WO2010000008A1 - Thiosemicarbazone compounds and use thereof - Google Patents

Abstract

A method of treating a proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of at least one compound of formula (I) (I) wherein R¹ is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; R² is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² are the same or different, or a salt, hydrate, or iron complex thereof, or a pharmaceutical composition comprising said compound, salt, hydrate or iron complex and a pharmaceutically acceptable carrier, diluent or excipient.

Description

Thiosemicarbazone Compounds and Use Thereof

Technical Field

The present invention relates to thiosemicarbazone compounds, more particularly 2-benzoylpyridine thiosemicarbazone compounds. The invention also relates to pharmaceutical compositions comprising these compounds and to their use in the treatment of diseases and disorders, including proliferative diseases and disorders, such as cancer.

Background to the Invention

Rapidly proliferating cancer cells have an increased requirement for Fe, compared with normal cells, as illustrated by the increased expression of transferring receptor 1 (TfR1). This membrane protein is necessary for Fe uptake from the serum Fe-transport protein, transferring (Tf). Therefore, cellular Fe pools and Fe uptake are a potential therapeutic target for inhibiting proliferation of rapidly proliferating cells, such as cancer cells.

The antiproliferative properties of bis(2-pyridyl)thiosemicarbazone and bis(2-pyridyl)hydrazone compounds are described in WO 2004/069801. However, this document does not specifically disclose 2-benzoylpyridine thiosemicarbazone compounds of formula (I) according to the present invention, or their use in treating proliferative diseases. Furthermore, this document does not suggest that 2-benzoylpyridyl thiosemicarbazone compounds according to the present invention would be selective for rapidly proliferating cells relative to cancer cells, show surprisingly high efficacy against cancerous cells, and have reduced toxicity relative to other antiproliferative agents.

There is a need for alternative therapies for treating proliferative diseases, such as cancer (including treatment of solid tumors), and related conditions. There is also a need for compounds that are capable of selectively targeting rapidly proliferating cells relative to normal cells. More particularly, there is a need for therapeutically acceptable compounds that are capable of selectively targeting and inhibiting rapidly proliferating cells in vivo. Summary of the Invention

In one aspect the present invention relates to a method of treating a proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of at least one compound of formula (I):

wherein

R¹ is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl;

R² is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² are the same or different; or a salt, hydrate or iron complex thereof; or a pharmaceutical composition comprising said compound, salt, hydrate or iron complex and a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect the present invention relates to a method of inhibiting proliferation of rapidly proliferating cells, comprising contacting said cells with a therapeutically effective amount of at least one compound of formula (I)

wherein

R¹ is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl;

R² is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² are the same or different; or a salt, hydrate or iron complex thereof; or a pharmaceutical composition comprising said compound, salt, hydrate or iron complex and a pharmaceutically acceptable carrier, diluent or excipient.

In a further aspect the present invention relates to the use of at least one compound of formula (I)

wherein

R¹ is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl;

R² is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² are the same or different; or a salt, hydrate or iron complex thereof; in the manufacture of a medicament for treating a proliferative disease.

In another aspect the present invention relates to the use of at least one compound of formula (I)

wherein

R¹ is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl;

R² is selected from H, Ci_-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² are the same or different; or a salt, hydrate or iron complex thereof; in the manufacture of a medicament for inhibiting cellular proliferation.

Typically, the amount of the compound of formula (I) or salt, hydrate or iron complex thereof, is a therapeutically effective amount.

In a further aspect the invention relates to a compound of formula (I) as defined herein for treating a proliferative disease. In another embodiment the invention relates to a compound of formula (I) as defined herein for inhibiting cellular proliferation.

Brief Description of the Figures

Figure 1. Chemical structures of the iron chelators desferrioxamine (DFO), 2-hydroxy-1- naphthaldehyde isonicotinoyl hydrazone (NIH), pyridoxal isonicotinoyl hydrazone (PIH), 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine®; 3-AP), di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone ("Dp44mT"), and members of the 2 benzoylpyridine thiosemicarbazone (BpT) series of compounds.

Figure 2. A)The effect of the Fe complexes of the BpT series on ascorbate oxidation. Chelators at iron-binding equivalent (IBE) ratios of 0.1 , 1 , and 3 were incubated in the presence of Fe"' (10 μM) and ascorbate (100 μM). The UV-Vis absorbance at 265 nm was recorded after 10 and 40 min and the difference between the time points calculated. Comparison of EDTA with BpT, Bp4mT, Bp44mT, Bp4eT, Bp4aT, Bp4pT. Results are mean ± SD (3 experiments). B) The effect of various Fe chelators on the hydroxylation of benzoate in the presence of Fe'" and hydrogen peroxide. Chelators at iron-binding equivalent (IBE) ratios of 0.1 , 1 , and 3 were incubated for 1 h at room temperature in the presence of Fe'" (30 μM), hydrogen peroxide (5 mM), and benzoate (1 mM). The fluorescence of hydroxylated benzoate was measured at 308 nm excitation and 410 nm emission. Comparison of EDTA with BpT, Bp4mT, Bp44mT, Bp4eT, Bp4aT and Bp4pT. Results are mean ± SD (3 experiments).

Figure 3. The effect of the chelators DFO, NIH, PIH and the BpT compounds on: (A) ⁵⁹Fe mobilization from prelabeled SK-N-MC neuroepithelioma cells and (B) ⁵⁹Fe uptake from ⁵⁹Fe-transferrin (⁵⁹Fe-Tf) by SK-N-MC neuroepithelioma cells. Results are mean + SD of 3 experiments with 3 determinations in each experiment.

Figure 4. A) Bp4eT (0.4 mg/kg) inhibits the growth of DMS-53 tumour xenografts in nude mice when given intravenously once per day, 5 days/week for up to 7 weeks. Each experimental point represents mean from 6-12 mice. B) Weight loss in nude mice when given Bp4eT (0.4 mg/kg) Dp44mT (0.4 mg/kg or 0.75 mg/kg) intravenously once per day, 5 days/week for up to 7 weeks. C) Bp4eT (0.4 mg/kg) is effective at inhibiting the growth of AT6.1 tumour xenografts in nude mice when given intravenously once per day, 5 days/week for 16 days. Each experimental point represents mean from 6-12 mice.

Figure 5. Haematologic indices of mice treated intravenously with vehicle (control), Dp44mT (0.75 mg/kg), or Bp4eT (0.4 mg/kg or 0.75 mg/kg) for 2 weeks once a day/5 days a week and bearing DMS-53 xenografts. Figure 6. Gomori Trichrome stain of cardiac tissue sections from nude mice bearing a DMS-53 lung carcinoma xenograft and treated intravenously with Dp44mT (2 weeks at 0.75 mg/kg) or Bp4eT (7 weeks at 0.4 mg/kg or 0.75 mg/kg) once per day, 5 days/week.

Definitions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

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.

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. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".

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, features, compounds and compositions.

As used herein, the term "C_1-6 alkyl" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight chain or branched chain saturated aliphatic groups having from 1 to 6 carbon atoms. In various embodiments the alkyl group may be C_1-4 alkyl, Ci_-3 alkyl, or C_1-2 alkyl. In other embodiments the alkyl group may be C_2-6 alkyl, C_2-4 alkyl or C_2-3 alkyl. Thus, for example, the term C_1-6 alkyl includes, but is not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1 ,2-dimethylpropyl, 1 ,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyI, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethyIbutyl, 1,2-dimethylbutyl, 1 ,3-dimethylbutyI, 1 ,2,2-trimethylpropyl, 1 ,1 ,2-trimethylpropyl, and the like.

As used herein, the term "C₂-₆ alkenyl" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 6 carbon atoms and at least one double bond any where in the chain. The alkenyl group may be C_2-4 alkenyl. The alkenyl group may be C_2-3 alkenyl. Unless indicated otherwise, the stereochemistry about each double bond may be independently cis or trans, or E or Z as appropriate. Examples of alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl- 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butentyl, 1 ,3-butadienyl, 1-pentenyl, 2-pententyl, 3-pentenyl, 4-pentenyl, 1 ,3-pentadienyl, 2,4-pentadienyl, 1,4-pentadienyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1 ,3-hexadienyl, 1 ,4-hexadienyl, and the like.

The present invention includes within its scope all isomeric forms of the compounds disclosed herein, including all diastereomeric isomers, racemates and enantiomers. Thus, formula (I) should be understood to include, for example, E, Z, cis, trans, (R), (S), (L), (D), (+), and/or (-) forms of the compounds, as appropriate in each case.

In the context of this invention the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.

In the context of this specification, the term "vertebrate" includes humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian. In a preferred embodiment, the vertebrate is a human.

In the context of this specification, the term "treatment", refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

In the context of this specification the term "therapeutically effective amount" includes within its meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

All references cited in this application are specifically incorporated by cross-reference in their entirety.

