analogues on Iron-Mediated Free Radical Damage The chemotherapeutic drugs, doxorubicin and bleomycin, partly mediate their cytotoxic effects by forming metal ion complexes that generate free radicals. The ascorbate oxidation, benzoate hydroxylation, and plasmid DNA cleavage assays were used to assess the redox-activity of the Fe-complexes of NT analogues which exhibited the greatest anti-proliferative activity in the SK-N-MC cells, namely, NNH, NT, and N4mT. The redox-active Fe complexes of EDTA and Triapine were used as internal controls (Dean & Nicholson, 1994).

For the ascorbate oxidation, benzoate hydroxylation, and plasmid DNA strand break assays, the term"iron-binding equivalents" (IBEs) is used to express the data. This was due to the different coordination modes of the ligands to Fe i. e. , DFO and EDTA are hexadentate and form 1 : 1 ligand: Fe complexes, while 311, Triapine, and NT and its analogues are tridentate resulting in 2: 1 ligand: Fe complexes. Thus, for a direct comparison of the tridentate and hexadentate ligands it was necessary to add twice as much tridentate as hexadentate chelator. In the present study, a range of ligand: Fe IBE ratio's were used, namely, 0.1, 1, or 3. An IBE of 1 is equivalent to the complete filling of the coordination shell of the Fe atom by the ligand (s). Thus, for a hexadentate chelator (i. e. , DFO or EDTA), an IBE ratio of 1 represents 1 ligand to 1 Fe atom, while for a tridentate chelator (i. e. , Triapine, NT, or 311) it is equal to 2 ligands to 1 Fe atom. An IBE of 0.1 represents an excess of Fe to chelator i. e., 1 hexadentate chelator or 2 tridentate chelators in the presence of 10 Fe atoms. An IBE of 3 represents an excess of chelator to Fe, and is equal to 3 hexadentate chelators or 6 tridentate chelators in the presence of 1 Fe atom.

Example 8 (a)-Ascorbate Oxidation Methodology : Ascorbate Oxidation Assay This assay was performed in accordance with methods known in the art (Dean & Nicholson, 1994). Briefly, ascorbic acid (0.1 mM) was prepared immediately prior to an experiment and incubated in the presence of Fe (III) (10 LM) and a 50-fold molar excess of citrate (500 pM) and the chelator (1-60 pM). Absorbance at 265 nm was measured after 10 and 40 min incubation at room temperature and the decrease between these time points calculated.

The ability of the Fe-complexes of NT ligands to reduce Fe (III) as determined by ascorbate oxidation is shown in Figure 15A. EDTA promoted the oxidation of ascorbate at all ligand: Fe (III) ratios (expressed as iron binding equivalents (IBEs). At IBE ratios of 0.1, 1, and 3, EDTA increased ascorbate oxidation to 145%, 382%, and 376% of the control respectively (Fig. 15A). In contrast, NNH, NT, and N4mT, at IBE ratios of 1 and 3, were protective against ascorbate oxidation (Fig. 15A). For instance, at an IBE ratio of 3, these chelators reduced ascorbate oxidation to 19%, 33% and 77% of the control respectively, (Fig. 15A). Triapine potentiated ascorbate oxidation at all IBE ratios (Fig.

15A).

Example 8tb)-Benzoate Hydroxelation Methodology : Benzoate Hydroxylation Briefly, this assay is based on the ability of hydroxyl radicals to hydroxylate benzoate to fluorescent products (308 nm excitation and 410 nm emission) (Dean & Nicholson, 1994). Benzoic acid (1 mM) was incubated for 1 h at room temperature in 10 mM sodium phosphate (pH 7.4) with 5 mM hydrogen peroxide, the chelator (3-180 M), and ferrous sulfate (30 iM). For the benzoate and plasmid degradation assays (see below) all solutions were prepared immediately prior to use and the Fe (II) added to water extensively degassed with nitrogen. The addition of Fe (II) was used to start the reaction and the solution was kept in the dark prior to measuring the fluorescence using a Perkin Elmer L550B fluorimeter. In these experiments, salicylate was implemented as a standard, and was also used to determine quenching by the chelators.

