WO2007088528A1 - Microtubule targeting agents - Google Patents

Abstract

The present invention provides for the use of a compound having the general formula (I), wherein: (i) A represents O or S; (ii) the cyclic group labelled F represents a benzyl group, a 2,3-naphthyl group or a benzyl group substituted with ethoxycarbonyl at the 2-position; (iii) and Y represents N(CH3)2, NHCH3, CH3 or pyridine; as an agent for one or more of the group consisting of an attenuating microtubule growth, depolymerising microtubules, arresting G2/M transition, activation of CDK1 activity, treatment of coronary heart disease, treatment of restenosis, treatment of arteriosclerosis and treatment of malaria.

Description

Title

Microtubule targeting agents Field of the Invention

The present invention relates to microtubule targeting agents and inhibitors of tubulin assembly. Background to the Invention

Microtubules are highly dynamic cytoskeletal fibres that are composed of α/β tubulin and play an important role in many physiological processes including phagosome movement (Blocker et al 1998), retinal development, fungal growth, embryonic development, cell motility, vesicle trafficking in the cytoplasm, maintenance of cell shape, organelle distribution, providing support for neuronal axons in the cytoplasm and especially in mitosis and cell division. Due to the fundamental importance of microtubules in so many biological processes, reagents that target microtubule activity or function are of great benefit in biological and biomedical research. Microtubules are involved in the separation of chromatids during mitosis and in the maintenance of cell shape during interkinesis. Various microtubule inhibitors are known. To date, these have in the main been obtained from microbial sources and chemical libraries. Due to the importance of microtubule function in mitosis, many microtubule disrupting agents have been used as chemotherapy agents.

The well characterized and clinically used antimitotic drugs, such as the taxanes (paclitaxel, docotaxel) and the Vinca alkaloids (vincristine, vinblastine etc.) bind to tubulin. Alternating α and β-tubulins polymerise to microtubules which constitute the mitotic spindles. Microtubule inhibitors disrupt the microtubule dynamics of tubulin polymerization and depolymerization, which results in the inhibition of chromosome segregation in mitosis and consequently the inhibition of cell division. The three major classes of agents that bind tubulin are the taxanes, which stabilize the microtubules by blocking disassembly, the Vinca alkaloids and lastly agents which bind to the colchicine site on tubulin. These latter two classes are microtubule-destabilizing agents that act by blocking assembly of tubulin heterodimers.

Nocodazole or methyl[5-(2-thienyl-carbonyl)-lH-benzimidazol-2-yl]-carbamate inhibits the polymerisation of free tubulin molecules by binding an arginine residue of the β-tubulin subunit. Paclitaxel is a natural (though quite toxic) substance typically derived via a semi-synthetic process from the yew tree to yield the chemical 5β,20-Epoxy-l,2α,4,7β,10β,13α-hexahydroxytax-l l-en-9- one 4,10-diacetate 2-benzoate 13-ester with (2 R,3iS)-N-benzoyl-3-phenylisoserine. The resultant drug is administered intravenously. It is used as a chemotherapy drug to treat patients with a wide variety of cancers, such as ovarian cancer, breast cancer and Kaposi's sarcoma. It is sold under the tradename Taxol®. Together with docetaxel, it forms the drug category of the taxanes. Despite the high cytotoxicity and strong anti-microtubule activity of taxanes, recent reports indicate that there may be additional side effects associated with these compounds. A recent report on clinical trials of taxanes in the treatment of breast cancer has disclosed that paclitaxel causes nerve damage, muscle pain and disturbances in heart rhythm, whereas docetaxel provokes mouth sores and a plunge in white blood cells. The exact cause of these side effects is unclear, and may not be entirely associated with the anti-microtubule affects of the drugs. Consequently, additional unknown biochemical effects potentially may compromise the use of these compounds in microtubule research; the side effects may furthermore be different depending on the cell type being researched. Therefore, there exists a need to develop new anti-microtubule compounds for use in research that do not provoke some or all of these additional biochemical reactions.

The underlying mechanisms by which many microtubule-targeting agents function remains largely unknown. For example, even amongst extensively studied chemotherapeutic agents known to target microtubules, the exact nature of the interaction is unknown in many cases and future studies are needed to identify how the drug functions. Such studies would yield important information for the development and refinement of the microtubule targeting agents in and of themselves, as well as provide insight into the nature of chemotherapeutic drugs and combination strategies. Much of such mechanistic work is performed on cell lines in culture. However, many cell lines are resistant to many microtubule-targeting agents in current use. This phenomenon is known as multidrug resistance (MDR). Most cell-based research makes use of cell lines due their immortality, but an associated problem is that these cell lines are by definition aberrant mutants, and a single cell's mutation under selective pressures can often give rise to a cell line displaying new found resistance. MDR is multifactorial, but the most consistent feature is amplification and over expression of genes encoding a plasma membrane protein known as the P-glycoprotein drug efflux pump (Aran et al., 1999). The vinca alkaloid group of microtubule targeting drugs which include vinblastine are extremely good substrates for P-glycoprotein and thus are of limited use in the study of drug resistant cell lines. Furthermore, resistance to paclitaxel has been shown to be mediated in part by P-glycoprotein. The search for new anti-microtubule agents has been vigorous with the goal of providing new agents that can circumvent cellular responses that confer MDR. There therefore exists a need for additional anti-microtubule agents to supplement the existing class of anti- microtubule compounds.

In the field of antineoplastic chemotherapy, antimicrotubule agents constitute an important class of compounds, with broad activity both in solid and in hematologic neoplasias. The taxanes are effective in the treatment of refractory ovarian cancer, metastatic breast cancer, non-small cell lung cancer and head and neck and bladder carcinomas (Crown et al., 2000; Rowinsky, 1997). The Vinca alkaloids have importance in the treatment of leukaemia, Hodgkin's disease, non-Hodgkin's lymphomas, testicular cancer, Kaposi's sarcoma, breast cancer and other malignancies (Jordan and Wilson, 2004). This antineoplastic activity is rooted in these compounds' ability to induce mitotic arrest and subsequent apoptotic cell death via the spindle assembly checkpoint. Anti-microtubule agent-induced mitotic arrest is associated with an upregulation and activation of cyclin B 1/cyclin- dependent kinase 1 (CDKl) activity in a variety of cell lines (Donaldson et al., 1994). Studies in both normal and transformed human cells, treated with anti-microtubule agents, have shown that apoptosis can be initiated rapidly and directly from mitotic arrest (Woods et al. 1995; Hayne et al. 2000; Fan et al. 2001; Zhou et al. 2002). In particular, a role for c-Jun-N-terminal kinase (JNK) signalling in the apoptotic response of cells to anti-microtubule agents has been suggested. JNK is a member of the mitogen-activated protein kinase family and becomes activated in response to a variety of stressful stimuli including UV-irradiation, growth factor deprivation, heat shock and chemotherapeutic drugs. JNK activity is also increased by anti-microtubule agents in a wide variety of cell types (Lee et al. 1998; Wang et al. 1998; Shtil et al. 1999; Wang et al. 1999), and inhibition of JNK inhibits paclitaxel- (Lee et al. 1998; Wang et al. 1999), vinblastine- (Brantley-Finley et al. 2003), nocodazole- and colchicine-induced apoptosis (Wang et al. 1998). Because JNK has been implicated primarily in stress responses, JNK activation by antimicrotubule agents may represent an acute response to microtubule damage. The phosphorylation and thus inactivation of the anti- apoptotic protein Bcl-2 has also been described as an important step from microtubule damage to apoptosis (Haldar et al. 1995; Blagosklonny et al. 1997) and many studies have implicated JNK in the upstream signalling pathway leading to Bcl-2 phosphorylation.

It has previously been shown that some members of a novel series of pyrrolo-l,5-benzoxazepine (PBOX) compounds potently induce apoptotic cell death in a variety of human chemotherapy resistant cancerous cells indicating their potential in the treatment of both solid tumours and tumours derived from the haematopoietic system (Zisterer et al. 2000; Mc Gee, et al. 2001; Mc Gee et al. 2002; Mc Gee et al. 2004). However, a fuller understanding of the molecular mechanisms underlying the apoptotic effects of these PBOX compounds would be essential to their development as therapeutic agents in the treatment of cancer. It has also been shown that activation of JNK is essential during PBOX induced apoptosis (McGee et al., 2001) and that Bcl-2 phosphorylation is a critical step in the apoptotic pathway induced by a representative PBOX compound, PBOX-6 (McGee et al., 2004). However, the precise method of action of these apoptotic compounds was not known.