Abbreviations

DFO - desferrioxamine

EDTA - ethylenediamine tetraacetic acid

MTT - [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium]

Tf - transferrin

Bp4mT - 2-Benzoylpyridyl 4-methyl-3-thiosemicarbazone

Bp44mT - 2-Benzoylpyridyl 4,4-dimethyl-3-thiosemicarbazone

Bp4eT - 2-Benzoylpyridyl 4-ethyl-3-thiosemicarbazone

Bp4aT - 2-Benzoylpyridyl 4-allyl-3-thiosemicarbazone

Bp4pT - 2-Benzoylpyridyl 4-phenyl-3-thiosemicarbazone

BpT - 2-Benzoylpyridyl 3-thiosemicarbazone

Bp4e4mT - 2-Benzoylpyridyl 4-methyl-4-ethyl-3-thiosemicarbazone

DpT - di-2-pyridylketone 3-thiosemicarbazone

Dp44mT - di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone

Dp4aT - di-2-pyridylketone 4-allyl-3-thiosemicarbazone

Dp4pT - di-2-pyridylketone 4-phenyl-3-thiosemicarbazone Detailed Description of Preferred Embodiments of the Invention

The present invention relates to 2-benzoylpyridine thiosemicarbazone compounds and their iron complexes. More particularly, the invention relates to the use of 2-benzoylpyridine thiosemicarbazone compounds and their iron complexes as antiproliferative agents.

Throughout this description, 2-benzoylpyridine thiosemicarbazone compounds of general formula (I) according to the present invention may be referred to as "BpT compounds", "BpT analogues", "BpT ligands" or "BpT chelators". Unless specifically indicated otherwise, these terms should be considered synonymous.

One aspect of the present invention relates to a method of treating a proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of at least one compound of formula (I) as defined herein, or a salt, hydrate, or iron complex thereof, or a pharmaceutical composition comprising said compound, salt, hydrate or iron complex and a pharmaceutically acceptable carrier, diluent or excipient.

A further aspect of the present invention relates to the use of at least one compound of formula (I) as defined herein, or a salt, hydrate or iron complex thereof, in the manufacture of a medicament for treating a proliferative disease in a vertebrate.

In preferred embodiments the vertebrate is a human. In preferred embodiments the proliferative disease is cancer, including a benign or malignant cancer, such as a solid tumour. For example, the cancer may be selected from the group including, but not limited to, carcinogenic tumours, tumours of epithelial origin, such as colo-rectal cancer, breast cancer, lung cancer, head and neck tumours, hepatic cancer, pancreatic cancer, ovarian cancer, gastric cancer, brain tumours, bladder cancer, prostate cancer and urinary/genital tract cancer; mesenchymal tumours, such as sarcoma; and haemopoietic tumours such as B cell lymphoma. For example, the cancer may be a haematological tumour, a solid tumour, a non-solid tumour, (e.g., leukaemia, lymphoma). In preferred embodiments, the cancer is selected from lung cancer, prostate cancer, and brain tumours. Another aspect of the present invention relates to a method of inhibiting proliferation of rapidly proliferating cells, comprising contacting said cells with a therapeutically effective amount of at least one compound of formula (I) as defined herein or a salt, hydrate or iron complex thereof, or a pharmaceutical composition comprising said compound, salt, hydrate or iron complex and a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect the present invention relates to the use of at least one compound of formula (I) as defined herein, or a salt, hydrate or iron complex thereof, in the manufacture of a medicament for inhibiting proliferation of rapidly proliferating cells.

Preferred embodiments relate to inhibition proliferation of rapidly proliferating cells in a vertebrate. In a preferred embodiment the vertebrate is a human. In preferred embodiments, compounds of formula (I) selectively target rapidly proliferating cells relative to normal cells. For example compounds of formula (I) may selectively target rapidly proliferating cells relative to MRC-5 fibroblasts. Thus, for example, compounds of formula (I) as defined herein may be taken up by rapidly proliferating cells at a higher rate than normal cells.

Compounds of formula (I) in accordance the present invention have the following chemical structure:

wherein

R¹ is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; R² is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² may be the same or different.

Also encompassed by the present invention are salts and hydrates of compounds of general formula (I).

In one embodiment of compounds of general formula (I) R¹ and R² are the same. In another embodiment, R¹ and R² are different. In one embodiment, R¹ is selected from H, C_1-4 alkyl, C_2-4 alkenyl and phenyl; and R² is selected from H, Ci_-4 alkyl, C_2-4 alkenyl and phenyl. In another embodiment, R¹ is selected from H, Ci_-3 alkyl and C_2-3 alkenyl; and R² is selected from H, Ci_-3 alkyl and C_2-3 alkenyl.

In one embodiment of compounds of formula (I) according to the present invention, R¹ and R² are both hydrogen. In another embodiment, R¹ and R² are not both hydrogen. In another embodiment R¹ is hydrogen and R² is methyl. In a further embodiment, R¹ is methyl and R² is methyl. In another embodiment R¹ is hydrogen and R² is ethyl. In a further embodiment R¹ is hydrogen and R² is allyl. In another embodiment R¹ is hydrogen and R² is phenyl. In a further embodiment, R¹ is methyl and R² is ethyl. In another embodiment R¹ is ethyl and R² is ethyl.

Another aspect of the invention relates to a subset of compounds of formula (I) having structural formula (Ia):

wherein R¹ is C_1-6 alkyl;

R² is selected from C_2-6 alkyl, C_2-6 alkenyl and phenyl; and R¹ and R² are different; and salts or hydrates thereof.

In various preferred embodiments of compounds of formula (Ia), R¹ is Ci_-4 alkyl; R² is selected from C_2-6 alkyl, C_2-6 alkenyl and phenyl. In other preferred embodiments, R¹ is Ci_-4 alkyl; R² is selected from C_2-4 alkyl, C_2-4 alkenyl and phenyl. In other preferred embodiments R¹ is methyl or ethyl; R² is selected from C_2-6 alkyl and C_2-6 alkenyl. In other preferred embodiments R¹ is methyl or ethyl; R² is selected from C_2-4 alkyl and C_2-4 alkenyl. In one embodiment, R¹ is methyl and R² is ethyl. In another embodiment R¹ is methyl and R² is propyl. In a further embodiment R¹ is methyl and R² is isopropyl. In another embodiment R¹ is methyl and R² is vinyl. In a further embodiment R¹ is methyl and R² is allyl. In another embodiment R¹ is ethyl and R² is propyl. In a further 12

corresponding thiosemicarbazone having a desired substitution pattern. A representative reaction is shown in Scheme 1 below:

Scheme 1

Schiff base condensation

where R¹ and R² are as defined in formula (I).

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. Generally, the reaction is carried out in the presence of a catalytic amount of acid. Suitable acids include, for example, glacial acetic acid. Compounds of formula (I) may be purified using standard techniques, such as recrystallisation from a suitable solvent system, or column chromatography.

Synthesis of Fe complexes

Fe complexes of compounds of formula (I) may be prepared using standard techniques known to those skilled in the art. For example, ferrous (Fe")complexes of compounds of formula (I) may be prepared by dissolving the ligand in a polar solvent, such as an alcohol (e.g., ethanol), followed by addition of a base (e.g., Et₃N) and an Fe(II) salt, e.g., Fe(CIO₄)₂.6H₂O, then heating the mixture at reflux to form the Fe" complex. The complex may be purified by filtering and washing with a suitable solvent, and/or recrystallisation. The Fe" complex may be in the form of a hydrate.

Ferric (Fe"¹) complexes of compounds of formula (I) may be prepared using a similar method which involives dissolving the ligand in a polar solvent, such as an alcohol (e.g., ethanol), then adding a base (e.g., Et₃N) and an Fe(III) salt, e.g., Fe(CIO₄)₃.6H₂O, then heating the mixture at reflux to form the Fe(II) complex. The complex may be purified by filtering and washing with a suitable solvent, and/or recrystallisation. The Fe¹" complex may be in the form of a salt and/or hydrate. 11

embodiment R¹ is ethyl and R² is isopropyl. In another embodiment R¹ is ethyl and R² is vinyl. In a further embodiment R¹ is ethyl and R² is allyl.

Compounds of formula (Ia) are a subset of general formula (I). Therefore, throughout the specification references to formula (I) should be understood to include compounds of formula (Ia).

Compounds of general formula (I) are tridentate Iigands that are able to chelate transition metal ions, including Fe(II) and Fe(III). The pyridyl nitrogen, the imino (C=N) nitrogen, and the thiocarbonyl (C=S) group may each serve as donor atoms to coordinate metal ions such as Fe(III) and Fe(II). Thus, the present invention also includes iron complexes of compounds of formula (I). The iron complex may be an Fe(II) or an Fe(III) complex. Those skilled in the art will appreciate that there may be cis/trans stereoisomerisation about the C=N double bond.

A preferred embodiment of the invention relates to Fe complexes of compounds of formula (Ia). In a particularly preferred embodiment the Fe complex is selected from Fe"[(Bp44mT)₂], Fe"'[(Bp44mT)₂], Fe"[(Bp4eT)₂] and Fe'"[(Bp4eT)₂], or a salt or hydrate thereof.

