WO2017058748A1

WO2017058748A1 - Thiosemicarbazones

Info

Publication number: WO2017058748A1
Application number: PCT/US2016/053882
Authority: WO
Inventors: John C. Warner; Jeffery A. GLADDING; Srinivasa R. Cheruku
Original assignee: Oncochel Therapeutics, Llc
Priority date: 2015-09-29
Filing date: 2016-09-27
Publication date: 2017-04-06

Abstract

Described herein are thiosemicarbazone compounds and their use in therapy. More particularly, described herein a selection of dipyridyl thiosemicarbazone compounds, pharmaceutical compositions containing those compounds, and methods of treating cancer comprising administering those compounds and compositions.

Description

THIOSEMICARBAZONES

BACKGROUND

[0001] Thiosemicarbazone iron chelators are a class of anti-cancer agents that have been found to be extremely potent and selective against a number of different neoplasms both in vitro and in vivo. These compounds function by targeting iron, an essential element for DNA synthesis, in cancer cells. Iron chelators were initially developed for iron-overload diseases such as β- thalassemia, with the chelator desferrioxamine (DFO) being the most widely used treatment for this disease. However, clinical trials examining DFO against neuroblastoma found that this agent was effective at inhibiting the progression of this cancer in some patients. These early studies were the first to identify the potential of iron chelators as anti-cancer agents. Since then, iron chelators designed specifically for the treatment of cancer have been developed, with the thiosemicarbazone iron chelator 3-aminopyridine-2-carboxaldehyde

thiosemicarbazone (Triapine^®) (Vion Pharmaceuticals, New Haven Conn., United States of America) entering a number of phase I and II clinical trials.

[0002] Thiosemicarbazone iron chelators function by binding iron and copper and forming redox-active complexes, leading to the production of reactive oxygen species (ROS) that induce cancer cell cytotoxicity. One highly active compound is a thiosemicarbazone class of iron chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, (abbreviated herein as

Dp44mT), which is described in WO 2004/069801. Dp44mT has been demonstrated to markedly and significantly reduce the growth of a number of different tumors in vitro and in vivo and was found to be more potent and less toxic than Triapine^®. However, studies using high, non-optimal doses of Dp44mT found that it induced some cardiotoxicity in nude mice.

[0003] Thiosemicarbazone compounds target the metastasis suppressor, NDRG1. NDRG1 inhibits both growth and metastasis as well as angiogenesis of pancreatic cancer in vivo leading to reduced tumor progression. Moreover, NDRG1 has also recently been correlated with increased differentiation of pancreatic cancers and its potential as a promising therapeutic target against pancreatic cancer has been reported. NDRG1 has a number of key molecular targets in pancreatic cancer including the tumor suppressors PTEN and SMAD4, both of which are up-regulated in response to NDRGl. Therefore, NDRGl may be a promising therapeutic target, especially for the treatment of pancreatic cancer. NDRGl was recently found to be up- regulated using iron-chelating anti-cancer agents in vitro and in vivo. Iron chelators increased NDRGl expression via hypoxia-inducible transcription factor (HIF-l)-dependent mechanisms, although HIF-l-independent pathways have also been observed. Iron-chelating anti-cancer agents therefore provide an important opportunity to target NDRGl expression in cancer cells by cellular iron depletion.

[0004] There is a need for new and alternative treatments for cancer. Described herein are novel thiosemicarbazone compounds that advantageously inhibit cellular proliferation and may be useful for the treatment of cancer.

SUMMARY

[0005] Provided herein are compounds of general formula (I):

wherein R is selected from the group consisting of

and salts, hydrates and solvates thereof.

[0006] In certain embodiments the salts are pharmaceutically acceptable salts, which may be acid addition salts, such as hydrochloride salts.

[0007] Also provided herein are pharmaceutical compositions comprising a compound of formula (I), or a salt, hydrate or solvate thereof, together with a pharmaceutically acceptable excipient, diluent or adjuvant.

[0008] Also provided herein is a method of treating cancer in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a compound of formula (I) or a salt, hydrate or solvate thereof, or a pharmaceutical composition thereof. The mammal may be a human.

[0009] Also provided herein is the use of a compound of formula (I) or a salt, hydrate or solvate thereof in the manufacture of a medicament for the treatment of cancer.

[0010] Also provided herein is a compound of formula (I) or a salt, hydrate or solvate thereof, or a pharmaceutical composition thereof, for the treatment of cancer.

[0011] The compounds provided herein are useful for the treatment of a wide variety of cancers, including solid and non-solid tumors, including but not limited to, melanoma, skin cancer, breast cancer, prostate cancer, bladder cancer, liver cancer, gastrointestinal cancer, colon and rectal cancer, brain tumor, head and neck cancer, bone cancer, pancreatic cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer as well as hematological tumors (e.g., leukemias and lymphomas).

[0012] Also provided herein are methods of inhibiting cellular proliferation, the method comprising contacting one or more cells with an effective amount of a compound of formula (I) or a salt, hydrate or solvate thereof. The cells may be in vitro or in vivo, and may be mammalian cells.

[0013] 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 element, inte.g.er or step, or group of elements, integers or steps, but not the exclusion of any other element, inte.g.er or step, or group of elements, integers or steps. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely."