This assay is based on the ability of hydroxyl radicals to hydroxylate benzoate to fluorescent products (308 nm excitation and 410 nm emission). Previous studies have shown that the EDTA-Fe complex increases benzoate hydroxylation (Dean & Nicholson, 1994). At IBE ratios of 0.1, 1, and 3 EDTA increased benzoate hydroxylation by 159%, 341% and 358% of the control respectively (Fig. 15B). NNH was mildly protective at an IBE ratio of 3 where it reduced benzoate hydroxylation to 73% of the control (Fig. 15B).

NT had little effect on the hydroxylation of benzoate, whereas N4mT elevated it to 157% of the control (Fig. 15B). In contrast Triapine greatly increased benzoate hydroxylation to 530% and 683% of the control, at IBE ratios of 1 and 3, respectively (Fig. 15B).

Example 8 (c)-Plasmid DNA Integrity <BR> <BR> <BR> <BR> Methodology : Measurement of DNA Integrity Using the Plasmid pGEM-7Zf (+) in the<BR> <BR> <BR> <BR> <BR> <BR> Presence of Fe and the Chelator Escherichia. coli (DH-5a) were transformed with the plasmid pGEM-7Zf (+) (Promega Inc.) and were grown in LB medium. The plasmid DNA was then purified using the Qiagen plasmid purification kit (Qiagen Inc. , USA). Reagents were added to sterile Eppendorf tubes in the following order: purified sterile water, chelator (1,10, and 30 1M), FeSO4 (10 I1M), H202 (1 mM), and plasmid DNA (10 llg/ml). Samples were incubated at room temperature for 30 min and 25 p1 aliquots were immediately loaded with 5 pl loading dye onto a 1% agarose gel containing ethidium bromide (Hermes-Lima

et al, 1998). As controls, plasmid linearised with BamHl, plasmid alone, plasmid incubated with H202, and plasmid incubated with Fe (II) and H202 were used.

The ability of NT analogues to prevent Fe-mediated hydroxyl radical damage to plasmid DNA was assessed. Untreated plasmid and plasmid treated with H202 that both run on gels as a single band of supercoiled (SC) DNA were used as one control (Fig. 16). Plasmid treated with the restriction enzyme BamHl was used as a another control. When plasmid was treated with Fe (II) and H202 (Fig. 11), SC DNA was partially converted to the open circular (OC) form (Fig. 16).

Although EDTA was redox active in both the ascorbate oxidation (Fig. 15A) and benzoate hydroxylation assays (Fig. 15B), this chelator was protective of SC DNA at IBE ratios of 1 and 3. NNH was not protective of SC DNA in the presence of Fe (II) and H202, resulting in the formation of OC DNA and also linearised DNA, particularly at IBE's of 1 and 3 (Fig. 16), despite NNH not being redox-active in the ascorbate oxidation and benzoate hydroxylation assays.

The Fe complexes of NT and N4mT at an IBE of 0.1 showed effects on SC DNA that were similar to that produced by the Fe complex of NNH. However, at IBE ratio's of 1 and 3, N4mT and NT, caused plasmid degradation. This latter effect was pronounced for NT at an IBE of 3 (Fig. 16). Triapine, was more aggressive than NT, completely degrading plasmid DNA at IBE ratios of 1 and 3 (Fig. 16).

Crystal Structure Data for NT (Figure 2) Crystal data collection: CAD-4 software (Enraf-Nonius, 1989) ; cell refinement: SET4 in CAD-4 software; data reduction: Xtal (Hall et al. , 1992); programs used to solve structure: SHELX86 (Sheldrick, 1990) ; Program used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphic: PLATON (Spek, 1990; software used to generate crystallographic data: SHELXL 97.

Data collection was obtained under same general conditions as per: D. R. Richardson, E. Becker and P. V. Bernhardt. (1999) The biologically active iron chelators 2-pyridylcarboxaldehyde isonicotinoyl hydrazone, 2-pyridylcarboxaldehyde benzoyl hydrazone and 2-furfural isonicotinoyl hydrazone. Acta Crystallogr C. 15; 55 (ptl2): 2102-5.