In addition to treatment of cancer, microtubule inhibitors or attenuators are useful in the treatment of a number of conditions, including but not limited to psoriasis, restenosis, inflammatory disease states, myocardial infarction, glomerular nephritis, transplant rejection, and infectious diseases, including viral and bacterial infections. In addition to the need for further anti-cancer drugs, there also exists a need for further research tools into the functioning of microtubules, based on their fundamental role in a wide variety of biological processes. Microtubule inhibitors have been used broadly across many research applications, including immunology, cell signalling, karyotyping, cell motility, cell adhesion, cell morphogenesis, synaptic plasticity, and muscle contractility. Malaria remains the major tropical disease with more than 300 million people being infected and 3.5 million deaths annually. The widespread development of Plasmodium Falciparum (the causative agents of malaria) resistance to classical anti-malarial agents such as chloroquine has strengthened the efforts to develop new chemotherapeutic strategies. Microtubules play a crucial role in the life cycle of malarial parasites. They are one of the major cytoskeletal components and are involved in key steps of the life cycle such as formation of the mitotic spindle during parasite division and the exflagellation process during microgametogenesis. The microtubule-targeting agent taxol is known to inhibit the intraerythrocytic development of Plasmodium falciparum, by binding to tubulin and inhibiting mitotic spindle formation (Sinou et al., 1998). Thus taxol has potential as an anti-malarial agent. However, the drawbacks associated with known anti- microtubule reagents apply equally to their use as anti-malarials; for example, the development of resistance and existence of side effects. In addition, given the severity of malaria and the fact that both the parasite and the vectors may alter in their susceptibility to treatments over time there will always exist a need for the development of additional drugs.

The treatment of coronary artery disease remains one of the paramount problems in cardiology. Stenting is rapidly becoming the preferred method for the percutaneous treatment of coronary artert disease. In patients with coronary artery disease, restenosis after coronary stenting necessitates repeated percutaneous or surgical revascularisation procedures. The advent of drug-coated stents to prevent restenosis is a promising area of research. Use of slow release taxol-eluting stents have shown in recent clinical trials to be safe and, as compared with bare metal stents, to reduce the rates of clinical and angiographic restenosis by inhibiting or delaying smooth muscle proliferation (Stone et al., 2004). However, the drawbacks associated with known anti-microtubule reagents apply equally to their use as stent coatings; for example, the development of resistance and existence of side effects.

There therefore exists a need for additional anti-microtubule agents to supplement the existing classes of compounds. Object of the Invention It is an object of the invention to provide additional agents for attenuating microtubule growth to supplement the existing class of anti-microtubule compounds. It also an object of the invention to provide additional agents for disrupting microtubule assembly to supplement the existing class of anti-microtubule compounds. It is a further object of the invention to provide novel methods to attenuate microtubule growth and/ or disrupt microtubule assembly. It is an object of the invention to provide agents for use as antimalarial compounds. It is a further object of the invention to provide an agent that can act assist in the treatment of coronary heart disease. It is an additional object of the invention to provide an agent for the inhibition of the G2/M checkpoint. Summary of the Invention

The invention provides for the use of a compound having the general formula (I):-

wherein: (i) A represents O or S;

(ii) the cyclic group labelled F represents a benzyl group, a 2,3-naphthyl group or a benayl group substituted with ethoxycarbonyl at the 2-position;

(iii) and Y represents N(CH₃)₂, NHCH₃, CH₃ or pyridine; as an agent for one or more of the group consisting of an attenuating microtubule growth, depolymerising microtubules, arresting G2/M transition, activation of CDKl activity, treatment of coronary heart disease, treatment of restenosis, treatment of arteriosclerosis and treatment of malaria. The invention also provides methods of attenuating microtubule growth, depolymerising microtubules, arresting G2/M transition, activating CDKl activity, treatment of coronary heart disease, treatment of restenosis, treatment of arteriosclerosis and treatment of malaria comprising administering a compound of the invention, optionally together with a suitable carrier or diluent. In some embodiments of the invention, compounds of the invention may be used as stent coatings in the treatment of coronary or vascular disorders such as restenosis, arteriosclerosis, and coronary heart disease.

Preferably, the method or use of the invention employs one or more compounds selected from :- 7-[(Dimethylcarbamoyl)oxy]-6-(l-naphthyl)pyrrolo[2,l-d][l,5]benzoxazepine (PBOX 6);

4-Acetoxy-5-(l-naphthyl)naphtho[2,3-b]pyrrolo[2,l-d][l,4]oxazepine (PBOX 15);

7-[(Isonicotinoyl)oxy]-6-(l-naphthyl)pyrrolo[2,l-d][l,5]benzothiazepine (PBOX 65);

3-(Ethoxycarbonyl)-7-((dimethylcarbamoyl)oxy)-6-(l-naphthyl)pyrrolo[2,l-d][l,5] benzoxazepine

(PBOX 70); 4-[(Dimethylcarbamoyl)oxy]-5-(l-naphthyl)naphtho[2,3-b] pyrrolo [2,l-d][l,4] oxazepine (PBOX

16)

7-Acetoxy-6-( 1 -naphthyl)pyrrolo[2, 1 -d] [ 1 ,5]benzoxazepine (PBOX 4);

7-[(Methylcarbamoyl)oxy]-6-( 1 -naphthyl)pyrrolo[2, 1 -d] [ 1 ,5]benzoxazepine (PBOX 7);

7-Acetoxy-6-(l-naphthyl)pyrrolo[2,l-d][l,5]benzothiazepine (PBOX 9); 7-[(Dimethylcarbamoyl)oxy]-6-(l-naphthyl)pyrrolo[l,2-d][l,5]benzothiazepine (PBOX-8); and 7-

Acetoxy-6-( 1 -naphthyl)pyrrolo [ 1 ,2-d]pyrido [3 ,2-b] [ 1 ,4]oxazepine (PBOX 12).

Particularly preferred are the compounds PBOX-15, and PBOX-6.

The present invention also provides for a method of modulating microtubule polymerisation in a subject, said method comprising administering a therapeutically effective amount of at least one of the compounds of the invention. In some embodiments, of the invention, the invention provides for a method of treatment of diseases and conditions associated with microtubule proliferation and/or fidelity, said method comprising administering a therapeutically effective amount of at least one of the compounds of the invention.

In some embodiments of the invention, the invention provides use of the compounds in the manufacture of a medicament for the treatment of one or more of the group consisting of coronary heart disease, restenosis, arteriosclerosis, malaria and disorders associated with excess microtubule proliferation or activity. Favourably, the medicament further comprises a suitable carrier or diluent.

Preferably, the compounds of the invention are administered together with a diluent or carrier.

Preferred compounds of the invention are detailed below. Synthesis of the compounds of the invention are described in Campiani et al, 1996a and 1996b and in PCTYIEO 1/00020. Name Structure Formula MW

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1. PBOX-6-induced apoptosis is accompanied by G2/M arrest ; MCF-7 and K562 cells were treated either with vehicle (Control) or 10μM PBOX-6 for up to 48 h. Cells were harvested at the time points indicated as described in the Methods section and the percentage of cells in each phase of the cell cycle, the pre-Gl phase (A), Gl phase (B), S phase (C) and G2/M phase analysed using flow cytometry. The pre-Gl phase represents apoptotic cells. The results represent the mean ± S.E.M of 6 separate experiments.

Fig 2.PBOX-6 treatment induces morphological features of prometaphase arrest in MCF-7 cells ; Microscopic analysis of MCF-7 cells was performed following treatment with either vehicle (0.5% (v/v) ethanol (A)), lOμM PBOX-6 (B), lOμM nocodazole (C) or lOμM paclitaxel (D) for 8 hours. Following treatment, cells were centrifuged onto a glass slide and stained using the Rapi Diff kit. The cells were visualised under a light microscope (Nikon) at a magnification of 4OX. The results shown are representative of 3 separate experiments.