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1. A Structural formulae of the compounds Dp44mT, DpC (di-2-pyridylketone 4- cyclohexyl-4-methyl-3-thiosemicarbazone), and WBI-OC-01 - 10, and antiproliferative activity (IC50) for each compound.

[0015] FIG. 2. Antiproliferative activity of novel thio-semicarbazones against SK-N-MC neuroepithelioma.

[0016] FIG. 3. Induction of methemoglobinemia in intact RBC by thio-semicarbazone compounds. New compounds WBI-OC-01, -02, and -04 did not induce methemoglobinemia.

[0017] FIG. 4. Antiproliferative activity of thio-semicarbazone compounds does not correlate with methemoglobinemia toxicity.

[0018] FIG. 5. Cytotoxicity of thiosemicarbazones (IC50) does not correlate with log P

(octanol/water partition ratio at equilibrium). [0019] FIG. 6. Methemoglobin induction by bis-thiosemicarbazones is negatively correlated with molecular weight but not calculated logP (octanol/water partition ratio at equilibrium) or ICso (p=0.0854).

[0020] FIG. 7 shows a comparison of the cytotoxicity of the thiosemicarbazone compounds against cancer cells (SK-N-MC) to that against a primary endothelial cell line (HUVEC).

[0021] FIG. 8 shows that three of the DpC analogs (WBI-OC-01, -02, and -04) have superior properties to DpC.

DETAILED DESCRIPTION

DEFINITIONS

[0022] The following 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.

[0023] 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.

[0024] 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 of any two or more of said steps, features, compositions and compounds.

[0025] The compounds of formula (I) are tridentate ligands capable of chelating transition metal ions, such as iron (Fe(ll) and Fe(lll)). Accordingly, throughout this specification the compounds of the invention may be referred to as "ligands," "chelators," or "iron chelators." Throughout this specification, a reference to "compound(s)," or "chelators" or "ligand(s)" is a reference to compounds of formula (I), including salts, hydrates and solvates thereof, unless expressly indicated otherwise. [0026] The present invention includes within its scope all isomeric forms of the compounds of formula (I) and salts, hydrates and solvates thereof disclosed herein, including all

diastereomeric isomers (including cis/trans isomers), racemates and enantiomers.

[0027] The term "administering" and variations of that term including "administer" and "administration" as used herein includes contacting, applying, delivering or providing a compound or composition to an organism, mammal, or a surface by any appropriate means.

[0028] In the context of this specification, the term "mammal" 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.

[0029] 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. Thus, for the avoidance of doubt, references herein to ^'treatment¹ include references to curative, palliative and prophylactic treatment.

[0030] In the context of this specification the term "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.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0031] Provided herein are thiosemicarbazone compounds that have antiproliferative properties and which may therefore be useful in the treatment of cancer. In particular, the present invention relates to the thiosemicarbazone compounds of general formula (I):

/ wherein R is selected from the group consisting of

and salts, hydrates and solvates thereof.

[0032] Compounds provided herein are iron chelators and can chelate Fe(ll) and Fe(lll).

Accordingly, iron complexes (i.e., Fe(ll) and Fe(lll) complexes) of the compounds of formula (I) are also encompassed by the present invention.

[0033] Compounds of formula (I) or salts, hydrates or solvates thereof, may be prepared by methods known to those skilled in the art, including for example, Schiff base condensation of an imine with a ketone, as described for example in Advanced Organic Chemistry, 4.sup.th Ed (John Wiley & Sons, New York, 1992) and Vogel's Textbook of Practical Organic Chemistry, 5.sup.th Ed (John Wiley & Sons, New York, 1989). An exemplary general synthetic scheme for preparing compounds of formula (I) is provided in U.S. Patent 8,927,580, the contents of which are incorporated herein by reference.

[0034] Salt forms of the compounds of formula (I) may be readily prepared using techniques known to those skilled in the art. For example, acid addition salts may be prepared by dissolving a compound of formula (I) in a suitable non-polar solvent, such as hexane, dichloromethane, etc., and stirring with an aqueous acid corresponding to the desired salt. For example, hydrochloric acid would yield the hydrochloride salt, nitric acid would yield the nitrate salt, sulfuric acid would yield the sulfate salt, etc.

[0035] Compounds of general formula (I) or their salts, hydrates and solvates may be purified using stand techniques known to those skilled in the art. In preferred embodiments, the compounds of formula (I) may be purified by crystallization from a suitable solvent or mixture of solvents. Suitable solvents would be known to those skilled in the art and include, for example, methanol, ethanol, acetonitrile, ethyl acetate, N,N-dimethylformamide,

dimethylsulfoxide, and mixtures thereof. In other embodiments the product may be

recrystallized from a solvent mixture comprising one or more organic solvents, such as those listed above, and water. After purification, compounds of general formula (I) may be substantially pure. For example, the compounds of formula (I) may be isolated in a form which is at least about 80%, 85%, 90%, 95%, 98%, or 99% pure. Therapy

[0036] Also described herein is the use of thiosemicarbazone compounds of general formula (I) and salts, hydrates and solvates thereof, in therapy. I n particular, the thiosemicarbazone compounds of formula (I) have antiproliferative properties and therefore may be useful in the treatment of cancer. The thiosemicarbazone compounds described herein are iron chelators. These compounds may be useful for the treatment of a wide variety of cancers (tumors), including but not limited to, melanoma, skin cancer, breast cancer, prostate cancer, bladder cancer, liver cancer, gastrointestinal cancer, colon and rectal cancer, brain cancer, head and neck cancer, bone cancer, pancreatic cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer as well as hematological tumors (e.g., leukemias and lymphomas).