Compounds of general formula (I) are capable of modifying cellular Fe levels e.g., by releasing or 'mobilizing' intracellular pools of Fe, and inhibiting Fe-uptake from transferring (Tf), which may contribute to the antiproliferative and cytotoxic properties of these compounds. Other properties of compounds of formula (I) and their Fe complexes that may contribute to the antiproliferative and cytotoxic properties include cellular uptake (e.g., the lipophilicity of the compounds which is linked to their ability to cross cell membranes), and redox cycling.

Synthesis of Compounds of formula (I)

Compounds of formula (I) may be prepared using methods well known to those skilled in the art and as described, for example, in J. March, Advanced Organic Chemistry, 4^th Edition (John Wiley & Sons, New York, 1992); and Vogel's Textbook of Practical Organic Chemistry, 5^th Edition (John Wiley & Sons, New York, 1989). For example, compounds of formula (I) may be prepared by a Schiff base condensation reaction in which a ketone or aldehyde is condensed with either an acid thiosemicarbazide to produce a Therapeutic properties

Compounds of formula (I) and/or their Fe complexes are particularly advantageous and show surprising in vitro and in vivo efficacy for rapidly proliferating cells and thus, may be useful for treating proliferative diseases such as cancer, e.g., solid tumours.

Advantageously, compounds of formula (I) and/or their Fe complexes may be selectively taken up by rapidly proliferating cells relative to normal cells. Compounds of general formula (I) and/or their Fe complexes may have up to 100-times, 500-times, 1000-times, 1500-times, 2000-times, 2500-times, 3000-times, or 3500-times greater selectivity for rapidly proliferating cells relative to normal cells. In particularly preferred embodiments compounds of formula (I) are approximately 2000-3500-times more selective for rapidly proliferating cells relative to normal cells. In a preferred embodiment, a compound of formula (I) is approximately 3000-times more selective for rapidly proliferating cells relative to normal cells.

A further particular advantage of compounds of formula (I) and/or their iron complexes is that they show surprising clinical efficacy in vivo. For example, compounds of formula (I) according to the present invention may be less cardiotoxic than other anti-cancer agents.

A further advantage of the Fe complexes of compounds of formula (I) is that they display increased redox activity relative to other anticancer agents.

Formulations

In accordance with the present invention, compound(s) of the invention may be administered alone. Alternatively, the compounds may be administered as a pharmaceutical or veterinarial formulation which comprises at least one compound according to the invention. The compound(s) may also be present as suitable salts, including pharmaceutically acceptable salts, or hydrates.

In accordance with the present invention, the thiosemicarbazone compounds of formula (I), and/or their iron complexes, may be used in combination with other known therapies, including surgery, chemotherapeutics and radiotherapeutics. Examples of chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin), chlorambucil), ethylenimines and methylmelamines (e.g. hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, streptozociπ), triazenes (e.g., dacarbazine (dimethyltriazeno-imidazolecarboxamide), temozolomide), folic acid analogues (e.g., methotrexate), pyrimidine analogues (e.g., 5-fluorouricil, floxuridine, cytarabine, gemcitabine), purine analogues (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin (2'-deoxycoformycin) cladribine, fludarabine), vinca alkaloids (e.g., vinblastine, vincristine), taxanes (e.g., paclitaxel, docetaxel), epipodophyllotoxins (e.g., etoposide, teniposide), camptothecins (topotecan, irinotecan), antiobiotics (e.g., actinomycin D, daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin C), enzymes (e.g., L-asparaginase), interferon-alpha, interleukin-2, cisplatin, carboplatin, mitoxantrone, hydroxyurea, procarbazine, mitotane, aminoglutethimide, imatinib, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate), oestrogens (e.g., diethylstilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen, anastrozole), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogens (e.g., flutamide), and gonadotropin-releasing hormone analogues (e.g., leuprolide), and combination therapeutic regimens such as COMP (cyclophosphamide, vincristine, methotrexate and prednisone), mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), PROMACE/MOPP (prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine).

Combinations of active agents may be synergistic.

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 compounds according to 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.

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, digluconate, cyclopentanepropionate, 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 non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine, and the like.

Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration. Depending on the route of administration, the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound. The compound may also be administered parenteral^ or intraperitoneal^.

Dispersions of the compounds according to the invention may 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 one embodiment of the invention, the compound(s) of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound(s) and other ingredients may 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 compound(s) may 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 compound(s) of formula (I) and/or (II) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may 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 compound 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 compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may 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 compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The compound(s) may be formulated 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 one embodiment, the carrier may be an orally administrable carrier.

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

Also included in the scope of this invention are delayed release formulations.

Compounds of the invention may also be administered in the form of a "prodrug". A prodrug is an inactive form of a compound which is transformed in vivo to the active form. Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.

In one embodiment, the compound may be administered by injection. 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 may 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 diseases and/or infections 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 a conventional course of treatment determination tests. Generally, an effective dosage 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 500mg/m². For example, generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m², about 25 to about 350mg/m², about 25 to about 300mg/m², about 25 to about 250mg/m², about 50 to about 250mg/m², and about 75 to about 150mg/m².

The following examples of the invention represent preferred embodiments and are not intended to be limiting to the overall scope of the invention.

Examples

Example 1 - Preparation of BpT Analogues

General Synthesis of BpT Analogues. 2-Benzoylpyridine (1.934g, 10 mmol) was dissolved in EtOH (15 mL), and a solution of 10 mmol of the appropriate thiosemicarbazide in water (15 mL) was added. Glacial acetic acid (5-6 drops) was added, and the mixture was gently refluxed for 4-5 h. The mixture was allowed to cool to room temperature and kept at 4°C overnight to complete precipitation. The yellow product was filtered off and washed with EtOH and diethyl ether (Reference 1).

2-Benzoylpyridyl 4-methyl-3-thiosemicarbazone ("Bp4mT"): (from 4-methyl thiosemicarbazide): Yield 92%. Anal, calcd for C₁₄H₁₄N₄S: C, 61.2; H, 5.3; N, 20.4; S, 11.7%. Found: C, 61.1 ; H, 5.1; N, 20.5; S, 12.3%. IR (crrϊ¹) 3296m, 1530s, 1468s, 1308m, 1224s, 1107s, 1042m, 998m, 816m, 797s, 767m, 730m, 694s, 644s, 598m. ¹H NMR (DMSO-cfe): δ 8.85 (d, 1H, pyr); 8.71 (d, 1 H, pyr); 8.01 (t, 1 H, pyr); 7.63 (m, 3H, -C₆H₅ or pyr); 7.45 (m, 3H, pyr or -C₆H₅); 7.31 (m, 1H, pyr or -C₆H₅), 3.04 (d, 3H, -CH₃). 2-Benzoylpyridyl 4,4-dimethyl-3-thiosemicarbazone ("Bp44mT"): (from 4,4-dimethyl thiosemicarbazide): Yield 86%. Anal, calcd. For C₁₅H₁₆N₄S: C, 63.4; H, 5.7; N, 19.7; S₁ 11.3%. Found: C₁ 62.8; H, 5.9; N, 19.5; S, 10.6%. IR (crrϊ¹) 2871w, 1571m, 1493m, 1461w, 1424w, 1368m, 1339m, 1305s, 1268w, 1234s, 1159s, 1117s, 1062m, 1002s, 961s, 902s, 788s, 720s, 698s, 656w, 645w. ¹H-NMR (DMSO-Cf₆): δ 8.70 (dd, 1 H), 8.45 (dd, 1H), 8.12-7.84 (dd, 2H), 7.66-7.44 (dd, 4H), 7.33 (dt, 1H), 3.35 (s, 3H), 3.25 (s, 3H).

2-Benzoylpyridyl 4-ethyl-3-thiosemicarbazone ("Bp4eT"): (from 4-ethyl thiosemicarbazide): Yield 91%. Anal, calcd. for C₁₅H₁₆N₄S: C, 63.4; H, 5.7; N, 19.7; S, 11.3%. Found: C, 62.9; H, 5.5; N, 19.5; S, 11.2%. IR (crrf¹) 329Ow, 1583m, 1529s, 1434s, 1416m, 1371w, 1304s, 1251m, 1208s, 1152w, 1089s, 1050s, 997w, 935m, 923m, 89Ow, 813s, 793s, 762w, 728s, 699s, 644s, 601 w. ¹H-NMR (DMSOd₆): δ 8.75 (t, 1 H), 8.03 (td, 1H), 7.63-7.56 (td, 1 H), 7.47-7.42 (m, 3H), 7.34 (dt, 1 H), 3.65 (m, 2H), 1.16 (t, 3H).

2-Benzoylpyridyl 4-allyl-3-thiosemicarbazone ("Bp4aT"): (from 4-allyl thiosemicarbazide): Yield 80%. Anal, calcd for C₁₆H₁₆N₄S: C, 64.8; H, 5.4; N, 18.9; S, 10.8%. Found: C, 64.7; H, 5.5; N, 19.5; S, 10.6%. IR: (crrϊ¹) 337Ow, 1642w, 1582w, 1516s, 1468w, 1442w, 1422w, 130Ow, 1247m, 1205m, 1179m, 115Ow, 1114m, 1072w, 1047w, 991w, 962w, 921s, 836m, 796s, 770s, 734m, 699s, 664m, 619s, 567m. ¹H-NMR (DMSO-c/₆): δ 8.03 (d, 1H), 8.00 (d, 4H), 7.98 (d, 2H), 7.64-7.55 (m, 4H), 7.47-7.44 (d, 4H), 7.35 (q, 1H), 5.94 (m, 1 H), 5.1 (dd, 1 H), 4.23 (t, 2H).