Fig 3. PBOX-6 induces an upregulation in CDKl kinase activity in a manner comparable with paclitaxel and nocodazole ; Whole cell extracts were prepared from MCF-7 cells following treatment with either lOμM PBOX-6 (A), paclitaxel (B) or nocodazole (C) for the indicated times. Protein (200-500μg) was ininiunoprecipitated with anti-CDKl antibody and Protein A beads and then incubated with Histone Hl substrate and γ³²P-ATP and resolved by SDS-PAGE. Densitometry was performed on the resultant bands using Scion Image software with 0 hours vehicle set to 100%. The results represent the mean ± S.E.M of 3 separate experiments. Statistical analysis was carried out using the Instat computer program. * p<0.05, **p<0.01 with respect to 0 hours vehicle, Students t-test. Fig 4.PBOX-6 induces an upregulation in cyclin Bl expression in a manner comparable with paclitaxel and nocodazole ; Whole cell extracts were prepared from MCF-7 cells following treatment with either vehicle (0.5% (v/v) ethanol) or lOμM PBOX-6 for the indicated times. Protein (50μg) was resolved using SDS-PAGE, transferred onto PVDF and probed with anti-cyclin B₁ antibody (A). Results were compared with an immunoblot of samples prepared from MCF-7 cells following treatment with either vehicle (0.5% (v/v) DMSO), paclitaxel or nocodazole for the indicated times (B). The results shown are representative of 4 separate experiments. Fig. 5. Effect of PBOX-6, PBOX-15, PBOX-21, paclitaxel and nocodazole upon the organisation of MCF-7 cellular microtubule network ; MCF-7 cells were treated with either vehicle (0.5% (v/v) ethanol (A), 10OnM, lμM or lOμM PBOX-6 (B, C and D respectively), lμM nocodazole (E), lμM paclitaxel (F), lμM PBOX-15 (G) or 25μM PBOX-21 for 16 hours. After this time, the medium was carefully removed and the cells fixed in cold methanol. Cells were incubated with monoclonal anti-α-tubulin antibody at room temperature for 1 hour at room temperature, and then incubated with FITC-conjugated anti-mouse secondary antibody for a further hour at room temperature. After washing, the cells were stained briefly with propidium iodide. The organisation of the microtubule network (green) and the cellular DNA (red) was visualised by Nikon PS200 fluorescence microscopy at a magnification of 10OX. The results shown are representative of 3 separate experiments.

Fig. 6. PBOX-6 causes a dose-dependent depolymerisation of tubulin, whilst PBOX-21 has a negligible effect upon polymerisation status of tubulin ; Purified bovine tubulin was incubated at 37⁰C in the presence of either vehicle (0.5% (v/v) ethanol or DMSO) or the indicated concentrations of PBOX-6 (A), nocodazole (B), paclitaxel (C), or PBOX-21 (D). Tubulin polymerisation was determined by measuring the increase in absorbance over time at 340 nm. The results shown are representative of three separate experiments. Fig. 7. PBOX-6 does not bind to the colchicine or vinblastine binding site on purified tubulin ; Each reaction mixture contained lmg/ml >99% purified bovine tubulin, 0.25mM PIPES buffer at pH 6.9, containing 0.05mM GTP, and 0.25mM MgCl_2, in the presence or absence of either excess unlabelled colchicine (lOOμM) (A) or vinblastine (lOOμM) (B). The colchicine and vinblastine binding assays were performed as described in Materials and Methods. Each reaction mixture contained either 0.5 μM FITC-labelled colchicine (A) or 0.5 μM [³H] vinblastine (B) and was incubated for 30 minutes at 37°C. When testing the ability of a test compound to bind to either of these sites the reaction was performed in the presence or absence of the indicated test compounds (lOμM) or the appropriate vehicle control (ethanol for PBOX-6 and DMSO for other compounds). The data presented is representative of 2 separate experiments. Figure 8. P-glycoprotein expressing A2780-Adr cells are more resistant to the anthracycline antibiotic adriamycin than parental cells ; (A) Parental and adriamycin-resistant A2780 cells were treated with a range of concentrations of adriamycin for 72 hrs at which point % cell viability was quantitified by AlamarBlue™ assay (Biosource). (B) Whole cell lysates from parental, adriamycin- resistant and cisplatin-resistant A2780 cells were prepared, proteins separated by SDS-PAGE and transferred to PVDF. Blots were then probed with a monoclonal anti-P-glycoproten antibody (Merck Biosciences) followed by a HRP-conjugated anti-mouse secondary (Promega). Blots were also probed for b-actin (Merck Biosciences) as a loading control.

Figure 9. A2780-Adr are more resistant to paclitaxel (A) and vincristine(B) than parental A2780 cells ; Parental and adriamycin-resistant A2780 cells were treated with a range of concentrations of MTA' s vincristine (A) and paclitaxel (B) for 72 hrs at which point % cell viability was quantitified by AlamarBlue assay.

Figure 10. PBOX-6 (A) & PBOX-16 (B) reduce cell viability with a similar efficacy in both parental and p-glycoprotein expressing adriamycin-resistant A2780 cells ; Parental and adriamycin- resistant A2780 cells were treated with a range of concentrations of PBOX-6 (A) and PBOX 16 (B) for 72 hrs at which point % cell viability was quantitified by AlamarBlue assay. EXPERIMENTAL PROCEDUItES

Mater ϊals-MCF-7 human breast carcinoma breast cells were obtained from European Collection of Cell Cultures (ECACC). K562 human chronic myelogenous leukaemia cells were gratefully received from Prof. Mark Lawler (St. James's Hospital, Dublin). The pyrrolobenzoxazepine 7- [(dimethylcarbamoyl)oxy]-6-(2-naphthyl)pyrrolo-[2,l-cf] (l,5)-benzoxazepine (PBOX-6) and pyrrolobenzoxazepine (PBOX-15) were synthesized as described previously. The ApopTag Plus Kit was obtained from Chemicon International Inc. (Temecula, California, USA), the RapiDiff kit was obtained from Diagnostic Developments (Burscough, Lancashire, UK) and the CytoD YNAMIX Screen™3 was from Cytoskeleton Inc. (Denver, Colorado, USA). Colchicine, nocodazole, paclitaxel, vincristine and vinblastine were purchased from Sigma (Poole, Dorset, UK). [³H]Vinblastine was acquired from Amersham Biosciences (Buckinghamshire, UK), and the fiuorescein-labelled colchicine was obtained from Cytoskeleton Inc. The PBOX compounds were dissolved in ethanol, whilst the remaining drugs were dissolved in dimethyl sulfoxide. Control samples contained equivalent amounts of solvent. Anti-α-tubulin and FITC-conjugated goat anti- mouse were purchased from Sigma, anti-cyclin A, anti-cyclin Bi and anti-CDKl antibodies were obtained from BD Pharmingen (Cowley, Oxford, UK). Anti-JNK antibody was purchased from Santa Cruz (Heidelberg, Germany) and anti-PARP antibody was obtained from Merck Biosciences (Beeston, Nottingham, UK). Histone Hl and GST-c-Jun substrates were purchased from Sigma and Cell Signaling Technology Inc. (Beverly, Massachusetts, USA) respectively. The ECL detection system and [γ³²P] ATP were purchased from Amersham Biosciences. Unless stipulated, all other reagents were from Sigma.

Cell Culture- MCF-7 and K-562 cells were grown at 37⁰C under a humidified atmosphere of 95% O₂ and 5% CO₂. The MCF-7 cells were maintained in Minimum Essential Medium (MEM) whilst the K562 cells were maintained in RPMI 1640 medium. Both media were supplemented with 10% (v/v) Foetal Calf Serum (FCS), 2mM L-glutamine and lOOμg/ml gentamicin (complete medium). The MEM was also supplemented with 1% (v/v) non-essential amino acids.

Flow Cytometric Analysis-Ceils were harvested by trypsinisation and following centrifugation were washed with PBS and incubated in ice-cold 70% ethanol in PBS at 4°C. The samples were then centrifuged to remove the ethanol, resuspended in ImI of staining solution (lOμg/ml RNase A, lOOμg/ml propidium iodide) and incubated in the dark for 30 min at 37°C. Flow cytometric analysis was performed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson). RapiDiff staining of cells- MCF-7 cells were seeded at a density of 1 x 10⁵ in a volume of ImI per well of a 24-well plate. Following treatment with the indicated compounds, a 500μl aliquot of the cells was treated with ImM EDTA so as to prevent clumping of the cells. The cells were then resuspended thoroughly and cytocentrifuged on to poly-L-lysine coated slides at 700xg for 2 minutes. The slides were stained with the RapiDiff kit (eosin/methylene blue) under conditions described by the manufacturer. Immunoprecipitation and Kinase Activity Assays-Cells were lysed in IP lysis buffer containing