[0037] Pancreatic cancer is a devastating disease being fatal in 98-100% of cases within the first 5 years of diagnosis, with the survival from this disease being the same today as it was 20 years ago. The best treatment currently available for pancreatic cancer is the anti-cancer agent, gemcitabine, which is an analogue of the nucleoside, deoxycytidine. Gemcitabine is a prodrug which is converted within the cell to the active metabolites difluorodeoxycytidine di- and triphosphate (dFdCDP, dFdCTP). The success of gemcitabine in pancreatic cancer treatment has been limited. In fact, clinical trials using this agent have found that on average it increases the life-span of patients by only about 3 months. Gemcitabine has been combined with other anticancer agents such as 5-fluorouracil (5-FU) resulting in some improvement of its activity. However, the prognosis for pancreatic cancer patients remains dismal.

[0038] Surprisingly, the thiosemicarbazone compounds described herein, or salts, hydrates or solvates thereof, advantageously show antiproliferative properties which are at least as good as, and preferably better than, known anticancer agents. For example, the thiosemicarbazone compounds described herein are more effective in inhibiting proliferation of cancer cells (e.g., pancreatic cancer cells) when compared to gemcitabine and 5-fluorouracil. The compounds of the present invention (used alone or in combination with other anticancer agents, or as part of a therapeutic regimen), are therefore alternative anticancer agents possessing one or more advantageous therapeutic properties compared to existing anticancer agents. [0039] Another surprising and advantageous feature of the thiosemicarbazone compounds described herein, or salts, hydrates or solvates thereof, is that the compounds may

substantially inhibit pancreatic tumor growth, which is well known to be a particularly aggressive form of cancer. The compounds may therefore be used, alone or in combination with other anticancer agents or as part of a therapeutic regimen, in the treatment of pancreatic cancer.

[0040] A further surprising and advantageous feature of the thiosemicarbazone compounds described herein is that the compounds may be more effective and less toxic than the potent antiproliferative thiosemicarbazone compound di-2-pyridylketone 4,4-dimethyl-3- thiosemicarbazone (Dp44mT).

[0041] The protein myoglobin (Mb) plays an important role in oxygen storage and donation to muscles and is a monomeric counterpart to hemoglobin. In cancer clinical trials involving Triapine^®, induction of methemoglobinemia and hypoxia has been noted as a dose-limiting side effect. In patients that are undergoing chemotherapy for cancer, this complication is undesirable as these patients often have reduced respiratory performance. Accordingly, the excessive production of metHb reduces the clinical utility of Triapine^®. The compounds described herein possess a further advantage over other anticancer agents because they do not induce methaemoglobin (metHb) and/or metmyoglobin (metMb) formation, or the compounds induce metHb and/or metMb formation to a significantly less extent than other anticancer agents, such as Dp44mT and Triapine^®, while maintaining anti-tumor activity.

Formulations

[0042] When used for the treatment or prevention of an infection, disease, or disorder, the compound(s) described herein may be administered alone or in combination with other agents as part of a therapeutic regimen. The compounds may be administered as a pharmaceutical or veterinarial formulation which comprises at least one compound described herein. The compound(s) may also be present as suitable salts, including pharmaceutically acceptable salts. [0043] Pharmaceutical compositions suitable for the delivery of compounds described herein and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

[0044] In other embodiments the compound(s) described herein may be formulated in combination with one or more other therapeutic agents.

[0045] In other embodiments, the compounds described herein may be included in

combination treatment regimens with surgery and/or other known treatments or therapeutic agents, such as other anticancer agents, in particular, chemotherapeutic agents,

radiotherapeutic agents, and/or adjuvant or prophylactic agents. Suitable agents are listed, for example, in the Merck I ndex, An Encyclopaedia of Chemicals, Drugs and Biologicals, 12.sup.th Ed., 1996, the entire contents of which are incorporated herein by reference.

[0046] For example, when used in the treatment of solid tumors, the compounds described herein may be administered with one or more chemotherapeutic agents or combinations thereof, such as: adriamycin, taxol, docetaxel, fluorouracil, melphalan, cisplatin, alpha interferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), PROMACE/MOPP (prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine), vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG and the like.

[0047] Other examples of anticancer agents include alkylating agents such as nitrogen mustards (e.g., mechlorethamine, melphalan, chlorambucil, cyclophosphamide, (L-sarcolysin), and ifosfamide), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin), 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, methramycin), 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).

[0048] Combination regimens may involve the active agents being administered together, sequentially, or spaced apart as appropriate in each case. Combinations of active agents including the compounds described herein may be synergistic.

[0049] 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.

[0050] For example, suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing the compounds described herein with a pharmaceutically acceptable acid (including inorganic and organic acids) or a pharmaceutically acceptable base (including inorganic and organic bases). Suitable pharmaceutically acceptable salts of the compounds therefore include acid addition salts and base salts.

[0051] Suitable pharmaceutically acceptable acids include but are not limited to acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, oxalic acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like.