Crystal Data and Details of the Structure Determination for: NT (C12 H11 N3 O S) P2 (1)/c Z=4 Crystal Data Empirical Formula C12 H11 N3 O S Formula Weight 245.31 Crystal System Monoclinic Space group P21/c (No. 14) a, b, c [Angstrom] 13.602 (8) 5.2510 (10) 16.247 (9) alpha, beta, gamma [deg] 90 94.84 (3) 90 V [Ang**3] 1156.3 (10) Z 4 D (calc) [g/cm**3] 1.409 F (000) 512 Mu (MoKa) [/mm] 0.3 Final Structure Factor Calculation for NT (C13 H11 N3 02) P2 (1)/c Z=4 Total number of l. s. parameters = 53 Maximum vector length = 511 Memory required = 1016/24017 wR2 = 0.7679 before cycle 21 for 2031 data and 0/53 parameters GooF = S = 4.446 ; Restrained GooF = 4. 446 for 0 restraints Weight = 1/ [sigma2 (Fo2) + (0.1000 * P) ^2 + 0. 00 * P] where P = ( Max ( Fo^2, 0) + 2 * Fc^2 ) / 3 R1 = 0. 4292 for 769 Fo > 4sig (Fo) and 0.5399 for all 2031 data

wR2 = 0.7679, GooF = S = 4. 446, Restrained GooF = 4. 446 for all data Occupancy sum of asymmetric unit = 13. 00 for non-hydrogen and 0.00 for hydrogen atoms Bond lengths and angles S1-Distance Angles C15 1.7372 (0.0378) S1- C2-Distance Angles C10 1.3100 (0.0364) C6 1.3644 (0.0354) 118.21 (2.64) C24 1.4561 (0.1219) 100.39 (4.63) 129.69 (5.45) C2-C10 C6 C3-Distance Angles C24 1.6612 (0.1180) C7 1.3537 (0.0356) 98.23 (3.96) C3-C24 C4-Distance Angles C7 1.2917 (0.0388) C10 1.3369 (0.0386) 124.65 (2.80) C9 1.5885 (0.0361) 116.91 (2.77) 118.43 (2.48) C4-C7 C10 C5-Distance Angles C13 1.3982 (0.0459) C9 1.4154 (0.0456) 123.64 (3.15) C5-C13 C6-Distance Angles C15 1.3557 (0.0378) C2 1.3644 (0.0354) 117.12 (2.62) C6-C15 C7-Distance Angles C12 1.5680 (0.0439) C4 1.2917 (0.0388) 126.36 (2.81) C3 1.3537 (0.0355) 108.76 (2.69) 124.73 (3.08) C7-C12 C4 C9-Distance Angles C5 1.4154 (0.0457) C4 1.5885 (0.0361) 116.20 (2.65) C9-C5 C10-Distance Angles C2 1.3100 (0.0363) C4 1.3369 (0.0385) 122.06 (2.78) C10-C2 C12-Distance Angles C7 1.5680 (0.0439) C13 1.3955 (0.0412) 113.62 (2.83) C12-C7

C13-Distance Angles C5 1.3982 (0.0459) C12 1.3955 (0.0412) 123.07 (3.35) C13-C5 C15-Distance Angles C6 1.3557 (0.0377) si 1.7372 (0.0376) 116.57 (2.54) C15-C6 C24-Distance Angles C3 1.6612 (0.1171) C2 1.4561 (0.1217) 112.98 (7.38) C24-C3 Final Coordinates and Equivalent Isotropic Displacement Parameters of the non-Hydrogen atoms for: NT (C12 Hll N3 0 S) P2 (1)/c Z=4 Atom x y z U (eq) [Ang2] si 0.54229-0. 37370 0.13483 0.0613 O1 0.20108 0.31028 0.00380 0.0579 Nl 0.33474 0.13744 0.11518 0.0434 N2 0.41172-0. 01059 0.14379 0.0431 N3 0.40826-0. 20927 0.01995 0.0624 C1 0.22862 0.48554 0.14167 0.0335 C2 0.17889 0.48374 0.06123 0.0458 C3 0.10624 0.65439 0.03816 0.0516 C4 0.07698 0.83280 0.09199 0.0510 C5 0.12299 0.84360 0.17533 0.0423 C6 0.09120 1.03152 0.22955 0.0525 C7 0.13638 1.04792 0.30727 0.0586 C8 0.21242 0.88018 0.33150 0.0613 C9 0.24300 0.69645 0.28157 0.0506 C10 0.19804 0.67370 0.19759 0.0376 Cil 0.30712 0.30924 0.16352 0.0364 C12 0.44893-0. 19717 0.09828 0.0515 U (eq) = 1/3 of the trace of the orthogonalized U Tensor Crystal Structure for N44mT (Figure 3) Crystal data collection: CAD-4 software (Enraf-Nonius, 1989) ; cell refinement: SET4 in CAD-4 software; data reduction : Xtal (Hall et al. , 1992); programs used to solve structure: SHELX86 (Sheldrick, 1990) ; Program used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphic: PLATON (Spek, 1990; software used to generate crystallographic data: SHELXL 97.