2-Benzoylpyridyl 4-phenyl-3-thiosemicarbazone ("Bp4pT"): (from 4-phenyl thiosemicarbazide): Yield 84%. Anal, calcd for C₁₉H₁₆N₄S C, 68.7; H, 4.9; N, 16.9; S, 9.6%. Found: C, 68.0; H, 4.8; N, 16.8; S, 9.6%. IR (ατT¹) 3299w, 3051w, 1592s, 1528s, 1466w, 1438m, 1366w, 1302m, 1252w, 1202w, 1171m, 1104s, 1027w, 998w, 92Ow, 835w, 785m, 755s, 730m, 691s, 634s, 591w. ¹H-NMR (DMSO-tf₆): δ 10.67(s, 1 H), 10.30 (s, 1H), 8.88 (d, 3H), 7.72-7.71 (td, 3H), 7.61-7.55 (dq, 4H), 7.54-7.40 (td, 2H), 7.23 (tt, 2H).

2-Benzoylpyridyl 3-thiosemicarbazone ("BpT"): (from thiosemicarbazide): Yield 90%. Anal, calcd. for C₁₃H₁₂N₄S: C, 60.9; H, 4.7; N, 21.9; S, 12.51%. Found: C, 60.3; H, 4.6; N, 21.7; S, 12.2%. IR: (cm^"1) 322Ow, 1590s, 1453m, 1417s, 1326m, 1273w, 1253w, 1103w, 1088W, 1072w, 1048m, 1024w, 998w, 971w, 943w, 921w, 844s, 797s, 769s, 703s, 652s, 630m, 605m. ¹H NMR (DMSO-c/₆): δ 8.79 (dq, 1H), 8.85 (dq, 1 H), 8.59 (s, 1 H), 8.05 (dd, 1 H), 7.97-7.54 (m, 3H), 7.45-7.33 (dt, 3H).

2-Benzoylpyridyl 4-ethyl-4-methyl-3-thiosemicarbazone ("Bp4e4mT"): (from 4-ethyl- 4-methyl thiosemicarbazide): Yield 71%. Anal, calcd for C₁₆H₁₈N₄S: C, 64.40; H, 6.08; N, 18.74. Found: C, 64.52; H, 6.37; N, 18.64.

Example 2 - Preparation of Fe"(BpT)? Complexes

General Synthesis of Fe"(BpT)₂ Complexes. Ferrous complexes of BpT compounds were prepared by the following general method. The appropriate 2-benzoylpyridine thiosemicarbazone (3.5 mmol) was dissolved in EtOH (15 mL), and Et₃N (0.35 g, 3.4 mmol) was added followed by Fe(CIO₄)₂.6H₂O (0.64 g, 1.7 mmol). The mixture was gently refluxed for 30 min. The reaction was done under nitrogen. When the mixture was cooled, the green product was filtered off and washed with ethanol and then diethyl ether.

[Fe(Bp4mT)₂].0.5H₂O: Yield 80%. Anal, calcd. for C₂₈H₂₇FeN₈O_{0 5}S₂: C, 55.7; H, 4.5; N, 18.6; S, 10.6%. Found: C, 56.0; H, 4.3; N, 18.9; S, 10.8%. IR (cm^"1) 3365w, 1589w, 1502s, 1476w, 1425s, 1391s, 1339s, 1232m, 1199m, 1152s, 1014m, 962w, 815w, 772s, 743s, 699s, 658m. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cm^"1) 640 (13600), 382 (40800).

Fe(Bp44mT)₂: Yield 76%. Anal, calcd. for C₃₀H₃₀FeN₈S₂: C, 57.9; H, 4.9; N, 18.0; S, 10.3%. Found: C, 57.1; H, 4.9; N, 18.0; S₁ 10.3%. IR (crrf¹) 2917w, 1586w, 1515s, 1446w, 1421w, 1386s, 1297s, 1234s, 1140s, 1012m, 963w, 920s, 817w, 770s, 741s, 695s, 632s. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cm^"1) 644 (7900), 395 (25900).

Fe(Bp4eT)₂: Yield 67%. Anal, calcd. for C₃₀H₃₀FeN₈S₂: C, 57.9; H, 4.9; N, 18.0; S, 10.3. Found: C, 57.5; H, 4.8; N, 18.0; S, 9.7%. IR (cm^"1) 3023w, 159Ow, 1527s, 1503w, 1449w, 1423m, 1380s, 1333w, 1283w, 1254s, 1198w, 115Ow, 1113w, 1087s, 1047m, 1014m, 963w, 92Ow, 874w, 826w, 771s, 741s, 691s, 645s. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cm^"1) 641 (12700), 382 (37600). [Fe(Bp4aT)J.3H₂O: Yield 77%. Anal, calcd. for C₃₂H₃₆FeN₈O₃S₂: C, 54.8; H, 5.2; N, 16.0; S, 9.1%. Found: C₁ 54.5; H, 4.4; N, 16.0, S, 9.1%. IR (cnT¹) 3201 w, 1642w, 1593w, 1538w, 1503s, 1407s, 1346w, 1327m, 1307w, 1255w, 1227m, 1189m, 1145w, 1080s, 1017w, 998w, 952w, 909w, 847w, 775s, 747s, 697s, 648w, 619s. Electronic spectrum (MeOH): λ_max (nm) (ε L moP¹ cm^"1) 642 (7700), 378 (40800).

[Fe(Bp4pT)₂].H₂O: Yield 68%. Anal, calcd. for C₃₈H₃₂FeN₈OS₂: C, 62.0; H, 4.4; N, 15.2; S, 8.7. Found: C, 61.9; H, 4.2; N, 15.1; S, 7.9. IR (crrT¹) 3051w, 1592m, 1520m, 1492w, 1470m, 1401s, 1335w, 1314w, 1245m, 1205w, 1176m, 1149w, 1112w, 1096w, 1071w, 1014w, 962w, 897w, 772m, 741s, 687s, 615m. Electronic spectrum (MeOH): λ_max (nm) (ε L mo!^"1 cm^"1) 656 (8630), 391 (21800).

Fe(BpT)₂: Yield 56%. Anal, calcd. for C₂₆H₂₂FeN₈S₂: C, 55.1 ; H, 3.9; N, 19.8; S, 11.3%. Found: C, 54.8; H, 3.9; N, 19.6; S, 11.4%. IR (cm^"1) 3109w, 1618s, 1580s, 1509w, 146Ow, 1407vs, 1335w, 1304s, 1199s, 115Ow, 1123s, 107Ow, 1054w, 1011 m, 945s, 877w, 815w, 771m, 742s, 694s, 663m. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cm-¹) 641 (7300), 367 (20600).

Example 3 - Preparation of Fe¹¹VBpT)I⁺ Complexes

General Synthesis of [Fe'"(BpT)]⁺ Complexes. Ferric complexes of BpT compounds were prepared by the following general method. The appropriate thiosemicarbazone (3.5 mmol) was dissolved in 15 mL of ethanol, and 0.36 g of Et₃N was added to the solution. An amount of 0.81 g (1.7 mmol) of Fe(CIO₄)₃.6H₂O was added, and the mixture was gently refluxed for 30 min. When the mixture was cooled, the dark-brown powder was filtered off and washed with ethanol and then diethyl ether.

[Fe(Bp4mT)₂]CIO₄.H₂O: Yield 91%. Anal, calcd. for C₂₈H₂₈CIFeN₈O₅S₂: C, 47.2; H, 4.0; N, 15.7; S, 9.0%. Found: C, 47.2; H, 3.7; N, 15.7; S, 9.1%. IR (cm^'1) 3364w, 1585w, 1552s, 1437s, 1395s, 1343m, 1163m, 1077vs, 783m, 749m, 700s, 620s. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cnrT¹) 476 (15700), 379 (37300).

[Fe(Bp44mT)₂]CIO₄.0.5H₂O: Yield: 88%. Anal, calcd. for C₃₀H₃₁CIFeN₈O₄₅S₂: C, 49.3; H, 4.3; N, 15.3; S, 8.8%. Found: C, 48.9; H, 4.0; N, 15.2; S, 8.7%. IR (cm^"1) 2919w, 1594w, 1542m, 1510m, 1458m, 1440m, 1395s, 1304s, 1249s, 1152w, 1077vs, 967w, 910s, 779m, 746m, 726m, 696s, 665w, 635w, 619s. Electronic spectrum (MeOH): λ_max (nm) (ε L mor¹ cm^"1) 482 (8300), 395 (21200).