5OmM (HEPES), pH 7.5, 15OmMNaCl, ImM EDTA, 2.5mM EGTA, 10% (v/v) glycerol, 0.1% (v/v) Tween 20, ImM DTT, ImM NaF, 1OmM β-glycerophosphate, and protease inhibitors (lOμg/ml leupeptin, lOμg/ml aprotinin, 0. ImM PMSF and 0.ImM sodium orthovanadate). The DTT, NaF, β-glycerophosphate and protease inhibitors were added fresh to the IP buffer. The protein content of the supernatant was determined by Bradford assay. The appropriate antibody (lμg) was added to equal amounts of protein (100-500μg) in each cell lysate sample. The samples were incubated for 3 hours at 4°C. A 50% slurry of protein A beads in PBS (50μl) was then added to the samples and incubated overnight at 4⁰C. The samples were then centrifuged for 2 minutes at 500xg and the beads washed. The precipitates were washed 3 times in ImI of ice-cold IP lysis buffer and then used either for kinase activity assay or resuspended in 20μl of 3X SDS sample buffer and resolved by SDS-PAGE for western blot analysis. For kinase activity assays, following washes with ice-cold IP lysis buffer, the immune-complex beads were washed 3 times with ice- cold kinase buffer which contained the following: 5OmM HEPES, pH 7.5, 2.5mM EGTA, 1OmM MgCl₂, ImM DTT, 1OmM β-glycerophosphate, ImMNaF and 0.ImM sodium orthovanadate. The kinase reactions were performed by incubating the immunoprecipitates in 30μl of kinase buffer supplemented with 20μM unlabelled adenosine triphosphate (ATP), 2μCi [γ³²P] ATP and either lOμg Histone Hl substrate for CDKl or lμg of GST-c-Jun substrate for JNKl, and incubating them at 30°C for 30 minutes. The kinase reactions were stopped by resuspending the samples in an aliquot of 3X SDS sample buffer (20μl) and resolved by SDS-PAGE. The gel was dried under a vacuum and exposed to Kodak X-Omat film at -70⁰C. Phosphorylation was quantitated using a densitometric scan using the Scion Image software package of the phosphorylated bands in the autoradiogram (© 2000 Scion Corporation, based on NIH Image for Macintosh). Western Blot analysis-Cells were lysed in lysis buffer containing 5OmM Tris-HCl, pH 7.5, 25OmM NaCl, 5mM EDTA, 0.1% (v/v) Triton, 5OmM sodium fluoride, protease inhibitors (lμg/ml aprotinin, lμg/ml leupeptin, lμg/ml pepstatin A, ImM sodium orthovanadate, lOOμg/ml phenylmethylsulfonyl fluoride (PMSF)) and ImM dithiothreitol (DTT), and the protein concentration of the resultant supernatants were determined by a Bradford assay. For Western blot analysis of PARP, the cells were resuspended in PARP sample buffer (200μl) (62.5mM Tris/HCL pH 6.8, 6M urea, 10% (v/v) glycerol, 2% (w/v) SDS, 0.00125% bromophenol blue and 5% (v/v) β- mercaptoethanol added immediately before use), sonicated for 15 seconds and heated at 65°C for 15 minutes. Equal amounts of lysate was resolved by SDS-PAGE and transferred onto 0.2μm PVDF membrane. The PVDF membrane was blocked in blocking buffer containing 5% (w/v) dry milk in TBST (0.1% (v/v) Tween 20 in TBS) at room temperature for 1-3 hours. Following blocking the immunoblot was incubated in primary antibody overnight at 4°C, and in horseradish peroxidase-labeled secondary antibody for 1 hour at room temperature according to manufacturer's instructions. The immunoblots were analysed using the ECL detection system. Indirect Immunofluorescence-Cells, seeded upon coverslips were washed gently with ImI of PBS and fixed in 100% methanol at -20⁰C for 10 minutes, after which time they were washed three times with PBS. The cells were then blocked at room temperature for 30 minutes in 5% BSA made up in PBST (0.1% (v/v) Triton in PBS). The cells were incubated for 1 hour at room temperature in primary antibody to α-tubulin, washed 3 times with PBS and then incubated for a further hour at room temperature in FITC-conjugated goat anti-mouse, after which time they were washed a further 3 times with PBS. The cells were then incubated with 0.2μg/ml PI made up in blocking buffer for 2 minutes. To each coverslip, 5μl of a 2μg/ml phenylethylenediamine in a 50:50 solution of PBS and glycerol was applied to the surface of each slide. The slides were then immediately mounted and viewed at a magnification of 100Ox with a Nikon microscope and images captured using ImageCapture software. Tubulin Polymerisation Assay-The, assembly of >99% purified bovine tubulin was monitored using the CytoD YNAMIX Screen™3. Purified bovine brain tubulin was resuspended on ice in ice-cold G-PEM buffer (8OmM PIPES pH 6.9, 0.5mM MgCl₂, ImM EGTA, ImM GTP, 5% (v/v) glycerol) and a lOOμl volume (300μg) was pipetted into the designated wells of a half area 96-well plate prewarmed to 37°C. Each compound tested was made up in G-PEM buffer. The assay was conducted at 37°C and tubulin polymerisation was followed turbidimetrically at 340nm in a

Spectramax 340PC spectrophotometer (Molecular Devices). The absorbance was measured at 30 second intervals for 1 hour.

Colchicine Tubulin Competition Binding Assay-Each, reaction was performed in 0.25mM PIPES buffer at pH 6.9, containing 0.05mM GTP, and 0.25mM MgCl₂, containing test compound at the appropriate concentration and >99% purified tubulin stock (lmg/ml). The reaction mixture was mixed gently and incubated at 37°C for 30 minutes, after which time, fluorescent colchicine was added to each reaction giving a final concentration of 0.5μM. This was then incubated for a further 30 minutes at 37°C. Each reaction was then applied to a G25 Sephadex™ chromatography column and the eluate from the columns collected in designated wells of a 96-well plate. When the eluate from the application of the reaction mixture had been collected, 160μl of buffer was applied to the top of the column and the eluate collected in a fresh well. This was continued until 12 wells were filled per reaction. The plate was then read on a Spectramax Gemini XS fluorescence 96-well spectrophotometer (Molecular Devices), at excitation and emission wavelengths of 485nm and 535nm respectively.

Vinblastine Tubulin Competition Binding Assay-The ability of a ligand to bind to the vinblastine binding site on tubulin was assayed as described for determination of binding to the colchicine binding site, utilizing a radio labeled instead of a fluorsecent analogue. The binding of radiolabeled [³H]-vinblastine was used and its presence within the collected fractions measured by liquid scintillation counting. Each reaction contained a final concentration of 0.5μM [³H]- vinblastine, and >99% purified bovine brain tubulin (lmg/ml). The separation of the bound [³H]- vinblastine from unbound [³H] -vinblastine was achieved using a G50 sephadex™ column. EXAMPLES

PBOX-6 Induced Apoptosis is Preceded by an Accumulation of Cells in G2/M- G2/M inhibition accompanied by persistent CDKl activation is a hallmark of blockage of mitosis. Some agents (such as taxol) that block cells in mitosis interact directly with tubulin but there are many others such as monastrol and 13-hydroxy-15-oxozoapatlin (OZ) that do not. Monastrol is an inhibitor of the kinesin protein. This protein along with tubulin and others is important in the formation of the mitotic spindle. The target of OZ is still unknown (it has been shown NOT to affect tubulin dynamics). It is thought to inhibit a spindle associated motor protein that is required for chromosome congression. DNA damaging agents (which are widely used as chemotherapeutic agents) such as SN-38 are known to induce G2/M arrest and apoptosis in many tumour cells but these agents do not bind to tubulin and do not interfere with microtubules.

Surprisingly, it was found that the compounds of the invention can also function to arrest cells in G2/M phase. Fig. 1 demonstrates that PBOX-6 induced apoptosis (as measured by the pre-Gl peak in FACS analysis) of human breast MCF-7 and CML K562 cells was preceded by an accumulation of cells in the G2/M phase. Tunel staining and PARP cleavage confirmed that the PBOX-6 induced increase in the pre-Gl peak was due to apoptotic cell death (results not shown). This detailed time course of the changes in distribution of cell cycle phases in response to PBOX-6 treatment also demonstrates that PBOX-6-induced apoptosis was temporally preceded by arrest in the G2/M phase of the cell cycle. PBOX-6 Induces Morphological Features of Prometaphase Arrest in MCF-7 Cells- There are many known agents (such as OZ) that do not target microtubules but do arrest cells in early mitosis i.e. prometaphase (see Rundle et al., 2001), although some microtubule targeting agents are known to arrest the cell cycle in early mitosis i.e. prometaphase/metaphase. The morphological features of MCF-7 cells were analysed following treatment with PBOX-6 and two representative antimicrotubule agents, paclitaxel and nocodazole as controls (Fig. 2). Cells treated with each of the compounds displayed distinct signs of arrest in the prometaphase stage of mitosis as the nuclear membrane has disappeared and the chromatin was condensed. This suggested the surprising fact that PBOX-6 acts as a mitotic inhibitor. Similar results were obtained in K562 cells, indicating that prometaphase arrest induced by PBOX-6 is not restricted to MCF-7 cells (data not shown). Effect of PBOX-6 on Cyclin Bl Expression and CDKl Activity- It was next necessary to determine what effect PBOX-6 treatment had upon CDKl kinase activity, as it is responsible, through association with its cyclin partner, cyclin B₁, for driving the cell through G2 and mediating both entrance into and exit out of the M phase of the cell cycle. Paclitaxel and nocodazole-induced M-phase arrest is associated with upregulation and activation of cyclin B/CDK1 kinase in a variety of cell lines (King et al. 1994; Wang et al 1998), thus treatment with these drugs served as a control. PBOX-6, paclitaxel and nocodazole treatment was found to cause a time-dependent increase in CDKl kinase activity relative to the vehicle control (Fig. 3A, B and C respectively). In order to address the mechanism by which CDKl activity was being stimulated, the expression levels of both CDKl and the cyclins known to associate with it, namely cyclin A and Bl, were next analysed. There was no alteration in protein expression of either cyclin A or CDKl prior to or coincident with the increase in CDKl activity following treatment with each of the compounds (results not shown). Analysis of expression of cyclin Bl did however reveal a marked increase in cyclin Bl after treatment with either PBOX-6 (Fig. 4A), paclitaxel or nocodazole (Fig. 4B), which directly correlated with the increase in CDKl activity for each of these compounds (see Fig 3). Immunoprecipitation experiments confirmed that the PBOX-6 induced CDKl activity was associated with cyclin Bl (data not shown). Similar results were also obtained in K562 cells, indicating that activation of cyclin B/CDK1 kinase caused by PBOX-6 is not restricted to MCF-7 cells (data not shown). Induction of cyclin B1/CDK1 kinase activity is a hallmark associated with mitotic arrest indicating that PBOX-6 arrests cells in the G2/M phase of the cell cycle.