Data collection was obtained under same general conditions as per: D. R. Richardson, E. Becker and P. V. Bernhardt. (1999) The biologically active iron chelators 2-pyridylcarboxaldehyde isonicotinoyl hydrazone, 2-pyridylcarboxaldehyde benzoyl hydrazone and 2-furfural isonicotinoyl hydrazone. Acta Crystallogr C. 15 ; 55 (ptl2): 2102-5.

Crystal Data and Details of the Structure Determination for: N44mT (C16 H21 N3 02 S2) P2 (1)/n Z=4 Empirical Formula C14 H15 N3 0 S, C2 H6 0 S Formula Weight 351.50 Crystal System Monoclinic Space group P21/n (No. 14) a, b, c [Angstrom] 12.018 (3) 7.8802 (7) 18.635 (4) alpha, beta, gamma [deg] 90 95.760 (10) 90 V [Ang**3] 1755.9 (6) Z 4 D (calc) [g/cm**3] 1.330 F (000) 744 Mu (MoKa) [/mm] 0.3 Final Structure Factor Calculation for N44mT (C16 H21 N3 02 S2) P2 (1)/n Z=4 Total number of l. s. parameters = 209 Maximum vector length = 511 Memory required = 2479/24017 wR2 = 0.2572 before cycle 7 for 3072 data and 0/209 parameters GooF = S = 1.037 ; Restrained GooF = 1. 037 for 0 restraints Weight = 1/ sigma2 (Fo^2) + (0.0926 * P) 2 + 4.60 * P] where P = ( Max ( Fo^2, 0) + 2 * Foc"2)/3 R1 = 0. 0764 for 1189 Fo > 4sig (Fo) and 0.2271 for all 3072 data wR2 = 0. 2572, GooF = S = 1. 037, Restrained GooF = 1. 037 for all data Occupancy sum of asymmetric unit = 23. 00 for non-hydrogen and 21.00 for hydrogen atoms Bond lengths and angles S1-Distance Angles C12 1.6872 (0.0069) S1- 01-Distance Angles C2 1.3617 (0.0078) 01- N1-Distance Angles C11 1.2703 (0.0078) N2 1.3724 (0.0074) 118.69 (0.55) N1-C11 N2-Distance Angles C12 1.3609 (0.0080) N1 1.3724 (0.0074) 118.90 (0.55) N2-C12 N3-Distance Angles C12 1.3172 (0.0086) C13 1.4595 (0.0086) 123.10 (0.59) C14 1.4708 (0.0088) 121.48 (0.63) 115.41 (0.63) N3-C12 C13 Cl-Distance Angles C2 1.3966 (0.0090) C11 1.4365 (0.0091) 121.15 (0.62) C10 1.4462 (0.0090) 117.27 (0.66) 121.48 (0.58) C1-C2 C11 C2-Distance Angles O1 1.3617 (0.0077) Ci 1.3966 (0.0090) 121.98 (0.67) C3 1.4067 (0.0099) 116.29 (0.62) 121.72 (0.67) C2-O1 C1 C3-Distance Angles C4 1.3558 (0.0106) C2 1.4067 (0.0098) 120.66 (0.71) C3-C4 C4-Distance Angles C3 1.3558 (0.0106) C5 1.4079 (0.0099) 121.08 (0.75) C4-C3 C5-Distance Angles C4 1.4079 (0.0099) C6 1.4171 (0. 0102) 121.61 (0.74) C10 1.4225 (0.0095) 119. 05 (0.66) 119.33 (0.69) C5-C4 C6 C6-Distance Angles C7 1.3381 (0.0108) C5 1.4171 (0. 0102) 120.83 (0.80) C6-C7 C7-Distance Angles C6 1.3381 (0.0108) C8 1.3671 (0.0107) 120.97 (0.76) C7-C6 C8-Distance Angles C7 1.3671 (0.0107) C9 1.3817 (0.0099) 121.71 (0.74) C8-C7 C9-Distance Angles C8 1.3817 (0.0099) C10 1.4278 (0.0091) 119. 