[Fe(Bp4eT)₂]CIO₄.0.5 H₂O: Yield 76%. Anal, calcd. for C₃₀H₃₁CIFeN₈O^₅S₂: C, 49.3; H, 4.3; N, 15.3; S, 8.8%. Found: C, 49.3; H, 4.5; N, 14.7; S, 8.2%. IR (cm^"1) 2976w, 1595w, 1537s, 1505s, 1421s, 1335s, 1297w, 1261w, 1239w, 1194m, 1151m, 1072vs, 998w, 966w, 781s, 747s, 697s, 672w, 657w, 619s. Electronic spectrum (MeOH): λ_max (nm) (ε L mor¹ cm^'1) 483 (11600), 381 (32600).

[Fe(Bp4aT)₂]CIO₄.2H₂O: Yield 89%. Anal, calcd. for C₃₂H₃₄CIFeN₈O₆S₂: C, 49.1; H, 4.4; N, 14.3; S, 8.2%. Found: C, 48.6; H, 4.1; N, 14.6; S, 7.9%. IR (cm^"1) 3306W, 1595w, 1537s, 1504s, 1414s, 1327m, 1251 m, 1193w, 1077s, 997w, 921 w, 781m, 747s, 696s, 646w, 618s. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cm^"1) 481 (10200), 382 (26700).

[Fe(Bp4pT)₂]CIO₄: Yield 90%. Anal, calcd. for C₃₈H₃₀CIFe₈O₄S₂: C, 55.8; H, 3.7; N, 13.7; S, 7.8%. Found: C, 55.2; H, 3.6%; N, 13.9%; S, 7.0%. IR (cm^"1) 3275w, 1594m, 152Ow, 1493w, 1472w, 1403s, 1336w, 1315w, 1249m, 1204w, 1177m, 1151w, 1096m, 1071s, 1016w, 964w, 898w, 876w, 843w, 823w, 773w, 743s, 688s, 659w, 618s. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cm^"1) 456 (13300), 402 (24900).

[Fe(BpT)₂]CIO₄: Yield 87%. Anal, calcd. for C₂₆H₂₂CIFe₈O₄S₂: C, 46.9; H, 3.3; N, 16.8; S, 9.6% Found: C, 46.5; H, 3.5; N, 16.9; S, 9.1%. IR (cm^"1) 3154w, 1613m, 1496m, 1426vs, 1322s, 1205s, 1075s, 953w, 780m, 746m, 722w, 697s, 657w, 621s. Electronic spectrum (MeOH): λ_max (nm) (ε L mol^"1 cm^"1) 474 (11200), 371 (23800).

Example 4 -Redox Properties of the BpT Analogues

Example 4.1 - Cyclic voltammetry

Methodology: Cyclic voltammetry was performed with a BAS100B/W potentiostat. A glassy carbon working electrode, an aqueous Ag/AgCI reference and Pt wire auxiliary electrode were used. All complexes were at ca. 1 mM concentration in DMF:H₂O 70:30 v/v. The supporting electrolyte was Et₄NCIO₄ (0.1 M) and the solutions were purged with nitrogen prior to measurement. All potentials are cited versus the normal hydrogen electrode (NHE) by addition of 196 mV to the potentials measured relative to the Ag/AgCI reference. The electrochemical properties of the Fe complexes of the BpT analogues may be significant to their anti-proliferative activity as anti-proliferative efficacy is linked with the capability of the chelator to undergo Fenton chemistry upon complexation with intracellular Fe. (References 2-4). In all cases, totally reversible Fe""" couples were identified at sweep rates between 50 and 500 mV s^"1. The redox potentials of the Fe complexes are presented in Table 1. The Fe^πι/" complexes of Bp4pT with a -NHPh terminal group showed by far the highest redox potential, while the remaining complexes cluster around a similar potential.

Table 1. Partition coefficients, Fe""" redox potentials, and ICsn (uM) values of the BpT compounds and their ferrous and ferric complexes at inhibiting the growth of SK-N-MC neuroepithelioma cells as determined by the MTT assay. Cells were seeded and allowed to attach to wells for 24 h and then incubated for 72 h at 37⁰C with control medium or the chelators. Results are mean + SD (3 experiments). The p values were determined using Student's ttest and compare the activity of the ligand to its complex. A value of p < 0.05 was considered significant. Comparable data for reference compounds DFO, NIH and 3-AP are also shown.

Bp4pT 1.92 +180 0.005 ± 0.002 0.21 ± 0.05 0.17 ± 0.01 P < 0.005

Example 4.2 - Ascorbate oxidation

Methodology: Ascorbate oxidation

Ascorbic acid (0.1 mM) was prepared immediately prior to an experiment and incubated in the presence of Fe'" (10 μM; added as FeCI₃), a 50-fold molar excess of citrate (500 μM) and the chelator (1-60 μM). The excess of citrate was used to prevent hydrolytic polymerization of Fe'". Absorbance at 265 nm was measured after 10 and 40 min at room temperature and the decrease between these time points calculated. The reaction was started by the addition of ascorbic acid. The absorbance of the chelator at 265 nm was accounted for by the addition of the compound in the blank. The Fe¹¹¹ stock solution was prepared in HCI (0.1 M) to prevent hydrolytic polymerization, and then immediately added to the chelators. Solutions were prepared using 18 MΩ water. In all experiments the results were expressed as a percentage of the control, i.e., a solution containing Fe'" (10 μM), citrate (500 μM) and ascorbic acid (0.1 mM), but no chelator.

In the ascorbate oxidation and benzoate hydroxylation experiments it was appropriate to express data as iron binding equivalents (IBEs). This was due to the different coordination modes of the various ligands to Fe, i.e., EDTA is hexadentate and forms 1 :1 ligand:Fe complexes, while the BpT chelators are tridentate resulting in 2:1 complexes. Thus, for a direct comparison of the hexadentate and tridentate ligands it was necessary to add twice as much tridentate as hexadentate chelator. In the present study, a range of ligand:Fe IBE ratios 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 (e.g., EDTA), an IBE ratio of 1 represents 1 ligand to 1 Fe atom, while for a tridentate chelator (e.g., Bp4mT) 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 or 3 bidentate chelators in the presence of 10 Fe atoms. An IBE of 3 represents an excess of chelator to Fe, and is equal to either 3 hexadentate or 6 tridentate ligands in the presence of 1 Fe atom.

The electrochemical data for the BpT compounds shown in Table 1 illustrate the facile interconversion between the ferric and ferrous states at potentials accessible to biological oxidants and reductants. Thus, the ability of the BpT compounds to catalyse the oxidation of a physiological substrate was important to determine if redox activity played a role in anti-proliferative activity. Accordingly, the oxidation of ascorbate mediated by the Fe complexes of the BpT compounds was examined. The positive control, EDTA, increased ascorbate oxidation to 357% and 382% of the control at an IBE of 1 and 3, respectively, while showing little activity at an IBE of 0.1 (Figure 2A). This is in agreement with previous studies demonstrating the ability of the EDTA/Fe complex to undergo facile redox cycling and catalyse ascorbate oxidation. (References 3-6). Some variation in the ability of the BpT compounds to catalyse Fe'"-mediated ascorbate oxidation was evident (Figure 2A). With the exception of Bp4pT, the BpT compounds markedly increased ascorbate oxidation to 249-454% and 287-469% of the control at an IBE of 1 and 3, respectively. Bp4pT mediated only 121% and 186% ascorbate oxidation of the control at an IBE of 1 and 3 (Figure 2A). When compared to their analogous di(2-pyridyl) thiosemicarbazone ("DpT") chelators at IBEs of 1 and 3, the BpT ligands were generally found to be significantly (p < 0.05) more effective at oxidizing ascorbate. The compounds Bp4aT and Bp4pT were comparable to Dp4aT and Dp4pT respectively (WO 2004/069801). While the compounds BpT, Bp4mT, and Bp4aT showed ascorbate oxidation activity comparable to that of EDTA, Bp4eT exhibited significantly (p < 0.01) higher ascorbate oxidative activity than EDTA at an IBE of 1 and 3. These data may explain the increased anti-proliferative activity of Bp4eT (IC₅₀ = 0.002 μM; Table 1), which showed the greatest anti-tumour effects of the BpT series of compounds. This result was also in good agreement with electrochemistry studies, which demonstrated that the Bp4eT Fe complex (+99 mV, Table 1) exhibited the lowest redox potential of the BpT series, enabling facile interconversion between the ferric and ferrous states to generate reactive oxygen species ("ROS"). Bp4pT, which possesses a 4-phenyl moiety, demonstrated the least ascorbate oxidation activity (Figure 2A). This could be related to the fact this compound exhibited the highest redox potential (+180 mV, Table 1) that would retard redox cycling between the Fe" and Fe'" states and prevent ascorbate oxidation. Plots analyzing the relationship between ascorbate oxidation activity and redox potential of the BpT series showed a linear relationship at an IBE of 1 (r = -0.90) and 3 (r = -0.89). This suggested that the oxidative half reaction is rate-limiting in this process.