PBOX-6 Causes a Dose-Dependent Disruption of MCF-7 Cellular Microtubule Network-Some anti-microtubule agents are known to target the cellular microtubule network, resulting in aberrant formation of the mitotic spindle, subsequent blockage of the cell cycle in G2/M-phase and apoptotic cell death (Wassmann and Benezra 2001). Because PBOX-6 markedly blocked the cell cycle in M-phase and induced apoptotic cell death, we tested whether PBOX-6 could directly affect the organization of the microtubule network of cells, as there were a number of alternate routes through which the action could be exerted, such as affecting spindle associated motor proteins. MCF-7 cells were treated with either a range of concentrations of PBOX-6 (10OnM to lOμM), paclitaxel (lμM), nocodazole (lμM) or with the same volume of vehicle as control. After 16 h of incubation the microtubule network was visualized by indirect immunofluoresence. The microtubule network in control cells exhibited normal arrangement with microtubules seen to traverse intricately throughout the cell and these cells displayed a normal compact rounded nucleus (Fig 5A). Surprisingly, in contrast, PBOX-6 caused a dose-dependent loss of microtubule network with only a diffuse stain visible throughout the cytoplasm (Fig 5B-D). These effects are similar to that exerted by nocodazole (Fig 5E) a known microtubule depolymerizer. Paclitaxel, on the other hand, exerts its effects by stabilizing the microtubule network, and resulted in a distinctive rigidity of the microtubules causing them to form microtubule 'bundles' (Fig 5F). PBOX-21 is a non-apoptotic member of the PBOX series of compounds and in fact induces Gl arrest within MCF-7 cells without any associated cytotoxicity (Mulligan et al., 2003).

To test the hypothesis that the differential response of the non- and pro-apoptotic subsets of PBOX compounds is based upon their ability to disrupt the microtubule network, the effect of PBOX-21, as well as a second potent pro-apoptotic PBOX compound, PBOX- 15 on the microtubule network of MCF-7 cells was examined. It was found that, similar to PBOX-6 (lOμM), PBOX-15 (lμM) (Fig 5G) appeared to induce a loss of the microtubule network of the cell. On the other hand, PBOX-21 (25μM) (Fig. 5H) had no effect upon the MCF-7 microtubule network and, as such, PBOX-21 may be considered a negative control. Collectively these results indicated the surprising result that the microtubule is the intracellular target for pro-apoptotic PBOX compounds such as PBOX-6.

Effect of PBOX-6 on Tubulin Polymerization in Vitro- Because PBOX-6 markedly disrupted the cellular microtubule network, we tested whether PBOX-6 could directly affect tubulin, the main component of this network. To test this hypothesis, tubulin polymerization and depolymerization in vitro were studied at 37°C in a reaction mixture containing purified tubulin and GTP. Results presented in Fig. 5 A show that PBOX-6 inhibited the polymerisation of tubulin in a dose- dependent manner, similar to the effect elicited by nocodazole (Fig. 5B). Similar results were obtained with the other representative pro-apoptotic PBOX compound, PBOX-15 (data not shown). Paclitaxel, in agreement with previous reports was shown to promote tubulin polymerization in a concentration-dependent manner (Fig. 5C). In contrast, PBOX-21 was found to have a negligible effect upon the polymerisation of tubulin in vitro relative to the vehicle control (Fig. 5D), in agreement with its lack of effect upon the cellular microtubule network. This again, surprisingly distinguishes the mechanism of action of the non- and pro-apoptotic subsets of the PBOX compounds, and for the first time identifies tubulin as the molecular target of the pro-apoptotic PBOX members.

Lack of Binding ofPBOX-6 to the Vinblastine and Colchicine Binding Sites on Tubulin- As PBOX-6 has been shown to directly bind to and cause depolymerisation of purified tubulin, it was necessary to gain information about the binding site of PBOX-6 on tubulin. To date, only the binding sites of the depolymerisers colchicine (Serrano et al. 1984; Uppuluri et al. 1993; Ravelli et al., 2004) and vinblastine (Rai and Wolff 1996) on tubulin have been well characterised. Compounds capable of depolymerising tubulin may bind to either of these two sites, or to an as yet uncharacterised novel site on tubulin. Assessment of the binding of PBOX-6 to the colchicine binding site involved a fluorescence-based assay. Excess unlabelled colchicine (lOOμM) or nocodazole (lOμM), which is known to bind to the colchicine binding site (Friedman and Platzer 1978; Uppuluri et al. 1993), displaced the binding of fluorescently-labelled colchicine relative to the vehicle control (Fig. 7A). PBOX-6 at a concentration of lOμM however did not displace the binding of labelled colchicine suggesting that PBOX-6 does not bind to the colchicine binding site on tubulin.

The ability of PBOX-6 to bind to the vinblastine binding site on tubulin was determined using a [³H]vinblastine displacement assay. Excess unlabelled vinblastine (lOOμM) and also vincristine (lOμM) (which binds to the vinblastine binding site) was able to displace [³H]vinblastine relative to their vehicle control (0.5% DMSO v/v), causing a reduction in the radioactive signal in the fractions containing tubulin, i.e. fractions 5-9 (Fig. 7B). In contrast, lOμM PBOX-6 did not cause a reduction of the radioactive signal relative to the PBOX-6 vehicle control in the fractions containing tubulin, suggesting that PBOX-6 does not bind to the vinblastine binding site. Any residual [³H]vinblastine that did not bind tubulin, or [³H]vinblastine that had been displaced from binding to tubulin by a vinblastine binding site competitor, was seen to elute between fractions 10 and 30. These combined results from the colchicine and vinblastine displacement assays indicate that PBOX-6 may have its own novel binding on tubulin. Multiple drug resistance

In order to explore the efficacy of compounds of the invention as alternative reagents for use with MDR cell lines, a comparative study was undertaken using paired human A2780 ovarian cells, a parental cell line and one over-expressing P-glycoprotein which makes these cells resistant to many chemotherapeutic agents such as adriamycin. Thus, the human ovarian cancer cell line, A2780, and its adriamycin-resistant sub-line A2780-Adr were treated with either PBOX-6, PBOX-16 or other microtubule targeting agents (MTAs) paclitaxel or vincristine and percent cell viability was quantified by AlamarBlue assay.

We confirmed that adriamycin-resistant A2780 cells (A2780-Adr) displayed 118-fold more resistance to the anthracycline antibiotic adriamycin than parental A2780 cells [Fig. 8A] and that this resistance was associated with expression of P-glycoprotein [Fig. 8B].

Table of IC50 Values

Common microtubule-targeting drugs paclitaxel and vincristine reduced cell survival to a much greater extent in parental A2780 cells in comparison to A2780-Adr cells [Fig 9]. In contrast, PBOX-6 and -16 reduced cell viability with a similar efficacy in both parental and p-glycoprotein expressing Adr-resistant A2780 cells [Fig. 10].