36 (0. 73) C9-C8 C10-Distance Angles C5 1.4225 (0.0095) C9 1.4278 (0.0091) 117.76 (0. 62) C1 1.4462 (0.0090) 120.14 (0.60) 122.08 (0.66) C10-C5 C9 C11-Distance Angles N1 1.2703 (0.0078) C1 1.4365 (0. 0091) 120.31 (0.59) C11-N1 C12-Distance Angles N3 1.3172 (0. 0086) N2 1. 3609 (0.0080) 115.36 (0.60) S1 1.6872 (0.0069) 123.62 (0.52) 121.02 (0.56) C12-N3 N2 C13-Distance Angles N3 1.4595 (0.0086) C13- C14-Distance Angles N3 1.4708 (0.0088) C14- S2-Distance Angles 02 1.4358 (0.0060) C34 1.7655 (0.0090) 110.62 (0.45) C33 1.7679 (0.0083) 108.99 (0.43) 98.49 (0.44) S2-02 C34 02-Distance Angles S2 1.4358 (0.0060) 02- C33-Distance Angles S2 1.7679 (0.0083) C33- C34-Distance Angles S2 1.7655 (0.0090) C34- Final Coordinates and Equivalent Isotropic Displacement Parameters of the non-Hydrogen atoms for: N44mT (C16 H21 N3 02 S2) P2 (1)/n Z=4 Atom x y z U (eq) [Ang2] si 0.18286 0.79160 0.40592 0.0565 O1 0.38976 0.94530 0.28684 0.0535 N1 0.42420 0.83577 0.41618 0.0392 N2 0.38583 0.76325 0.47596 0.0409 N3 0.24623 0.64213 0.53099 0.0487 C1 0.57216 0.93294 0.35370 0.0337 C2 0.50131 0.97996 0.29295 0.0418 C3 0.54172 1.06376 0.23416 0.0534 C4 0.65190 1.10124 0.23469 0.0510 C5 0.72941 1.05225 0.29267 0.0415 C6 0.84539 1.08450 0.29250 0.0559 C7 0.91818 1.03400 0.34740 0.0633 C8 0.88254 0.95319 0.40584 0.0588 C9 0.77063 0.92045 0.41084 0.0461 C10 0.69000 0.96924 0.35301 0.0387 C11 0.52858 0.86071 0.41590 0.0379 C12 0.27489 0.72760 0.47495 0.0384 C13 0.32659 0.58810 0.59053 0.0582 C14 0.12937 0.59376 0.53716 0.0690 S2 0.13413 0.79601 0.12571 0.0818 02 0.08911 0.86022 0.05674 0.0835 C33 0.28108 0.77997 0.12722 0.0775 C34 0.12858 0.95257 0.19303 0.0790 U (eq) = 1/3 of the trace of the orthogonalized U Tensor Crystal Structure Data for N4eT (Figure 4) Crystal data collection: CAD-4 software (Enraf-Nonius, 1989) ; cell refinement: SET4 in CAD-4 software; data reduction : Xtal (Hall et al. , 1992); programs used to solve structure: SHELX86 (Sheldrick, 1990) ; Program used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphic: PLATON (Spek, 1990; software used to generate crystallographic data: SHELXL 97.

Data collection was obtained under same general conditions as per: D. R. Richardson, E. Becker and P. V. Bernhardt. (1999) The biologically active iron chelators 2-pyridylcarboxaldehyde isonicotinoyl hydrazone, 2-pyridylcarboxaldehyde benzoyl hydrazone and 2-furfural isonicotinoyl hydrazone. Acta Crystallogr C. 15 ; 55 (ptl2) : 2102-5.