In summary, the results of this ascorbate oxidation study indicates that the increased anti-proliferative activity of the BpT compounds is due, at least in part, to their profound redox activity that can induce oxidative damage. Example 4.3 - Benzoate hydroxylation Methodology: Benzoate hydroxylation

Briefly, this procedure is based on the ability of hydroxyl radicals to hydroxylate benzoate to give fluorescent products (308 nm excitation and 410 nm emission). 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 Fe chelator (3-180 μM), and ferric chloride (30 μM). All solutions were prepared immediately prior to use and the Fe'" added to water that had been extensively degassed with nitrogen. The addition of Fe was used to start the reaction and the solution was kept in the dark prior to measuring the fluorescence using a Perkin-Elmer L550B fluorometer (Perkin-Elmer, USA). In these experiments, salicylate was implemented as a standard and was also used to determine quenching by the chelators. In all experiments the results are expressed as a percentage of the control, i.e., a solution containing benzoic acid (1 mM), sodium phosphate (10 mM; pH 7.4), hydrogen peroxide (5 mM), and ferric chloride (30 μM) but no chelator.

As a further assessment of redox activity of the Fe complexes, it was of interest to examine benzoate hydroxylation in the presence of Fe'" and BpT ligands (Figure 2B). These experiments gauge the ability of an Fe complex to catalyse the breakdown of hydrogen peroxide to generate hydroxyl radicals via so-called Fenton chemistry leading to benzoate hydroxylation. (References 5 and 6). The Fe¹" complex of EDTA (the positive control) increased benzoate hydroxylation to 218% and 281% at IBEs of 1 and 3, respectively (Figure 2B). Consistent with the ascorbate oxidation results, all the BpT analogues except Bp4pT significantly (p <0.05) increased benzoate hydroxylation, especially at an IBE of 3. The greatest hydroxylation of benzoate was observed with BpT, while Bp44mT, Bp4aT, Bp4eT, and Bp4mT had intermediate ability, increasing benzoate hydroxylation to 151%, 159%, 170%, and 197% of the control, respectively, at an IBE of 3 (Figure 2B). Bp4pT showed the least benzoate hydroxylation activity, being similar to the control at an IBE of 3 (Figure 2B).

Example 5 - Partition Coefficients of the Free Ligands

The octanol/water partition coefficients for the BpT analogues (Table 1) were determined using methods reported previously (Reference 1). This was important, as lipophilicity of chelators is known to be a relevant factor in terms of their anti-proliferative activity. The log P values varied from 0.74 (Bp4mT) to 4.01 (Bp4eT). Edward et a/, reported that the PIH class of chelators have maximum activity at mobilizing Fe from cells when they have values of log P of 2.8. (Reference 7). Many of the BpT ligands examined have log P values within this "optimal" range, which may in part explain their high Fe chelation efficacy and anti-proliferative activity.

Example 6 - Antiproliferative Activity of BpT Compounds

Methodology:

Presentation of Compounds

All compounds were dissolved in N,N-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).

Cell Culture Preparation

The human SK-N-MC neuroepithelioma cell line, MIAPaCa-2, PANC-1 and AsPC-1 prostate cancer cells were purchased from the American Type Culture Collection (ATCC), Rockville, MD, USA. The normal cell type MRC-5 fibroblasts were also obtained from the American Type Culture Collection (ATCC), Rockville, MD, USA. The cells were grown in Eagle's modified minimum essential medium (MEM; Gibco BRL, Sydney, Australia) containing 10% FCS (Commonwealth Serum Laboratories, Melbourne, Australia), 1% (v/v) non-essential amino acids (Gibco), 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO, USA), 100 μg/ml of streptomycin (Gibco), 100 U/ml penicillin (Gibco), and 0.28 μg/ml of fungizone (Squibb Pharmaceuticals, Montreal, Canada). This growth medium is subsequently referred to as "complete medium". Cells were grown in an incubator (Forma Scientific, OH, USA) at 37⁰C in a humidified atmosphere of 5% CO₂/95% air and subcultured. Cellular growth and viability were carefully assessed using phase-contrast microscopy and trypan blue staining.

MTT cellular proliferation assay

Cellular proliferation was examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium) assay. Cells were seeded in 96-well microtitre plates at 15,000 cells/well in 0.1 mL of MEM medium containing all supplements as described above (complete medium) and also 1.25 μM human diferric Tf. This seeding density resulted in exponential growth of the cells for the duration of the assay. The cells were grown overnight and the compounds to be tested were then added in 0.1 mL of complete medium containing 1.25 μM diferric transferrin. The final concentration of the compounds was 0.39-50 μM. Control samples contained complete medium and 1.25 μM diferric Tf. Cells were incubated with the compounds for 90 h at 37⁰C in a humidified atmosphere containing 95% air and 5% CO₂. After this incubation, 0.01 mL of MTT was added to each well and the plates incubated for 2 h at 37⁰C. The cells were then solubilised by adding 0.1 mL of 10% SDS-50% isobutanol in 0.01 M HCI and the plates then read at 570 nm on a scanning multiwell spectrophotometer (Titertek Multiscan; Beckman Instruments Inc, California). MTT colour formation was directly proportional to the number of viable cells. The results of the MTT assays are expressed as a percentage of the control value.

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 2-5 separate experiments.

Example 6.1 - Anti-proliferative Activity of BpT Ligands against Tumour Cells The ability of the BpT compounds to inhibit cellular proliferation was assessed using SK-N-MC neuroepithelioma cells. The BpT compounds were compared to a number of relevant positive controls, including DFO, which is used for the treatment of Fe overload, the ligand 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (NIH), and the clinically trialed chelator 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine^®; 3-AP) that has been designed for cancer therapy (Figure 1). These results are presented in Table 1.

This study demonstrated the BpT analogues have high anti-proliferative activity (Table 1). In particular, the chelators Bp4mT, Bp44mT, Bp4eT, Bp4aT, and Bp4pT demonstrated particularly potent anti-proliferative effects (IC₅₀ = 0.002-0.005 μM; Table 1). Thus, the BpT compounds are a surprisingly effective series of chelators having anti-proliferative activity. All members of the BpT compounds tested, excluding BpT, demonstrated significantly (p < 0.05) greater efficacy than the controls DFO (IC₅₀ = 4.51 μM; Table 1), NIH (IC₅₀= 0.71 μM; Table 1), and Triapine^® (IC₅₀= 0.26 μM; Table 1). In addition, the majority of the BpT compounds demonstrated significantly (p<0.05) greater anti-proliferative effects than their corresponding DpT compounds (WO 2004/069801). The more hydrophilic chelator BpT (IC₅₀ = 4.66 μM; Table 1) demonstrated the lowest anti-proliferative activity of the series.

Additionally, the compound Bp4eT was shown to have marked activity against the human prostate cancer cell lines MIAPaCa-2, PANC-1 and AsPC-1 with IC₅₀ values < 0.002 μM; Table 2). Significantly, the activity of Bp4eT was far greater than that observed for gemcitabine and 5-flurouracil, current treatments of prostate cancer, with IC₅₀ values for these agents ranging from 0.012-76 μM and 24.3-112 μM respectively.

Table 2. ICgn (uM) values of Bp4eT compared to other chemotherapeutic agents against various prostate cancer cell lines as determined by the MTT assay. Cells were seeded and allowed to attach to wells for 24 h and then incubated for 72 h at 37⁰C with control medium or the chelators. Results are mean + SD (3 experiments).

MIAPaCa-2 PANC-1 AsPC-1

DFO 38.7 9.46 10.9

311 0.86 0.39 0.043 gemcitabine 0.012 10.99 76.2

5-flurouracil 24.27 96.8 112

Dp44mT 0.003 0.002 0.0001

Dp4eT 0.003 0.0007 0.0003

Example 6.2 - Anti-proliferative Activity of Fe Complexes of BpT

To determine the effect of complexation on the anti-proliferative behavior of the BpT compounds, their Fe" and Fe'" complexes were synthesized and their anti-proliferative activity was examined using SK-N-MC neuroepithelioma cells. In comparison with their free ligands, the Fe" and Fe'" complexes of all members of the BpT series, excluding BpT, demonstrated significantly (p <0.005) decreased anti-proliferative activity (Table 1). In fact, complexation resulted in a 2- to 200-fold increase of the IC₅₀ value. Both the Fe" and Fe'" complexes of BpT resulted in significantly (p < 0.05) enhanced anti-tumour activity (Table 1) when compared to the free ligand, decreasing the IC₅₀ value by a factor of 4.

Without intending to be limited to any particular mechanism of action, it is possible that the formation of the Fe complex may result in a more lipophilic species because of the inaccessibility of the donor atoms to the solvent, and is therefore better able to penetrate cellular membranes in comparison to the free ligand. Therefore, pre-complexation may lead to higher concentrations of the redox-active Fe complexes within cells, resulting in greater anti-proliferative effects. Also, lipophilic metal complexes may act as transport vehicles, dissociating after entry into the cell and as a consequence, delivering the toxic metal ion to the cell, acting as lipid soluble delivery shuttles. Considering that BpT was found to be one of the most hydrophilic members of the BpT series, its Fe complex may act as a lipophilic shuttle, allowing intracellular access of the ligand and metal, mediating their cytotoxic effects simultaneously.