These results suggest that while the efficacy of paclitaxel and vincristine appear to be affected by the expression of P-glycoprotein, the efficacy of PBOX-6 and -16 do not. This indicates that pro- apoptotic PBOX compounds may have the potential to treat p-glycoprotein-associated MDR cancers. These results also show that PBOXs may have an advantage over many currently used microtubule targeting agents such as paclitaxel and vincristine which are limited by the fact that they are good substrates for P-glycoprotein and can thus be rapidly removed from cells over- expressing this MDR protein.

Stents In vitro tests for cardioactivity/toxicity of PBOX-6, namely calcium channel antagonist inhibition analysis and cardiac vasodilation analysis, demonstrate a lack of cardioactivity/ toxicity of our PBOX compounds.

Table 1. Lack of inhibition of ³f] E] nitrendipine binding to rat heart by PBOX-6

Ki values for displacement of binding to rat heart L-type calcium channels. PBOX-6 is compared to two positive controls (Diltiazem and Verampil) of calcium antagonist activity.

TABLE 2. LACK OF CARDIOVASCULAR ACTIVITY OF PBOX-6

Vasorelaxing activity, which is a measure of calcium antagonism, was assessed on depolarised guinea pig aorta strips. Diltiazem was used as a positive control. Both compounds were tested at a concentration of 10^"4 M.

In addition, results from an in vivo toxicity study demonstrated that healthy Balb/c mice treated with PBOX-6 exhibited no lethality, no difference in weight and no effect on haematology parameters (including white bloods cells) at the end of the treatment regimen (McGee et al., 2005).

In order to assess the potential of the PBOX compounds in the treatment of restenosis a study is required to characterise the effects of PBOXs on proliferation and migration of human arterial smooth muscle cells (haSMCs) in vitro as previously described (Axel et al., 1997). For the in vitro study, initially monocultures of haSMCs will be used. Cell growth will be determined by an MTT assay after 4, 8 and 14 days in the presence or absence of the PBOX compound. If the PBOXs are shown to have antiproliferative activity a further study will be performed with cocultures of haSMCs with human arterial endothelial cells (haECs). Cell growth in the absence or presence of various mitogens such as PDGF and bFGF will again be determined after 4, 8 and 14 days in the presence or absence of the PBOX compound. The additional studies with growth-factor stimulated haSMCs and with the coculture system will be performed to imitate the complexity of the in vivo cell interaction, which can certainly influence the efficacy of antiproliferative drugs. Despite promising early results with paclitaxel-coated stents, there is some concern about the long- term outcomes. While decreasing the rate of restenosis, histologic analysis of the vessels with paclitaxel-coated stents showed less healing of vessel walls after stenting. There was chronic low grade inflammation, poor healing of the endothelium and intra-intimal hemorrhage (Winslow et al., 2005). Therefore, there exists a need to develop new anti-microtubule compounds for use in coating of stents that do not provoke some or all of these additional reactions. The results from the in vivo toxicity study demonstrated that healthy Balb/c mice treated with PBOX-6 exhibited no effect on haematology parameters (including white bloods cells) at the end of the treatment regimen suggesting that these PBOX compounds may provoke less side-effects than paclitaxel. Malaria

The causative agent in malaria in humans is lof 4 species of Plasmodium with Plasmodium falciparum causing both the most severe and common forms of the diseases. Of the limited number of drugs available more and more are becoming obsolete as the parasites rapidly develop resistance. Much hope has been placed in the development of vaccines, but the high degree of antigenic variation exhibited by the parasites is a difficult hurdle to surmount. In the absence of effective, long term vaccines, drug development remains fundamental in the fight against malaria. The present invention provides evidence that the compounds of the invention have activity against P. falciparum in culture suggesting that they may have potential as novel anti-malarial agents.

Table 3: PBOX-6 and PBOX-16 induces cytotoxicity of Plasmodium falciparum

Cytotoxicity of P. falciparum was measured with a range of concentrations of each compound for 48 and 72h using an LDH parasite assay as previously described (Monaghan et al., 2005) and IC50 values were determined. Values represent the mean of triplicate determinations. N.D.: not determined.

P. falciparum 3D7, a chloroquine-sensitive strain were maintained in continuous culture. The effect of two representative PBOX compounds, PBOX-6 and -16, and an unrelated microtubule targeting agent, vinblastine, was examined. Asynchronous parasitized human erythrocytes at 0.8% parasitemia and 2% hematocrit were grown, for 48 or 72 hours, in RPMI medium supplemented with the compounds or its vehicle (ethanol) in 96-well microtiter plates. The drug was diluted from stock solutions in ethanol into wells of the microtiter plate, down to subinhibitory concentrations. After incubation, the effect of the compounds on parasite growth was determined by the use of the parasite lactate dehydrogenase asasay as previously described. Dose response curves were constructed and IC50 values were obtained. The vehicle had no effect. IC-50 values in the low micromolar range were determined for the compounds tested. Results obtained with the PBOXs compared favourably to the result obtained with the other microtubule targeting agent tested, vinblastine.

Other microtubule targeting agents, such as colchicines, taxol and nocodazole have previously been demonstrated to show activity against the growth of P. falciparum in culture (Schrevel et al., 2002), in a similar assay to that described above, and have been described as promising candidates in the treatment of malaria. Schrevel and his co-workers determined IC50 values in the range of 10-20 micromolar for colchicines and nocodazole and a lower value of 7OnM for taxol. Results with two representatives of the present invention, PBOX-6 and -16, although not tested in exactly the same assay, compare favourably with those of colchicine and nocodazole, suggesting that it may have potential in the treatment of malaria. DISCUSSION Although PBOX-6 has previously been shown to induce apoptosis in many human tumor cell lines, the mechanism of action by which PBOX-6 induces apoptosis remained incomplete. It has been previously shown that activation of JNK is essential during PBOX induced apoptosis (McGee et al., 2001) and that Bcl-2 phosphorylation is a critical step in the apoptotic pathway induced by a PBOX-6 (McGee et al., 2004), but the molecular target of PBOX-6 remained to be identified. The present invention surprisingly demonstrates that apoptosis induced by PBOX-6 in human MCF-7 and K562 cells, is preceded by a marked G2/M arrest. The cells displayed morphological features that distinguished a mitotic arrest specifically in prometaphase. During prometaphase, the DNA chromatin network which was duplicated in the S period of interphase begins to twist and fold, eventually forming compacted chromosomes. The nuclear envelope breaks down allowing the nucleoplasms contents to come into contact with the spindle network, as the cell prepares to align its duplicated chromosomes during metaphase for separation by the mitotic spindle during anaphase (Crespo et al. 2001). The morphological effects elicited by PBOX-6 were similar to that initiated by two microtubule-targeting drugs, paclitaxel and nocodazole, a representative polymeriser and depolymeriser respectively. This in turn is in agreement with reports which show that anti-microtubule agents arrest the cell cycle in prometaphase (Woods et al. 1995; Li and Broome 1999).

In eukaryotic cells, cell cycle progression is regulated through the activation and inactivation of cyclin-dependent kinases, cyclins, and other regulatory factors. Inappropriate alteration in the expression and/or activation of cyclin-dependent kinases and regulators can lead to blockade of cell cycle progression and induction of apoptosis. It is widely reported that CDKl/cyclin B complexes are involved in the regulation of the G2/M phase and the M-phase transition. In the present study we show that PBOX-6 treatment surprisingly led to an increase in cyclin B level and stimulation of CDKl activity with a pattern similar to both paclitaxel and nocodazole. As the G2/M arrest profile paralleled the profile of induction of cyclin Bl expression and CDKl activity following treatment with PBOX-6, this would suggest that sustained activation of CDKl kinase activity by this compound is essential for the cessation of the cell cycle at the G2/M phase. The cell cycle is subject to many regulatory processes including checkpoints. Such checkpoints monitor DNA status and integrity and prevent inappropriate transitions into S phase (DNA synthesis) and M phase when problems are detected. Although the Gl/S checkpoint pathway is quite well understood, the G2/M checkpoint operates through a different mechanism and details of this are only beginning to emerge. Thus any compounds that can act as G2/M checkpoint inhibitors have the potential to uncover molecular mechanisms associated with this checkpoint. As a G2/M checkpoint inhibitor, PBOX acts in the low micromolar range, comparing favourably to other checkpoint inhibitors which act in the mM range e.g. caffeine and 2-aminopurine.