Crystal Data and Details of the Structure Determination for: N4eT (C14 H15 N3 O S) c2/c (15) Z = 4 Empirical Formula C14 H14 N3 O S Formula Weight 272.35 Crystal System Monoclinic Space group P2/n (No. 13) a, b, c [Angstrom] 26.608 (8) 7.0551 (6) 18.918 (5) alpha, beta, gamma [deg] 90 129.710 (10) 90 V [Ang**3] 2732.0 (12) Z 4 D (calc) [g/cm**3] 1.324 F (000) 572 Mu (MoKa) [/mm] 0.1 Final Structure Factor Calculation for N4eT (C14 H15 N3 0 S) c2/c (15) Z = 4 Total number of l. s. parameters = 173 Maximum vector length = 511 Memory required = 2025/24017 wR2 = 0.1462 before cycle 10 for 2410 data and 0/173 parameters GooF = S = 1.096 ; Restrained GooF = 1. 096 for 0 restraints Weight = 1/ sigma'2 (Fo^2) + (0.1000 * P) 2 + 0. 00 * P] where P = (Max (Fo^2, 0) + 2 * Fc^2 ) / 3 R1 = 0. 0422 for 1847 Fo > 4sig (Fo) and 0.0611 for all 2410 data wR2 = 0. 1462, GooF = S = 1. 096, Restrained GooF = 1. 096 for all data Occupancy sum of asymmetric unit = 19. 00 for non-hydrogen and 15.00 for hydrogen atoms Bond lengths and angles S1-Distance Angles C12 1.6837 (0.0020) S1- N1-Distance Angles C11 1.2831 (0.0023) N2 1.3748 (0.0021) 116.94 (0.16) N1-C11 N2-Distance Angles C12 1.3505 (0.0024) N1 1.3748 (0.0021) 120.49 (0.16) N2-C12 Cl-Distance Angles C2 1.3848 (0.0026) C10 1.4485 (0.0025) 118.46 (0.17) Cil 1.4497 (0.0026) 121.13 (0.16) 120.41 (0.16) C1-C2 C10 01-Distance Angles C2 1.3468 (0.0023) 01- Cll-Distance Angles N1 1.2831 (0.0023) Ci 1.4497 (0.0026) 121.73 (0.17) Cll-N1 N3-Distance Angles C12 1.3246 (0.0026) C13 1.4516 (0.0027) 124.79 (0.17) N3-C12 C12-Distance Angles N3 1.3246 (0.0026) N2 1.3505 (0.0024) 117.11 (0.18) si 1.6837 (0.0020) 123.47 (0.15) 119.42 (0.15) C12-N3 N2 C5-Distance Angles C6 1.4120 (0.0030) C4 1.4123 (0.0033) 121.30 (0.21) C10 1.4235 (0.0028) 119.43 (0.22) 119.28 (0.18) C5-C6 C4 C3-Distance Angles C4 1.3521 (0.0030) C2 1.4021 (0.0028) 120.25 (0.19) C3-C4 C2-Distance Angles O1 1.3468 (0.0023) C1 1.3848 (0.0026) 122.95 (0.17) C3 1.4021 (0.0028) 115.28 (0.17) 121.77 (0.18) C2-O1 C1 C10-Distance Angles C9 1.4009 (0.0029) C5 1.4235 (0.0028) 117.36 (0.18) C1 1.4485 (0.0025) 123.85 (0.18) 118.79 (0.18) C10-C9 C5 C13-Distance Angles N3 1.4516 (0.0027) C14 1.4959 (0.0034) 110.19 (0.19) C13-N3 C4-Distance Angles C3 1.3521 (0.0030) C5 1.4123 (0.0033) 121.41 (0.19) C4-C3 C9-Distance Angles C8 1.3728 (0.0030) C10 1.4009 (0.0029) 121.32 (0.22) C9-C8 C14-Distance Angles C13 1.4959 (0.0034) C14- C6-Distance Angles C7 1.3520 (0.0040) C5 1.4120 (0.0030) 121.17 (0.24) C6-C7 C8-Distance Angles C9 1.3728 (0.0030) C7 1.3847 (0.0039) 120.92 (0.23) C8-C9 C7-Distance Angles C6 1.3520 (0.0040) C8 1.3847 (0.0039) 119.77 (0.21) C7-C6 Final Coordinates and Equivalent Isotropic Displacement Parameters of the non-Hydrogen atoms for: N4eT (C14 H15 N3 O S) c2/c (15) Z = 4 Atom x y z U (eq) [Ang2] si 0.28225 1.05423 0.67351 0.0526 O1 0.00164 0.78653 0.38921 0.0500 N1 0.10766 0.85550 0.55506 0.0362 N2 0.17147 0.91306 0.61986 0.0406 N3 0.18042 0.93221 0.50803 0.0474 C1 0.00825 0.75873 0.52142 0.0321 C2-0.02551 0.74343 0.42800 0.0361 C3-0.09105 0.68494 0.36699 0.0452 C4-0.12240 0.63697 0.39883 0.0475 C5-0.09130 0.64682 0.49318 0.0424 C6-0.12409 0.59526 0.52637 0.0575 C7-0.09445 0.60983 0.61694 0.0672 C8-0.03075 0.67807 0.67849 0.0624 C9 0.00288 0.72716 0.64898 0.0478 C10-0. 02537 0.71184 0.55619 0.0354 C11 0.07610 0.81814 0.58381 0.0342 C12 0.20712 0.96147 0.59473 0.0374 C13 0.20940 0.98810 0.46725 0.0513 C14 0.16564 0.93338 0.36788 0.0630 U (eq) = 1/3 of the trace of the orthogonalized U Tensor