[0052] Suitable base salts may be formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

[0053] Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.

[0054] S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J.

Pharmaceutical Sciences, 1977, 66:1-19 and a review on suitable salts is provided by Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002), both of which are incorporated herein in their entirety.

[0055] 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 compound 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. [0056] Convenient modes of administration of compounds of the invention include parenteral (e.g., subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intrathecal, intraocular, intranasal, intraventricular injection or infusion techniques), intraperitoneal, 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.

[0057] 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.

[0058] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (for suitably water soluble active agents) 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 stabilize the composition against the contaminating action of microorganisms such as bacteria and fungi.

[0059] In one embodiment, the compound(s) described herein 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 the invention in pharmaceutical

compositions and preparations may, of course, be varied. For example, the amount 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 can be obtained. Suitable does may be obtained by single or multiple administrations.

[0060] The term "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.

[0061] The term "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) 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. I n 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.

[0062] In one embodiment, the carrier may be an orally administrable carrier.

[0063] Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration.

[0064] Also provided herein are delayed or extended release formulations.

[0065] The compound(s) described herein may be administered by injection. In the case of injectable solutions, the carrier may be a solvent or dispersion medium containing, for example, water (e.g., water-for-injection), saline, 5% glucose solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene 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 (e.g., polysorbate 80). 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, and/or 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.

[0066] Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0067] Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum tragacanth, 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 flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. 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 flavoring such as cherry or orange flavor. 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 compound can be

incorporated into sustained-release preparations and formulations. [0068] The pharmaceutical composition may further include a suitable buffer to minimize 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.

[0069] Single or multiple administrations of the pharmaceutical compositions 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 and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable.

[0070] 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.

[0071] 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.

[0072] Alternatively, an effective dosage may be calculated according to the Body Surface Area (BSA) of the patient to be treated. The BSA of a patient may be readily calculated using methods known to those skilled in the art. A suitable dose generally may be up to about 500 mg/m². For example, generally, an effective dosage any be in the range of about 10 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 m g/m², and about 75 to about 150 mg/m². EXAMPLES nthesis of Thiosemicarbazone Compounds

[0073] All these new compounds (1-5) have an alkyl, or alkyl-aryl, or alkyl-heteroaryl, or fused cycloalkyi replacement moiety for the cyclohexyl group in the DpC structure. The structural analogs 2- 4 have Rl and R2 substitutions at the thioamide nitrogen of the di-2-pyridylketone-3-thiosemicarbazone 6. Compound 5 has an 3 substitution of the di-2-pyridylketone-3-thiosemicarbazone 7, such that the thioamide nitrogen is an integral member of a heterocyclic group.

[0074] All reagents were purchased from commercial suppliers and used as supplied unless stated otherwise. Reactions were carried out in air unless stated otherwise. 400 MHz ¹H NM R spectra were obtained on a Jeol AS 400 spectrometer. Low-resolution mass spectra (LRMS) were obtained on a Jeol JMS-T100LC DART/ AccuTOF mass spectrometer.

[0075] Compounds 1 - 3, and 5 were synthesized by the steps outlined in Scheme 1, which is adapted from Molecular Pharmacology 2011, 80, 598-609 and WO 2012/079128 Al.

Synthetic Scheme 1:

[0076] The first step of Synthetic Scheme 1 involves the formation of a carboxymethyl dithiocarbamate from the reaction of a secondary amine with carbon disulfide in the presence of a sodium hydroxide, and, without isolation of the intermediate, reaction with sodium chloroacetate.

[0077] The second step of Synthetic Scheme 1 involves the reaction of the carboxymethyl

dithiocarbamate with hydrazine to form a thiosemicarbazide.

[0078] The third step of Synthetic Scheme 1 involves reaction of the thiosemicarbazide with di-2-pyridyl ketone to form the dipyridyl thiosemicarbazone compound.

[0079] In the fourth step of Synthetic Scheme 1, each of the compounds 1 - 3, and 5 is further isolated as the hydrochloric acid salt, as shown in Scheme 1. SYNTHETIC EXAMPLE 1

[0080] Synthesis of Compound 1, N-benzyl-2-(di(pyridin-2-yl)methylene)-N-methylhydrazine-l- carbothioamide, and its hydrochloride salt:

The procedure of Synthetic Scheme 1 is described in full for derivative 1, N-benzyl-2-(di(pyridin-2- yl)methylene)-N-methylhydrazine-l-carbothioamide and its hydrochloride salt:

[0081] 2-{{Benzyl{methyl)carbamothioyl)thio)acetic acid: Prepared as in Step 1 of Synthetic Scheme 1:

N-benzylmethylamine (1.0 mL) was added to 0.8M aqueous sodium hydroxide (9.7 mL). Carbon disulfide

(0.46 mL) was added dropwise and the reaction stirred at ambient temperature for 19 hours. Sodium chloroacetate (0.90 g) was added in one portion and the reaction stirred at ambient temperature for 24 hours. The reaction was quenched with 3M hydrochloric acid (3.87 mL) and the resulting precipitate collected via filtration. The precipitate was washed with water (3 ^χ 10 mL) and dried overnight at 50 °C under vacuum to afford 2 ((benzyl(methyl)carbamothioyl)thio)acetic acid (1.68 g).