Compounds of the present invention have been assessed for their ability to interact with the microtubule network of the cell. Indirect immunofluroescence allowed visualization of both the microtubule network and the DNA of the cell. This technique allowed detection of morphological changes in the microtubule network, such as alterations in microtubule organization and arrangement. It also allowed for detection of disruption to the DNA of the cell. It revealed that PBOX-6 actually possesses anti-microtubule activity, as PBOX-6 treatment disrupted the microtubule network of the cell in a dose-dependent manner. This provides some explanation for the dose-dependent effect of PBOX-6 upon the induction of mitotic arrest and apoptosis, and suggests that these effects are a direct result of the abrogation mediated by PBOX-6 upon the microtubule network of the cell. The disruption to the microtubules following treatment with lOμM PBOX-6 appeared similar to that elicited by the known microtubule depolymeriser, nocodazole. Increasing concentrations of nocodazole and other known tubulin depolymerisers are known to kinetically 'cap' the actively growing plus end of microtubules, preventing growth and thus leading ultimately to disassembly of the microtubules (Dumontet and Sikic 1999). Both PBOX-6 and nocodazole resulted in a dramatic destruction of the microtubule complex network of the cell, relative to the intricate mesh of microtubules witnessed in the vehicle control cells. The effect of PBOX-6 upon the microtubule network was distinct to that elicited by treatment with paclitaxel. Paclitaxel is a known microtubule polymeriser, which with increasing concentration causes an increase in microtubule mass and a consequential distinctive 'bundling' of the microtubules.

Surprisingly, the present invention demonstrates compounds of the invention target the microtubule network of the cell, causing its dissolution via microtubule depolymerisation. In order to address whether the differential response of the non- and pro-apoptotic subsets of PBOX compounds is based upon the ability to distort the microtubule network, the effect upon the microtubule network in MCF-7 cells of PBOX- 15, another potent member of the pro-apoptotic subset of PBOX compounds, and PBOX-21, a member of the non-apoptotic PBOX compounds, was assessed. This revealed that PBOX- 15 also disrupted the microtubule network of the cell in a manner similar to PBOX-6. In contrast, PBOX-21 had no effect upon the microtubule network of the cell, for the first time suggesting that the ability of a PBOX compound to inhibit microtubule polymerisation may determine its ability to induce apoptotic cell death.

The disassembly of the microtubule network following treatment with PBOX-6 and PBOX- 15 suggested that they act as microtubule depolymerising agents. In order to examine this hypothesis, the effect of PBOX-6 and PBOX- 15 upon tubulin assembly in a cell-free system in vitro was assessed and compared with the effects of nocodazole and paclitaxel. The effect of PBOX-21 upon the assembly of tubulin in vitro was also determined as a negative control. These assays revealed that both PBOX-6 and PBOX-15 inhibits the assembly of tubulin in a dose-dependent manner, confirming them as novel microtubule depolymerisers. As expected, and in agreement with the literature, nocodazole inhibited and paclitaxel induced tubulin assembly, both in a dose-dependent fashion. PBOX-21 had a negligible effect upon the assembly of tubulin relative to the vehicle control, once again suggesting that the differential response of members of the anitproliferative and pro-apoptotic subsets of PBOX compounds depends upon their ability to directly bind to and inhibit the polymerisation of cellular microtubules.

It is notable that both 10OnM and lμM PBOX-6 were capable of inhibiting the assembly of tubulin in the cell-free assay system, whilst these concentrations were found to elicit no effect upon either the microtubule network of the cell or the cell cycle progression. This discrepancy between the concentrations of PBOX-6 that elicit an effect in a cell-free system and cell-based assay is possibly due to compound depletion and/or cell penetration effects. Discrepancies between the concentrations of anti-microtubule agents that elicit effects in cell-based and cell-free systems have been widely reported (Jiang et al. 1998; Jiang et al. 1998; Ling et al. 2002). Anti-tubulin compounds can be classified into categories depending on their binding sites on tubulin. Through the use of techniques such as photoaffinity labelling (Bai, et al. 1996; Bai et al. 1996; Rai and Wolff 1996) and protein footprinting experiments (Chaudhuri et al. 2000) as well as the crystal structures identified by Nogales et. al. (1998), and more recently by Ravelli et al., (2004) specific binding residues and binding domains upon tubulin have been at least partially characterised for paclitaxel, vinblastine and colchicine. Vinca alkaloids, rhizoxin, dolastetins and spongistatin react with the domain for vinblastine. Colchicine, nocodazole, podophyllotoxin and curacin A bind to the colchicine domain (Uppuluri, Knipling et al. 1993). These are the only two defined binding sites of tubulin depolymerisers. The present invention usefully determined whether PBOX-6 mediated its antimicrotubule activity via binding to either of these sites. The results from the competitive binding experiments revealed the surprising fact that PBOX-6 did not inhibit binding of either colchicine or vinblastine to tubulin, indicating that PBOX-6 has its own, novel, binding site upon tubulin. Other antimicrotubule agents such as estramustine (Panda et al. 1997), arsenic trioxide (Ling et al. 2002), naphthopyran (Dell 1998) and cemadotin (Jordan et al. 1998) have also been shown to bind to as yet uncharacterised novel sites on tubulin. hi conclusion, tubulin has been identified as the molecular target of pro-apoptotic PBOX compounds such as PBOX-6. The ability of PBOX-6 both to bind tubulin and cause microtubule depolymerisation suggest it as a novel candidate for antineoplastic therapy. This is all the more significant, as both the clinical success of, and increasing resistance to antimicrotubule agents in the treatment of a variety of cancers has prompted the search for new agents with a similar mechanism.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

REFERENCES

Axel, D.I., Kunert, W., Goggelmann, C, Oberhoff, M., Herdeg, C, Kuttner, A., Wild, D.H., Brehm, B.R.,

Riessen, R., Koveker, G and Karsch, K.R. (1997) Circulation, 96, 636-645.

Bai, R., Pei, X. F., Boye, O., Getahun, Z., Grover, S., Bekisz, J., Nguyen, N. Y., Brossi, A., and Hamel, E.

(1996) J. Biol. Chem. 271, 12639-12645

Bai, R., Schwartz, R. E., Kepler, J. A., Pettit, G. R., and Hamel, E. (1996) Cancer Res. 56, 4398-4406

Blagosklonny, M. V., Giannakakou, P., el-Deiry, W. S., Kingston, D. G., Higgs, P. I., Neckers, L., and

A Blocker, G Griffiths, JC Olivo, AA Hyman and FF Severin (1998) Journal of Cell Science, VoI 111, Issue

3 303-312

Fojo, T. (1997) Cancer Res. 57, 130-135

Brantley-Finley, C, LyIe, C. S., Du, L., Goodwin, M. E., Hall, T., Szwedo, D., Kaushal, G. P., and

Chambers, T. C. (2003) Biochem. Pharmacol. 66, 459-469

Chaudhuri, A. R., Seetharamalu, P., Schwarz, P. M., Hausheer, F. H., and Luduena, R. F. (2000) J. MoI. Biol.

303, 679-692

Crespo, N. C, Ohkanda, J., Yen, T. J., Hamilton, A. D., and Sebti, S. M. (2001) J. Biol. Chem. 276, 16161-

16167

Crown, J., and O'Leary, M. (2000) Lancet 355, 1176-1178

Dell, C. P. (1998) Curr. Med. Chem. 5, 179-194

Donaldson, K. L., Goolsby, G. L., Kiener, P. A., and Wahl, A. F. (1994) Cell. Growth Differ. 5, 1041-1050

Dumontet, C, and Sikic, B. I. (1999) J. Clin. Oncol. 17, 1061-1070

Fan, M., Goodwin, M. E., Birrer, M. J., and Chambers, T. C. (2001) Cancer Res. 61, 4450-4458

Friedman, P. A., and Platzer, E. G. (1978) Biochim. Biophys. Acta. 544, 605-614

Greene, L.M., Fleeton, M., Mulligan, J., Chikkanna, G., Sheahan, B.J., Atkins, GJ., Campiani, G., Nacci, V.,

Lawler, M., Williams, CD. and Zisterer, D. M. Oncology Reports In Press

Haldar, S., Jena, N., and Croce, C. M. (1995) Proc. Natl. Acad. Sd. U. S. A. 92, 4507-4511

Hayne, C, Tzivion, G., and Luo, Z. (2000) J. Biol. Chem. 275, 31876-31882

Hayot, C, Farinelle, S., De Decker, R., Decaestecker, C, Darro, F., Kiss, R. & Van Damme, M. (2002) Int.