Crystal Structure Data for N4pT (Figure 5) Crystal data collection: CAD-4 software (Enraf-Nonius, 1989) ; cell refinement: SET4 in CAD-4 software; data reduction: Xtal (Hall et al. , 1992); programs used to solve structure: SHELX86 (Sheldrick, 1990) ; Program used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphic: PLATON (Spek, 1990; software used to generate crystallographic data: SHELXL 97.

Data collection was obtained under same general conditions as per: D. R. Richardson, E. Becker and P. V. Bernhardt. (1999) The biologically active iron chelators 2-pyridylcarboxaldehyde isonicotinoyl hydrazone, 2-pyridylcarboxaldehyde benzoyl hydrazone and 2-furfural isonicotinoyl hydrazone. Acta Crystallogr C. 15; 55 (ptl2): 2102-5.

Crystal Data and Details of the Structure Determination for: N4PT (CIS H15 N3 O S) c2/c (15) Z = 4 Empirical Formula C18 H15 N3 0 S Formula Weight 321.40 Crystal System Monoclinic Space group P2/n (No. 13) a, b, c [Angstrom] 19.243 (4) 6.7948 (6) 24.4710 alpha, beta, gamma [deg] 90 95.4800 90 V [Ang**3] 3185.0 (7) Z 4 D (calc) [g/cm**3] 1.340 F (000) 672 Mu (MoKa) [/mm] 0.1 Final Structure Factor Calculation for N4PT (C18 H15 N3 0 S) c2/c (15) Z = 8 Total number of l. s. parameters = 210 Maximum vector length = 511 Memory required = 2267/24017 wR2 = 0.1608 before cycle 12 for 2801 data and 0/210 parameters GooF = S = 0.927 ; Restrained GooF = 0. 927 for 0 restraints Weight = 1/ sigma2 (Fo^2) + (0.1000 * P) A2 + 0. 00 * P] where P = (Max (Fo2, 0) + 2 * Foc 2)/3

R1 = 0. 0405 for 1426 Fo > 4sig (Fo) and 0.1112 for all 2801 data wR2 = 0. 1608, GooF = S = 0. 927, Restrained GooF = 0. 927 for all data Occupancy sum of asymmetric unit = 23. 00 for non-hydrogen and 15.00 for hydrogen atoms Bond lengths and angles S1-Distance Angles C12 1.6684 (0.0027) S1- N2-Distance Angles C12 1.3491 (0.0031) N1 1.3712 (0.0029) 122.39 (0.21) N2-C12 C5-Distance Angles C4 1.4017 (0.0041) C6 1.4174 (0.0037) 121.25 (0.27) C10 1.4266 (0.0036) 119.30 (0.25) 119.44 (0.27) C5-C4 C6 N1-Distance Angles C11 1.2856 (0.0031) N2 1.3712 (0.0029) 116.06 (0.21) N1-C11 C12-Distance Angles N3 1.3312 (0.0033) N2 1.3491 (0.0031) 115.15 (0.23) si 1.6684 (0.0027) 125.94 (0.20) 118.88 (0.20) C12-N3 N2 C13-Distance Angles C14 1.3648 (0.0040) C18 1.3727 (0.0038) 120.01 (0.26) N3 1.4301 (0.0031) 116.89 (0.25) 123.00 (0.26) C13-C14 C18 N3-Distance Angles C12 1.3312 (0.0033) C13 1.4301 (0.0031) 131.09 (0.23) N3-C12 C3-Distance Angles C4 1.3574 (0.0038) C2 1.4034 (0.0038) 119.60 (0.26) C3-C4 01-Distance Angles C2 1.3488 (0.0030) 01- C6-Distance Angles C7 1.3492 (0.0047) C5 1.4174 (0.0037) 121.34 (0.29)