^-NM (400 MHz, DMSO-d6) Major isomer: δ 7.43-7.22 (m, 5H), 5.30 (s, 2H), 4.09 (s, 2H), 3.29 (s, 3H).

Minor isomer: δ 7.43-7.22 (m, 5H), 5.07 (s, 2H), 4.06 (s, 2H), 3.39 (s, 3H).

[0082] N-Benzyl-N-methylhydrazinecarbothioamide: Prepared as in Step 2 of Synthetic Scheme 1: 2 ((benzyl(methyl)carbamothioyl)thio)acetic acid (1.68 g) was suspended in water (0.82 mL) and hydrazine monohydrate (1.64 mL). This mixture was heated at 120 °C until fuming and then cooled to ambient temperature. The heating and cooling procedure was repeated four more times. The solid material was dissolved in dichloromethane (100 mL) and washed with water (25 mL) and saturated sodium bicarbonate solution (25 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to give N benzyl-N methylhydrazinecarbothioamide (1.21 g).

¹H-NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 7.34-7.28 (m, 2H), 7.26-7.18 (m, 3H), 5.01 (s, 2H), 4.75 (broad s, 2H), 2.93 (s, 3H).

[0083] N-benzyl-2-(di(pyridin-2-yl)methylene)-N-methylhydrazine-l-carbothioamide, Compound 1:

Prepared as in Step 3 of Synthetic Scheme 1: A 100 mL flask fitted with a reflux condenser was charged with N benzyl-N-methylhydrazinecarbothioamide (1.21 g) and di-2-pyridylketone (1.14 g). The reaction vessel was purged with nitrogen for 20 minutes, and then 200 proof ethanol (18.8 mL) and glacial acetic acid (0.04 mL) were added. The reaction was heated at reflux for 24 hours then cooled to room temperature. The solvent was removed in vacuo and the residue redissolved in dichloromethane (50 mL). The organic layer was washed with water (20 mL) and saturated sodium chloride solution (20 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified via chromatography on silica gel (0 to 100% ethyl acetate in hexanes) to afford N benzyl-2 (di(pyridin-2-yl)methylene)-N- methylhydrazine-l-carbothioamide (1.18 g).

^XH -NMR (400 M Hz, DMSO-d6) δ 8.59 (d, 1H), 8.01-7.83 (m, 3H), 7.62-7.43 (m, 3H), 7.39 7.24 (m, 6H), 5.21 (broad s, 2H), 3.32 (s, 3H).

LRMS: 362.07 (M+H)+

[0084] N-Benzyl-2-(di(pyridin-2-yl)methylene)-N-methylhydrazine-l-carbothioamide hydrochloride, the hydrochloric acid salt of Compound 1: Prepared as in Step 4 of Synthetic Scheme 1: N-Benzyl-2- (di(pyridin-2-yl)methylene)-N-methylhydrazine-l-carbothioamide (1.18 g) was dissolved in hot 1,4- dioxane (80 mL) and the solution cooled to ambient temperature. Hydrochloric acid in 1,4-dioxane (4N, 0.82 mL) was added dropwise and the reaction stirred at ambient temperature for 18 hours. The precipitate was collected via filtration, washed with tert-butylmethyl ether, and dried under vacuum at 60°C to afford N benzyl-2 (di(pyridin-2 yl)methylene)-N-methylhydrazine-l carbothioamide

hydrochloride (1.3 g).

^XH -NMR (400 M Hz, DMSO-d6) δ 8.74-8.51 (broad s, 1H), 8.61 (d, 1H), 7.98 (t, 2H), 7.93-7.85 (m, 1H), 7.61-7.46 (m, 3H), 7.39-7.24 (m, 5H), 5.20 (broad s, 2H), 3.32 (s, 3H).

SYNTHETIC EXAMPLE 2:

[0085] Synthesis of Compound 2, N -Benzyl-2-(di(pyridin-2-yl)methylene)- N -ethylhydrazine-1- carbothioamide, and its hydrochloride salt: Prepared according to Scheme 1 and as detailed in the synthesis for Compound 1. Intermediates, Compound 2 and its hydrochloric acid salt were characterized as follows:

[0086] 2-{(Benzyl(ethyl)carbamothioyl)thio)acetic acid: ^XH -NMR (400 MHz, DMSO-d6) Major isomer: δ

12.72 (s, 1H), 7.41-7.23 (m, 5H), 5.28 (s, 2H), 4.14 (s, 2H), 3.73 (q, 2H), 1.19 (t, 3H). Minor isomer: δ

12.72 (s, 1H), 7.41-7.23 (m, 5H), 5.03 (s, 2H), 4.09 (s, 2H), 3.93 (q, 2H), 1.13 (t, 3H).

[0087] N-Benzyl-N-ethylhydrazinecarbothioamide: ^:H -NMR (400 M Hz, DMSO-d6) δ 8.93 (s, 1H), 7.34-

7.27 (m, 2H), 7.26-7.19 (m, 3H), 4.94 (s, 2H), 4.75 (broad s, 2H), 3.46 (q, 2H), 0.99 (t, 3H).