Example 6.3 - Anti-proliferative Activity against Normal Cells

Selectivity for neoplastic cells relative to normal cells is important for anti-cancer therapies. Therefore, for a BpT compound to be an effective anti-tumour agent in vivo, it must exhibit potent anti-proliferative activity against neoplastic cells while leaving normal cells unaffected. A selection of the most potent cytotoxic analogues, namely, Bp4mT, Bp44mT, Bp4eT, Bp4aT and Bp4pT were examined for their effect on the proliferation of mortal MRC-5 fibroblasts (Table 3).

It is immediately apparent that when SK-N-MC neuroepithelioma cells and MRC-5 fibroblasts were compared, there was a surprising and significant (p < 0.001) difference (more than 3 orders of magnitude) in IC₅₀ values of all tested analogues except Bp4pT. This indicates a surprising therapeutic index in targeting cancer cells over normal cells.

Table 3. ICsn (uM) values of Bp4mT, Bp44mT, Bp4eT, Bp4aT and Bp4pT at inhibiting the growth of mortal MRC-5 fibroblasts in comparison to SK-N-MC neuroepithelioma cells as determined by the MTT assay. Cells were seeded and allowed to attach to wells for 24 h and then incubated for 72 h at 37⁰C with control medium or the BpT chelators. Results are mean + SD (3 experiments). The p values were determined using Student's f-test and compare the activity of the ligand in normal and neoplastic cells. A value of p < 0.05 was considered as significant.

Example 7 - Cellular Fe Efflux and Inhibition of Fe uptake from Transferrin by BpT Chelators

Presentation of Compounds

All compounds were dissolved in N,N-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).

Ce// Culture Preparation

The human SK-N-MC neuroepithelioma cell line was purchased from the American Type Culture Collection (ATCC), Rockville, MD, USA. The cells were grown in Eagle's modified minimum essential medium (MEM; Gibco BRL, Sydney, Australia) containing 10% FCS (Commonwealth Serum Laboratories, Melbourne, Australia), 1 % (v/v) nonessential amino acids (Gibco), 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO, USA), 100 μg/ml of streptomycin (Gibco), 100 U/ml penicillin (Gibco), and 0.28 μg/ml of fungizone (Squibb Pharmaceuticals, Montreal, Canada). This growth medium is subsequently referred to as "complete medium". Cells were grown in an incubator (Forma Scientific, OH, USA) at 37⁰C in a humidified atmosphere of 5% CO₂/95% air and subcultured. Cellular growth and viability were carefully assessed using phase-contrast microscopy and trypan blue staining. MTT cellular proliferation assay

Cellular proliferation was examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium) assay by the same method described in Example 6.

Example 7.1 - Cellular Fe Efflux Mediated by BpT Chelators

In an effort to understand the mechanisms of action of the BpT series of compounds, experiments characterised the ability of these chelators to mobilize intracellular ⁵⁹Fe from prelabeled SK-N-MC neuroepithelioma cells (Figure 3A). The release of ⁵⁹Fe mediated by these ligands was compared to a number of positive controls, including desferoxamine (DFO), NIH, and pyridoxal isonicotinoyl hydrazone (PIH) (Figure 1).

Both NIH and PIH showed high ⁵⁹Fe mobilization activity, releasing 45 ± 4% and 47 ± 3% of intracellular ⁵⁹Fe, respectively. In comparison, the control, which was culture media alone, mediated 4 ± 1% of intracellular ⁵⁹Fe release. The clinically used chelator, DFO, demonstrated poor ⁵⁹Fe mobilization efficacy, resulting in the release of only 7 + 1 % of intracellular ⁵⁹Fe (Figure 3A). All members of the BpT series of compounds led to ⁵⁹Fe mobilization activity, resulting in the release of 23-41% of cellular ⁵⁹Fe. The ligand Bp4eT showed the greatest Fe chelation efficacy releasing 41% of intracellular Fe (Figure 3A). Together with Bp44mT, these chelators demonstrated ⁵⁹Fe mobilization activity comparable to that of PIH and NIH. All the BpT analogues were significantly (p < 0.001) more effective than DFO.

The chelator-mediated increase in cellular Fe mobilization was not mediated by their cytotoxic effects as the cells remained viable within the short 3 h of incubation used.

Example 7.2 - Inhibition of Cellular ⁵⁹Fe Uptake from ⁵⁹Fe Transferrin by BpT Chelators The ability of the BpT series of compounds to inhibit ⁵⁹Fe uptake from the serum Fe-binding protein transferrin (Tf) in SK-N-MC neuroepithelioma cells was also assessed. This is important, as inducing Fe deprivation and anti-proliferative activity involves both increasing Fe mobilization and preventing Fe uptake from Tf. The positive controls NIH and PIH were found to effectively reduce ⁵⁹Fe uptake to 9 ± 1% and 21 ± 1% of the control, respectively (Figure 3B). In contrast, the hydrophilic chelator, DFO, exhibited a poor ability to decrease ⁵⁹Fe uptake to only 64 ± 2% of the control (Figure 3B). With the exception of Bp4pT, all members of the BpT series markedly reduced ⁵⁹Fe uptake to between 11-17% of the control (Figure 3B). The lack of activity of Bp4pT may be due to its high lipophilicity (Table 1). In this case, it is possible that compounds that are too lipophilic may result in retention in cellular membranes, leading to reduced Fe chelation efficacy. Excluding BpT, most of the BpT series were significantly (p< 0.05) less effective than NIH at inhibiting ⁵⁹Fe uptake from ⁵⁹Fe-Tf. However, besides Bp4pT all analogues of the BpT series displayed significantly (p < 0.05) greater ability than PIH to inhibit ⁵⁹Fe uptake (Figure 3B). DFO demonstrated significantly (p < 0.005) less ability to prevent ⁵⁹Fe uptake from ⁵⁹Fe-Tf than the majority of the BpT compounds.

Example 8 - Relationship between Anti-proliferative Activity and the Chemical and

Biological Properties of the Chelators

Regression analyses comparing the ⁵⁹Fe efflux and anti-proliferative activity of BpT compounds was examined to determine if a relationship existed between these factors. A linear relationship was observed between ⁵⁹Fe efflux and anti-proliferative activity of the BpT series (r = -0.77). This suggested that the Fe mobilization efficacy of the BpT series of compounds may play an important role in their anti-proliferative activity. Plots comparing the ability of the BpT ligands to reduce ⁵⁹Fe uptake and induce antiproliferative activity were also analyzed, excluding BpT. These data illustrated a linear relationship within the BpT series (r =0.57). Analysis of the relationship between measured redox potentials and anti-proliferative activity determined that a linear relationship existed for the BpT series (r = 0.74). These results indicated that no single factor was responsible for the observed cytotoxicity. Rather, a combination of properties including lipophilicity, ⁵⁹Fe mobilization activity, the ability to inhibit ⁵⁹Fe uptake from Tf, and redox-cycling may contribute to the anti-proliferative effects of these chelators.

Example 9 - In-vivo Inhibition of Tumour Growth by Bp4eT

Methodology:

Female BALB/c nu/nu mice were housed in filter-top cages in groups of 6 with food and water supplied ad libitum. Mice were used at 8-10 weeks of age. All protocols were conducted in accordance with the University of Sydney Animal Ethics Committee

Guidelines.

Exponentially growing DMS-53 human lung carcinoma cells were harvested and a 1 :1 mixture of cell-containing medium and Matrigel^® (BD Biosciences, MA, USA) prepared using 1 x 10⁷ cells/100 μL The cell/matrigel mixture was injected s.c. into the right flank of methoxyflurane-anesthetised mice. Post-engraftment, tumour size was monitored and measurements made using Vernier callipers. Tumour volumes were calculated in mm³ according to the equation: d^z x D/2, where d and D are the shortest and longest diameters, respectively. When tumour volumes reached an average of 100 mm³, i.v. treatment was initiated (time point designated as day 0; Figure 4). Test compounds were dissolved in 15% propylene glycol in 0.9% saline and injected via the tail vein once per day over 5 consecutive days/week for up to 48 days. Control mice were treated with vehicle alone in parallel with drug treatments. Mice were checked every two days for changes in weight and tumour growth.

Example 9.1 - Effect of Bp4eT on the in vivo Inhibition of DMS-53 Human Lung

Carcinoma

An unexpectedly potent response to Bp4eT was observed in the DMS-53 xenograft model in nude mice (Figure 4A). After 49 days, average net tumour size in control vehicle-treated mice was 740% of the initial tumour volume, while in mice treated with

0.4 mg/kg Bp4eT, net tumour size was 313% of the initial volume.

Example 10 - Effect of Bp4eT on Various Indicators of Toxicity in Mice

Methodology:

Assessment of hematology, biochemistry and tissue histology

Upon termination of the tumour growth studies, blood was collected by cardiac puncture from mice under methoxyflurane-induced anaesthesia and the animals sacrificed by cervical dislocation. Hematologic parameters were determined using a Sysmex K-4500 analyser (TOA Medical Electronics, Kobe, Japan).