C6-C7 C4-Distance Angles C3 1.3574 (0.0038) C5 1.4017 (0.0041) 121.80 (0.26) C4-C3 Cl-Distance Angles C2 1.3867 (0.0037) C10 1.4380 (0.0033) 118.63 (0.23) Cil 1.4475 (0.0035) 121.20 (0.23) 120.15 (0.23) C1-C2 C10 C10-Distance Angles C9 1.4134 (0.0039) C5 1.4266 (0.0036) 117.26 (0.24) Ci 1.4380 (0.0033) 123.96 (0.25) 118.77 (0.25) C10-C9 C5 C2-Distance Angles O1 1.3488 (0.0030) Ci 1.3867 (0.0037) 122.34 (0.23) C3 1.4034 (0.0038) 115.83 (0.24) 121.83 (0.24) C2-O1 C1 C18-Distance Angles C13 1.3727 (0.0038) C17 1.3938 (0.0038) 118.79 (0.30) C18-C13 C9-Distance Angles C8 1.3693 (0.0037) C10 1.4134 (0.0039) 121.07 (0.28) C9-C8 C11-Distance Angles N1 1.2856 (0.0031) Ci 1.4475 (0.0035) 122.95 (0.24) C11-N1 C8-Distance Angles C9 1.3693 (0.0037) C7 1.3940 (0.0048) 121.26 (0.30) C8-C9 C7-Distance Angles C6 1.3492 (0.0047) C8 1.3940 (0.0048) 119.61 (0.28) C7-C6 C17-Distance Angles C16 1.3525 (0.0046) C18 1.3938 (0.0038) 121.10 (0.32) C17-C16 C15-Distance Angles C16 1.3601 (0.0048) C14 1.3872 (0.0040) 119.95 (0.33) C15-C16

C14-Distance Angles C13 1.3648 (0.0040) C15 1.3872 (0.0040) 120.25 (0.30) C14-C13 C16-Distance Angles C17 1.3525 (0.0046) C15 1.3601 (0.0048) 119.89 (0.31) C16-C17 Table S2-Final Coordinates and Equivalent Isotropic Displacement Parameters of the non-Hydrogen atoms for: N4PT (C18 H15 N3 0 S) c2/c (15) Z = 4 Atom x y z U (eq) [Ang2] S1-0. 01912 0.99109 0.08845 0.0882 O1 0.29920 1.00514 0.08166 0.0692 N1 0.16854 0.97433 0.04141 0.0462 N2 0.09748 0.97127 0.04282 0.0526 N3 0.11104 0.93871 0.13523 0.0546 C1 0.26375 1.00136-0. 01491 0.0418 C2 0.31460 1.01378 0.02909 0.0501 C3 0.38546 1.03841 0.02153 0.0620 C4 0.40523 1.05375-0. 03008 0.0625 C5 0.35699 1.03880-0. 07657 0.0506 C6 0.37854 1.05039-0. 13027 0.0644 C7 0.33253 1.02962-0. 17507 0.0749 C8 0.26217 0.99821-0. 16869 0.0764 C9 0.23861 0.98876-0. 11770 0.0623 C10 0.28493 1.00979-0. 06965 0.0472 C11 0.19050 0.98658-0. 00647 0.0462 C12 0.06722 0.96346 0.09028 0.0500 C13 0.09856 0.92185 0.19172 0.0520 C14 0.14852 0.99607 0.22948 0.0680 C15 0.14142 0.97844 0.28515 0.0851 C16 0.08515 0.88355 0.30226 0.0839

C17 0.03559 0.80979 0.26491 0.0755 C18 0.04152 0.82673 0.20879 0.0660 U (eq) = 1/3 of the trace of the orthogonalized U Tensor

References 1. D. R. Richardson, E. Becker and P. V. Bernhardt, Acta Crystallogr C.

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