[0088] N-Benzyl-2-(di(pyridin-2-yl)methylene)-N-ethylhydrazine-l-carbothioamide, Compound 2: ^XH -

NMR (400 M Hz, DMSO-d6) δ 8.60 (d, 1H), 8.01-7.85 (m, 3H), 7.62-7.45 (m, 3H), 7.40-7.24 (m, 6H), 5.18

(broad s, 2H), 3.76 (broad s, 2H), 1.25 (t, 3H). LRMS: 376.09 (M+H)+ [0089] N-Benzyl-2-(di(pyridin-2-yl)methylene)-N-ethylhydrazine-l-carbothioamide hydrochloride, the hydrochloric acid salt of Compound 2: ^XH -NM (400 M Hz, DMSO-d6) δ 8.72-8.34 (broad s, IH), 8.62 (d, IH), 8.06-7.84 (m, 3H), 7.64-7.44 (m, 3H), 7.93-7.22 (m, 5H), 5.16 (broad s, 2H), 3.76 (broad s, 2H), 1.23 (t, 3H).

SYNTHETIC EXAMPLE 3:

[0090] Synthesis of Compound 3, 2-(Di(pyridin-2-yl)methylene)- N -isopropyl- N -methylhydrazine-1- carbothioamide, and its hydrochloride salt: Prepared according to Scheme 1 and as detailed in the synthesis for Compound 1. Intermediates, Compound 3 and its hydrochloric acid salt were characterized as follows:

[0091] 2-((lsopropyl(methyl)carbamothioyl)thio)acetic acid: ^XH -NMR (400 M Hz, DMSO-d6) Major isomer: δ 12.73 (s, IH), 5.61 (sept, IH), 4.08 (s, 2H), 3.15 (s, 3H), 1.13 (d, 6H). Minor isomer: δ 12.73 (s, IH), 4.76 (sept, IH), 4.08 (s, 2H), 3.24 (s, 3H), 1.23 (d, 6H).

[0092] N-isopropyl-N-methylhydrazinecarbothioamide: ^:H -NMR (400 MHz, DMSO-d6) δ 8.66 (s, IH), 5.25 (sept., IH), 4.68 (broad s, 2H), 2.77 (s, 3H), 1.03 (d, 6H).

[0093] 2-(Di(pyridin-2-yl)methylene)-N-isopropyl-N-methylhydrazine-l-carbothioamide, Compound

3: ^XH -NMR (400 MHz, DMSO-d6) δ 8.82 (d, IH), 8.59 (d, IH), 8.03-7.83 (m, 3H), 7.63-7.55 (m, 2H), 7.47 (ddd, IH), 5.40 (broad s, IH), 3.13 (s, 3H), 1.18 (d, 6H). LRMS: 314.14 (M+H)+

[0094] 2-(Di(pyridin-2-yl)methylene)-N-isopropyl-N-methylhydrazine-l-carbothioamide

hydrochloride, the hydrochloric acid salt of Compound 3: ^XH -NMR (400 M Hz, DMSO-d6) δ 8.84 (d, IH), 8.64 (d, IH), 8.08-7.91 (m, 3H), 7.65-7.49 (m, 3H), 5.39 (broad s, IH), 3.14 (s, 3H), 1.19 (d, 6H).

SYNTHETIC EXAMPLE 4:

[0095] Synthesis of Compound 5, N '-(di(pyridin-2-yl)methylene)octahydro-lH-indole-l- carbothiohydrazide: Prepared according to Scheme 1 and as detailed in the synthesis for Compound 1. Intermediates, Compound 5 and its hydrochloric acid salt were characterized as follows:

[0096] 2-{{Octahydro-lH-indole-l-carbonothioyl)thio)acetic acid: ^-NMR (400 MHz, CDCI3) Multiple isomers: δ 4.60 (m, IH), 4.18-4.01 (m, 8H), 3.94-3.79 (m, 2H), 3.74-3.53 (m, 2H), 2.53-0.94 (m, 28H). LRMS: 260.07 (M+H)+. [0097] Octahydro-lH-indole-l-carbothiohydrazide: ^-NMR (400 M Hz, DMS0-d6) δ 8.48 (s, 1H), 4.52 (d, 2H), 4.19-4.10 (m, 1H), 3.46 (t, 1H), 2.25-0.91 (m, 12H).

[0098] N'-(di{pyridin-2-yl)methylene)octahydro-lH-indole-l-carbothiohydrazide, Compound 5: ^XH -

NMR (400 M Hz, DMSO-d6) δ 8.80 (s, 1H), 8.58 (s, 1H), 8.04-7.85 (m, 3H), 7.63-7.54 (m, 2H), 7.46 (dd, 1H), 3.87-3.63 (m, 2H), 2.42-1.07 (m, 12H).

LRMS: 366.18 (M+H)+

[0099] N'-(Di(pyridin-2-yl)methylene)octahydro-lH-indole-l-carbothiohydrazide hydrochloride, the hydrochloric acid salt of Compound 5: ^:H -NM R (400 MHz, DMSO-d6) δ 8.80 (broad s, 1H), 8.61 (s, 1H), 8.00 (t, 2H), 7.91 (d, 1H), 7.65-7.55 (m, 2H), 7.50 (dd, 1H), 4.51 (broad s, 2H), 4.17 (broad s, 1H), 2.42- 1.07 (m, 12H).