J. Oncol. 21, 417-25.

Jiang, J. D., Davis, A. S., Middleton, K., Ling, Y. H., Perez-Soler, R., Holland, J. F., and Bekesi, J. G. (1998)

Cancer Res. 58, 5389-5395

Jiang, J. D., Wang, Y., Roboz, J., Strauchen, J., Holland, J. F., and Bekesi, J. G. (1998) Cancer Res. 58,

2126-2133

Jordan, M. A., and Wilson, L. (2004) Nat. Rev. Cancer 4, 253-65

Jordan, M. A., Walker, D., de Arruda, M., Barlozzari, T., and Panda, D. (1998) Biochemistry 37, 17571-

17578

King, R. W., Jackson, P. K., and Kirschner, M. W. (1994). Cell 79, 563-571

Lee, L. F., Li, G., Templeton, D. J., and Ting, J. P. (1998) J. Biol. Chem. 273, 28253-28260 Li, Y. M., and Broome, J. D. (1999) Cancer Res. 59, 776-780

Ling, Y. H., Jiang, J. D., Holland, J. F., and Perez-Soler, R. (2002) MoI. Pharmacol. 62, 529-538

Mc Gee, M. M., Campiani, G., Ramunno, A., Fattorusso, C, Nacci, V., Lawler, M., Williams, D. C, and

Zisterer, D. M. (2001) J. Pharmacol. Exp. Ther. 296, 31-40

Mc Gee, M. M., Greene, L. M., Ledwidge, S., Campiani, G., Nacci, V., Lawler, M., Williams, D. C, and

Zisterer, D. M. (2004) J. Pharmacol. Exp. Ther., 310, 1084-1095

Mc Gee, M. M., Hyland, E., Campiani, G., Ramunno, A., Nacci, V., and Zisterer, D. M. (2002) F.E.B.S. Lett.

515, 66-70

Mc Gee, M.M., Gemma, S., Butini, S., Ramunno, A., Zisterer, D.M., Fattorusso, C, Catalanotti, B., Kukreja,

G., Fiorini, L, Pisano, C, Cucco, C, Novellino, E., Nacci, V., Williams, D.C. and Campiani, G. (2005; J.

Med. Chem 48, 4367-4377

Monaghan, P., Fardis, M., Revill, W.P., Bell, A. (2005) Journal of infectious diseases. 191, 1342-1349.

Mulligan, J. M., Campiani, G., Ramunno, A., Nacci, V., and Zisterer, D. M. (2003)Biochim. Biophys. Acta.

1639, 43-52

Nogales, E., Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199-203

Panda, D., Miller, H. P., Islam, K., and Wilson, L. (1997) Proc. Natl. Acad. Sci. U. S.A 94, 10560-10564

Pietras, R.J., Pegram, M.D., Finn, R.S., Maneval, D.A., Slamon, DJ. (1998) Oncogene, 17, 2235-2249.

Rai, S. S., and Wolff, J. (1996) J. Biol. Chem. 271, 14707-14711

Ravelli, R.B.G., Gigant, B., Curmi, P.A., Jourdain, I., Lachkar, S., Sobel, A. and Knossow, M. (2004) Nature

428, 198-202

Rowinsky, E. K. (1997) Annu Rev Med 48, 353-374

Rundle et al., 2001. Journal Biological Chemistry. VoI 276, p48231.

Schrevel, J., Sinou, V., Grellier, P., Frappier, F., Guenard, D., and Potier, P. (1994) Proc. Natl. Acad. Sci, 91,

8472-8476.

Serrano, L., Avila, J., and Maccioni, R. B. (1984) J Biol Chem 259, 6607-6611

Shtil, A. A., Mandlekar, S., Yu, R., Walter, R. J., Hagen, K., Tan, T. H., Roninson, I. B., and Kong, A. N.

(1999) Oncogene 18, 377-384

Sinou, V., Boulard, Y., Grellier, P., Schrevel, J. (1998) J. Eukaryot. Microbiol. 45, 171-183.

Stone, G. W., Ellis, S.G., Cox, D.A., Hermiller, J., Russel, M.E. (2004) N. Engl. J. Med. 350, 211-2.

Uppuluri, S., Knipling, L., Sackett, D. L., and Wolff, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11598-

11602

Wang, T. H., Popp, D. M., Wang, H. S., Saitoh, M., Mural, J. G., Henley, D. C, Ichijo, H., and Wimalasena,

J. (1999) J. Biol. Chem. 274, 8208-8216

Wang, T. H., Wang, H. S., Ichijo, H., Giannakakou, P., Foster, J. S., Fojo, T., and Wimalasena, J. (1998) J.

Biol. Chem. 273, 4928-4936

Wassmann, K., and Benezra, R. (2001). Curr. Opin. Genet. Dev. 11, 83-90

Winslow, R.D., Sharma, S.K., Kim, M.C. (2005) Mount Sinai Journal of Medicine, 72, 81-89. Woods, C. M., Zhu, J., McQueney, P. A., Bollag, D., and Lazarides, E. (1995) MoI. Med. 1, 506-526

Yu, D (2001) Semin Oncol, 28, 12-17

Zhou, J., Gupta, K., Yao, J., Ye, K., Panda, D., Giannakakou, P., and Joshi, H. C. (2002) J. Biol. Chem. 277,

39777-39785

Zisterer, D. M., Campiani, G., Nacci, V., and Williams, D. C. (2000) J. Pharmacol.Exp. Ther. 293, 48-59

Zisterer, D..M., Hance, N., Campiani, G., Garofalo, A., Nacci, V., and Williams D. C. (1998) Biochem.

Pharmacol. 55, 397-403

Claims

Claims

1. Use of a compound having the general formula (I):-

wherein:

(i) A represents O or S;

(ii) the cyclic group labelled F represents a benzyl group, a 2,3-naphthyl group or a benzyl group substituted with ethoxycarbonyl at the 2-position;

(iii) and Y represents N(CH₃)₂, NHCH₃, CH₃ or pyridine; as an agent for one or more of the group consisting of an attenuating microtubule growth, depolymerising microtubules, arresting G2/M transition, activation of CDKl activity, treatment of coronary heart disease, treatment of restenosis, treatment of arteriosclerosis and treatment of malaria.

2. A method of one or more of the group consisting of attenuating microtubule growth, depolymerising microtubules, arresting G2/M transition, activating CDKl activity, treatment of coronary heart disease, treatment of restenosis, treatment of arteriosclerosis and treatment of malaria; comprising administering a compound having the general formula (I):-

wherein:

(i) A represents O or S;

(ii) the cyclic group labelled F represents a benzyl group, a 2,3-naphthyl group or a benzyl group substituted with ethoxycarbonyl at the 2-position;

(iii) and Y represents N(CH₃)₂, NHCH₃, CH₃ or pyridine;

3. Use of a compound having the general formula (I):-

wherein:

(i) A represents O or S;

(ii) the cyclic group labelled F represents a benzyl group, a 2,3-naphthyl group or a benzyl group substituted with ethoxycarbonyl at the 2-position;

(iii) and Y represents N(CH₃)₂, NHCH₃, CH₃ or pyridine; in the manufacture of a medicament for the treatment of one or more of the group consisting of coronary heart disease, restenosis, arteriosclerosis, malaria and disorders associated with excess microtubule proliferation or activity.

4. Use as claimed in claim 1 or 3 or a method as claimed in claim 2 wherein the compound is one or more selected from those having the formulae:-

7-[(Dimethylcarbamoyl)oxy]-6-( 1 -naphthyl)pyrrolo[2, 1 -d] [ 1 ,5]benzoxazepine (PBOX 6); 4-Acetoxy-5-( 1 -naphthyl)naphtho[2,3-b]pyrrolo[2, 1-d] [l,4]oxazepine (PBOX 15); 7-[(Isonicotinoyl)oxy]-6-( 1 -naphthyl)pyrrolo[2, 1 -d] [ 1 ,5]benzothiazepine (PBOX 65); 3-(Ethoxycarbonyl)-7-((dimethylcarbamoyl)oxy)-6-( 1 -naphthyl)pyrrolo[2, 1 -d] [ 1 ,5] benzoxazepine (PBOX 70);

4-[(Dimethylcarbamoyl)oxy]-5-(l-naphthyl)naphtho[2,3-b] pyrrolo [2,l-d][l,4] oxazepine (PBOX 16)

7-Acetoxy-6-(l-naphthyl)pyrrolo[2,l-d][l,5]benzoxazepine (PBOX 4); 7-[(Methylcarbamoyl)oxy]-6-(l-naphthyl)pyrrolo[2,l-d][l,5]benzoxazepine (PBOX 7); 7-Acetoxy-6-( 1 -naphthyl)pyrrolo [2, 1 -d] [ 1 , 5]benzothiazepine (PBOX 9); 7-[(Dimethylcarbamoyl)oxy]-6-(l-naphthyl)pyrrolo[l,2-d][l,5]benzothiazepine (PBOX-8); and PBOX- 12 (7-Acetoxy-6-( 1 -naphthyl)pyrrolo[ 1 ,2-d]pyrido [3 ,2-b] [1,4] oxazepine).

5. A use or method substantially as herein described with reference to the accompanying figures and tables.