Liver, spleen, kidney, heart, brain and tumour tissues were collected and weighed, a sample fixed in formalin for histological staining. Tissue samples were also taken for Fe assays (see below).

Tissue Fe assay

Mouse tissues were subjected to nitric acid digestion and Fe levels were measured using inductively coupled plasma atomic emission spectrometry (ICP-AES) by standard techniques. Example 10.1 Biologic assessment following treatment with Bp4eT: Weight loss and hematological analyses

Since Dp44mT and Bp4eT are Fe chelators, potential systemic effects these chelators on physiological processes were assessed.

In the DMS-53 lung carcinoma study mice treated with 0.4 mg/kg Dp44mT or 0.4 mg/kg Bp4eT experienced weight loss of 18% and 12% of initial body weight, respectively, (Figure 4B) after 49 days of treatment. Control mice in this experiment lost 8% of initial body weight (Figure 4B).

In terms of haematology, after 2 weeks of treatment with Dp44mT at 0.75 mg/kg or Bp4eT at 0.75 mg/kg and 1.0 mg/kg, there was no significant change in red blood cell count, haemoglobin or white blood cell count (Figure 5A), indicating that at the doses and administration schedule used the chelators were well tolerated.

Example 10.2 - Tissue Fe levels following Bp4eT administration

Examination of Fe levels from the DMS-53 lung carcinoma study revealed no significant change in the Fe content of control liver, spleen, kidney, brain, heart or tumour compared to 0.4 mg/kg Dp44mT, 0.4 mg/kg Bp4eT or 0.75 mg/kg Bp4eT treated mice. A previous experiment had found a significant increase in splenic Fe in mice treated with a higher dose of Dp44mT (0.75 mg/kg per day) (Reference 8). However, mice treated with 0.4 mg/kg Dp44mT, 0.4 mg/kg Bp4eT or 0.75 mg/kg Bp4eT over 7 weeks showed a slight, but not significant, increase in heart Fe relative to control mice (Table 4). These results indicated that while the chelators were able to interact with intracellular Fe to inhibit tumour growth at the doses used they did not induce systemic Fe deprivation.

Table 4. Tissue Fe levels from Balb/C nude mice bearing DMS-53 lung carcinoma as measured by Inductively Coupled Plasma Atomic Emission Spectrophotometry (ICP-AES) after 7 weeks intravenous administration of control vehicle. Dp44mT (0.4 mg/kg) or Bp4eT (0.4 mg/kg or 0.75 mg/kg) once a day/5 days a week. Mean +/- SD of Tissue Iron Levels in Indicated Experimental Group (N=7)

Organ Control Dp44mT Dp4eT Bp4eT (0.4 mg/kg (0.4 mg/kg (0.75 mg/kg per day) per day) per day)

Liver 729 ± 44 760 ± 46 891 ± 140 801 ± 49

Spleen 2877 ± 482 3074 ± 368 3132 + 1178 3120 ± 395

Kidney 372 ± 114 303 ± 11 300 ± 28 276 ± 12

Brain 107 ± 3 97 ± 1 114 ± 3 108 ± 4

Heart 368 ± 51 463 ± 14 484 ± 25 462 ±16

Tumour 147 + 25 182 ± 20 243 ± 67 175 ± 42

Example 10.2 - Effects of Bp4eT on tissue histoloαv

Histological assessment was performed on tissues from the DMS-53 human lung carcinoma experiment. Results are discussed comparing tissues from DMS-53 xenografted mice treated with vehicle, Dp44mT (2 weeks at 0.75 mg/kg) or Bp4eT (7 weeks at 0.4 mg/kg and 0.75 mg/kg). In hematoxylin and eosin (H & E) stained sections, no significant differences were found in the histology of the liver, spleen, kidney, brain or tumour from control and chelator treated mice.

However, myocardial lesions were observed in mice treated with Dp44mT (0.75mg/kg; Figure 6B). Such lesions consisted of poorly differentiated foci of necrosis, being replaced with immature fibrous tissue which was obvious using Gomori-Trichrome stain (arrow; Figure 6B) and were consistent with those described previously for Dp44mT (Reference 8). However, surprisingly, Bp4eT did not induce myocardial lesions at 0.4 mg/kg or 0.75 mg/kg even after 7 weeks of administration (Figure 6B). References

1) Kalinowski, D. S.; Yu, Y.; Sharpe, P. C; Islam, M.; Liao,Y-T.; Lovejoy, D. B.; Kumar, N.; Bernhardt, P. V.; Richardson, D. R. (2007) Design, synthesis, and characterization of novel iron chelators: structure-activity relationships of the 2-benzoylpyridine thiosemicarbazone series and their 3-nitrobenzoyl analogues as potent anti-tumour agents. J. Med. Chem. 50:3716-29.

2) Chaston, T. B.; Watts, R. N.; Yuan, J.; Richardson, D. R. (2004) Potent anti-tumour activity of novel iron chelators derived from di-2-pyridylketone isonicotinoyl hydrazone involves fenton-derived free radical generation. Clin. Cancer Res. 10:7365-7374.

3) Bernhardt, P. V.; Caldwell, L. M.; Chaston, T. B.; Chin, P.; Richardson, D. R. (2003) Cytotoxic iron chelators: characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues. J. Biol. Inorg. Chem. 8:866-880.

4) Richardson, D. R.; Sharpe, P. C; Lovejoy, D. B.; Senaratne, D.; Kalinowski, D. S. et al. (2006) Dipyridyl thiosemicarbazone chelators with potent and selective anti-tumour activity form iron Complexes with redox activity. J. Med. Chem. 49:6510-6521.

5) Dean, R. T.; Nicholson, P. (1994) The action of nine chelators on iron-dependent radical damage. Free Radio. Res. 20: 83-101

6) Chaston, T. B.; Richardson, D. R. (2003) Interactions of the pyridine-2- carboxaldehyde Isonicotinoyl hydrazone class of chelators with iron and DNA: implications for toxicity in the treatment of iron overload disease. J. Biol. Inorg. Chem. 8:427-438.

7) Edward, J. T.; Chubb, F. L; Sangster, J. (1997) Iron chelators of the pyridoxal isonicotinoyl hydrazone class. Relationship of the lipophilicity of the apochelator to its ability to mobilize iron from reticulocytes in vitro: reappraisal of reported partition coefficients. Can. J. Physiol. Pharmacol. 75:1362-1368. 8) Whitnall, M., Howard, J. Ponka, P.; Richardson, D.R. (2006) A class of iron chelators with a wide spectrum of potent anti-tumor activity that overcome resistance to chemotherapeutics. Proc. Natl. Acad. ScL USA 103:14901-6.

Claims

CLAIMS:

1. A method of treating a proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of at least one compound of formula (I)

wherein

R¹ is selected from H, C_1-6 alky!, C_2-6 alkenyl and phenyl;

R² is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² are the same or different, or a salt, hydrate, or iron complex thereof, or a pharmaceutical composition comprising said compound, salt, hydrate or iron complex and a pharmaceutically acceptable carrier, diluent or excipient.

2. A method of inhibiting proliferation of rapidly proliferating cells, comprising contacting said cells with a therapeutically effective amount of at least one compound of formula (I)

wherein

R¹ is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl;

R² is selected from H, C_1-6 alkyl, C_2-6 alkenyl and phenyl; wherein R¹ and R² are the same or different, or a salt, hydrate, or iron complex thereof, or a pharmaceutical composition comprising said compound, salt, hydrate or iron complex and a pharmaceutically acceptable carrier, diluent or excipient.

3. The method according to claim 1 or 2, wherein the compound of formula (I) is selected from the group consisting of:

and

4. The method according to claim 3, wherein the compound of formula (I) is

5. The method according to claim 1 or 2, comprising administering a therapeutically effective amount of an Fe complex of a compound of formula (I).

6. The method according to claim 5, wherein the Fe complex is selected from Fe"[(Bp44mT)₂]_I Fe'"[(Bp44mT)₂], Fe"[(Bp4eT)₂] and Fe"^l[(Bp4eT)₂], or a salt or hydrate thereof.

7. The method according to any one of claims 1 to 6, wherein the vertebrate is a human.

8. The method according to any one of claims 1 to 7, wherein the proliferative disease is cancer.

9. The method according to claim 8, wherein the cancer is a solid tumour.

10. The method according to claim 9, wherein the cancer is selected from prostate cancer, lung cancer and brain tumours.

11. A compound of structural formula (Ia):

wherein R¹ is C_1-6 alkyl;

R² is selected from C_2-6 alkyl, C_2-6 alkenyl and phenyl; and R¹ and R² are different; or a salt or hydrate thereof.

12. An Fe(II) or Fe(III) complex of a compound of formula (Ia) as defined in claim 11.

13. An Fe complex of a compound of formula (I) as defined in claim 1 , selected from the group consisting of Fe"[(Bp44mT)₂], Fe^lll[(Bp44mT)₂], Fe"[(Bp4eT)₂] and Fe'"[(Bp4eT)₂], or a salt or hydrate thereof.