SYNTHETIC EXAMPLE 5

[0100] Synthesis of Compound 4, 2-(Di(pyridin-2-yl)methylene)- N methyl- N (pyridin-2

ylmethyl)hydrazine-l carbothioamide, and its hydrochloride salt: A basic amine on any or all of the

Rl, R2, or R3 substituents results in interference with the first step of Synthetic Scheme 1 in which a carboxymethyl dithiocarbamate is synthesized. A second synthetic method was used to prepare semithiocarbazone derivatives that contain a basic amine on the N-l substituent or substituents. The method is outlined in Synthetic Scheme 2 for the synthesis of Compound 4, 2 (di(pyridin-2- yl)methylene)- N methyl- N (pyridin-2 ylmethyl)hydrazine-l carbothioamide and its hydrochloride salt.

[0101] Synthetic Scheme 2:

[0102] The first step of Synthetic Scheme 2 is the reaction of a secondary amine with

thiocarbonyldiimidazole, and, without isolation of the intermediate, reaction of the intermediate with hydrazine to form the thiosemicarbazide.

[0103] The second step of Synthetic Scheme 2 involves reaction of the thiosemicarbazide with di-2- pyridyl ketone to form the dipyridyl thiosemicarbazone. Step 2 is carried out as detailed for Step 3 in Synthetic Scheme 1.

[0104] In the third step of Synthetic Scheme 2, the thiosemicarbazone is further isolated as the hydrochloric acid salt. Step 3 is carried out as detailed for Step 3 in Synthetic Scheme 1.

[0105] The synthetic procedure of Step 1 of Scheme 2 is described in full for Compound 4 and its hydrochloride salt.

[0106] N-Methyl-N-(pyridin-2-ylmethyl)hydrazinecarbothioamide: To a solution of

thiocarbonyldiimidazole (1.60 g) in acetonitrile (75 mL) was added triethylamine (3.14 mL) and 2

[(methylamino)methyl]pyridine (0.92 mL). The reaction was stirred at ambient temperature for 3 hours then hydrazine monohydrate (0.55 mL) was added. The reaction was quenched after 17 hours by the addition of saturated sodium bicarbonate solution (50 mL). The layers were separated and the aqueous extracted with dichloromethane (20 50 mL). The combined organic layer was washed with sodium chloride brine (50 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified via chromatography on silica gel (0 to 5% methanol in dichloromethane) to afford N methyl-N (pyridin-2 ylmethyl)hydrazinecarbothioamide (1.03 g). ^XH-NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.50 (d, 1H), 7.75 (td, 1H), 7.26 (dd, 1H), 7.18 (d, 1H), 5.05 (s, 2H), 4.73 (s, 2H), 3.06 (s, 3H).

[0107] 2-(Di(pyridin-2-yl)methylene)-N-methyl-N-(pyridin-2-ylmethyl)hydrazine-l carbothioamide, Compound 4: Compound 4 was prepared by reaction of N methyl-N (pyridin-2

ylmethyl)hydrazinecarbothioamide with di 2 pyridyl ketone, similarly to the method described in full for synthesis of Compound 1, Step 3 of Synthetic Scheme 1. ¹H-NMR (400 MHz, DMSO-d6) δ 8.92-8.33 (broad s, 1H), 8.58 (d, 1H), 8.48 (d, 1H), 8.03-7.83 (m, 3H), 7.78 (dt, 1H), 7.65-7.43 (m, 3H), 7.34-7.23 (m, 2H), 5.21 (broad s, 2H), 3.42 (s, 3H). LRMS: 363.13 (M+H)+

[0108] 2-(Di(pyridin-2-yl)methylene)-N-methyl-N-(pyridin-2-ylmethyl)hydrazine-l-carbothioamide hydrochloride: The hydrochloric acid salt of compound 4 was prepared by reaction of N methyl-N (pyridin-2 ylmethyl)hydrazinecarbothioamide with HCI, similarly to the method described in full for synthesis of the hydrochloric acid salt of Compound 1, Step 4 of Synthetic Scheme 1. ^-NM R (400 MHz, DMSO-d6) δ 8.91-8.48 (broad s, 1H), 8.60 (d, 2H), 8.11-7.80 (m, 4H), 7.65-7.42 (m, 5H), 7.93-7.22 (m, 5H), 5.32 (broad s, 2H), 3.46 (broad s, 3H).

Example 1: Anti-Proliferative Activity of Novel Thio-Semicarbazones

[0109] Methodology: SK-N-MC human neuroepithelioma cells were obtained from the

American Type Culture Collection (ATCC; Rockville, Md., USA) and cultured according to the methods described previously (Richardson, D R et al (1995). Blood 86: 4295-306). The cells were maintained in minimum essential media (MEM; Invitrogen, CA, USA) which contained 10% fetal calf serum (FCS; JRH Biosciences, Kansas, USA), 1% non-essential amino acids (Gibco, Victoria, Australia), 1% penicillin/streptomycin/glutamine (Gibco, VIC, Australia) and fungizone (0.28 ng/mL; Squibb Pharmaceuticals, Montreal, Canada).

[0110] The pancreatic cancer cell lines, including MIAPaCa-2, PANC 1, CAPAN-2 and CFPAC-1 were a generous gift from Prof. Andrew Biankin (Garvan Institute, NSW, Australia). The

MIAPaCa-2, PANC 1 and CFPAC-1 cell lines were grown in DMEM medium (Invitrogen) and CAPAN-2 cells were grown in McCoy's medium (Invitrogen). All media was supplemented with 10% (v/v) fetal calf serum (Invitrogen), 1% (v/v) non-essential amino acids (Invitrogen), 1% (v/v) sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 .mu.g/mL of streptomycin (Invitrogen), 100 U/ml penicillin (Invitrogen), and 0.28 .mu.g/mL of fungizone.

Results:

[0111] The ability of the compounds to inhibit cellular proliferation was assessed initially in SK- N-MC cells by the MTT assay. Compounds of Formula I where R= formula II (WBI-OC-01), formula II (WBI-OC-02), and formula III (WBI-OC-04) exhibited significant anti-proliferative activity in SK-N-MC cells (FIG. 2).

Example 2: In vitro Modulation of the Levels of Methemoglobin (metHb) by Iron Chelators

Methodology [0112] Chemicals: Triapine^® was synthesized and characterized according to published methods (Liu MC, Lin T C, et al (1992). J Med Chem 35: 3672-3677). Dp44mT, Bp4eT, DpC, di-2- pyridylketone-4-ethyl-4-methyl-3-thiosemicarbazone (Dp4e4mT), di-2-pyridylketone-4-phenyl- 3-thiosemicarbazone (Dp4pT) and di-2-pyridylketone-2-methyl-3-thiosemicarbazone (Dp2mT) were also synthesized and characterized using published procedures. Richardson DR, Sharpe P C, et al (2006). J Med Chem 49:6510-6521; Kalinowski D S, Yu Y, et al, (2007). J Med Chem 50: 3716-3729). All other chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA). For use in the assays described below, compounds were freshly prepared in DMSO and diluted (final [DMSO]<0.05°/o).

[0113] Red Blood Cell Isolation. Whole blood samples were collected from healthy human donors or mice in suitable blood collection tubes containing EDTA and used immediately. Red blood cells (RBCs) were isolated by centrifugation (480 χ g/5 min/4⁰ C.) then washed in Hank's balanced salt solution (HBSS). RBCs were resuspended 1:1 in HBSS and whole RBC assays were carried out at 37 °C.

[0114] Myoglobin Preparation: Mouse heart tissue was exhaustively perfused with ice-cold HBSS to remove blood and homogenized in ice-cold HBSS containing protease inhibitor cocktail (Roche, Basel, Switzerland). Heart homogenates were centrifuged (16,000.x g/45 min/4⁰ C.) and the supernatant ([oxyMb]=50 μΜ) used immediately.

[0115] Measurement of metHb by UV-Vis Spectrophotometry. The levels of metHb in RBC lysates were determined using a Shimadzu UV-Vis spectrophotometer (UV-1800; Shimadzu Corporation, Kyoto, Japan) at 577 nm and 630 nm for metHb and imetMb in accordance with published methods (Winterbourn CC and Carrell R W (1977). Biochem J 165: 141-148).

[0116] MetHb-Formation in Mice. C57BL6 mice (7-8 weeks-old) were used under a protocol approved by the University of Sydney Animal Ethics Committee. Dp44mT, DpC, or WBI-OC-01 - - 10 (all at 6 mg/kg) were dissolved in 30% propylene glycol/saline and administered iv via the tail vein. Subsequently, 30 min after administration, mice were anesthetized with isoflurane and blood samples obtained by cardiac puncture. Blood samples were lysed with 2.5 volumes of ultrapure water for metHb estimation. Mice were sacrificed with isoflurane, the heart exhaustively perfused with HBSS and Mb isolated.

[0117] Statistics. Data were compared using the Student's t-test. Results were considered significant when p<0.05. Results are mean ± SD.

Ability of Iron Chelators to Induce MetHb-formation in Human RBCs

[0118] The effect of chelator concentration (1-25 μΜ) on metHb-generation with intact RBCs was assessed (FIG. 3). A significant (p<0.001) dose-dependent increase in metHb relative to the control was detected at all ligand concentrations in intact RBCs treated with Dp44mT after 3 h/37° C. (FIG. 3). Significantly, WBI-OC-01, Dp4cycH4mT did not potentiate metHb formation in vitro (FIG. 12). This may constitute a clear advantage of DpcycH4mT therapy relative to

Triapine^®.

Claims

1. A compound of formula (I):

wherein R is selected from the group consisting of

and salts, hydrates and solvates thereof.

2. A compound according to claim 1, wherein the compound is a pharmaceutically acceptable salt.

3. A compound according to claim 2, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

4. A composition comprising a compound according to claim 1.

5. A composition according to claim 4, further comprising a pharmaceutically acceptable excipient, diluent or adjuvant.

6. A method of treating cancer in a mammal, comprising administering to a mammal in need thereof an effective amount of a compound according to claim 1.

7. A method of treating cancer in a mammal, comprising administering to a mammal in need thereof an effective amount of a composition according to claim 4.

8. The use of a compound according to claim 1 in the manufacture of a medicament for the treatment of cancer.

9. A method of inhibiting cellular proliferation, comprising contacting one or more cells with an effective amount of a compound according to claim 1.

10. The method of claim 9, wherein the cells are in vitro.

11. The method of claim 9, wherein the cells are mammalian cells.