THERAPEUTIC COMPOUNDS AND METHODS

Abstract: The present invention relates to bi-aryl compounds of formulae I-IV and their use in cancer treatment where substituents R, A, B and E are variables as defined.

Description: THERAPEUTIC COMPOUNDS AND METHODS FIELD OF THE INVENTION The present invention generally contemplates compounds which are useful in therapy. More particularly, the present invention relates to compounds which are useful in the treatment of cancer. In particular, the present invention relates to bi-aryl compounds and their use in the treatment of cancer. BACKGROUND OF THE INVENTION Despite intensive efforts of the medical and scientific community directed towards the search for a cure for cancer, this disease remains today one of the major causes of human mortality. Whilst a number of different treatments are currently employed in clinical practice, chemotherapy remains a commonly utilized and much investigated and researched therapeutic protocol in the treatment of cancerous conditions. A group of chemotherapeutic agents which is currently receiving considerable attention is Taxol ® and analogues which contain the fused tricyclic taxane ring system. Since its first isolation in the 1960's from the bark of the Pacific Yew tree, Taxol ® and its analogues have been viewed as leading target candidates in the search for an effective treatment for cancer, in particular breast and ovarian cancer. However, whilst demonstrating significant cytotoxic activity, Taxol ® itself, and the taxanes in general, are poorly soluble in the solvents considered acceptable for human administration. This has prompted sigmficant activity in the search for more soluble second generation derivatives, and for suitable formulation processes which provide pharmaceutical preparations which can effectively administer the required therapeutic dosages. Furthermore, although synthetic routes to the taxanes have been developed, their challenging structural complexity has thus far dictated that natural rather than synthetic sources remain the most viable means of obtaining commercially effective quantities for medical use, albeit by semi-synthetic routes. There remains, therefore, a continued need for alternative therapeutic compounds for use in the treatment of cancerous conditions. SUMMARY OF THE INVENTION Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers but not the exclusion of any other integer or step or group of integers. In a first aspect, the present invention provides for use in therapy a compound according to one of Formulae: wherein is an optional single bond; is an optional double bond; is either a hydrogen atom or a hydroxy group; p and q are independently selected from 0 to 5; and each R is independently selected from, a C, .10 alkyl group, a phenyl C,_ 10 alkyl group, a hydroxy group, a C 0 alkoxy group, a phenyl C l 0 alkoxy group, an amino group a C,_ 6 alkylamino group, a di-C 0 alkylamino group, a halogen atom, a -O- C(O)-C, .10 alkyl group, a -C(O)-C .10 alkyl group, a formyl group, a carboxy group, a carboxyC j.jQ alkylester, a carboxamido group, or where p and/or q are greater than 1, any two adjacent R groups together may optionally form the group -W-CH 2 -Z- where W and Z are independently selected from O or NH; each A may be the same or different and is a group CO 2 Q wherein Q is selected from hydrogen, C,-C 10 alkyl, phenyl C r C 10 alkyl, amino, C,-C 10 alkylamino or di-C,-C 10 alkylamino; or the two A groups together form a group -Y-X-C(O)-, wherein Y is selected from C(O) or (CH 2 )n, with n selected from 0, 1 or 2, and X is selected from CH 2 , NH or oxygen; B is a group selected from a hydrogen atom, a hydroxy group, a C 0 alkyl group, a C 0 alkoxy group, an amino group a C x _ 10 alkylamino group, a di-C 0 alkylamino group, or a halogen atom, a -O-C(O)-Cι .10 alkyl group, a -C(O)-C, .10 alkyl group, a formyl group, a carboxy group, a carboxyC,_ 10 alkylester, a carboxamido group; or the two B groups together form a group -CH 2 -CH 2 - or -CH=CH-, wherein one or more H atom may be replaced by a group selected from a hydroxy group, a Cι_ 10 alkyl group, a C, .10 alkoxy group, an amino group, a C 0 alkylamino group, a di-C, .10 alkylamino group, a halogen atom, a -O- C(O)-C, .10 alkyl group, a group, a formyl group, a carboxy group, a carboxy C, .10 alkylester, a carboxamido group; or a pharmaceutically acceptable derivative thereof. In another aspect, the present invention provides a method of treating cancer comprising the administration of an effective amount of a compound according to any one of Formulae I-IV to a subject in need thereof. In still yet a further aspect, the present invention provides a composition comprising a compound according to any one of Formulae I-IV together with a pharmaceutically acceptable carrier or excipient. In another aspect, the present invention provides an agent for the treatment of cancer, said agent comprising a compound according to any one of Formulae I-IV. In yet a further aspect, the present invention also provides a novel compound according to any one of general Formulae I-IV or a pharmaceutically acceptable derivative thereof. In a preferred embodiment of the invention, the compound is selected from Formula I or II, preferably from Formula I. BRIEF DESCRIPTION OF THE FIGURES Figure 1 graphically depicts the effect of Compound I-A on human cell lines. Figures 2-5 graphically depict dose response curves of Compound I-A, Taxol and Colchicine with Hep G2/A2 and Hep 3B/T2 cell lines in serum-free and culture media. Figure 6 graphically depicts the cytotoxicity of Compound I-A, Taxol and Colchine on human fibroblast cells. Figures 7-10 graphically depict cell survival dose response curves following removal of the administered drug ( Compound I-A, Taxol and Colchine) after 2 days. Figures 11-12 graphically depict cell cycle analysis of Hep G2/A2 and Hep 3B/T2 cell lines when treated with Compound I-A, Taxol or Colchine. Figures 13-14 photographically depict the DNA fragmentation analysis of Hep G2/A2 and Hep 3B/T2 eel lines treated with Compound I-A and Taxol. Figures 15-16 illustrate the micronucleation in Hep G2/A2 and Hep 3B/T2 cells treated with Compound I-A and Taxol. Figure 17 depicts an immunoblot of Hep G2/A2 and Hep 3B/T2 cells treated with Compound I-A, Taxol and Colchicine. Figures 18-21 graphically depict the weight and volume of mmours in nude mice following implantation of Hep 3B/T2 cells and treatment with Compound I-A or Taxol. Figure 22 depicts the effects of Compound I-A and Taxol on microtable polymerization. Figure 23 graphically deptics the ability of Compound I-A and Taxol to promote microtubule polymerization as monitored by change in absorbance at 350nm. Figure 24 depicts the effect of Compound I-A, taxol or colchicine tubule polymerization. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present inventors have now discovered that certain bi-aryl compounds exhibit cytotoxic activity useful in the treatment of cancer. Accordingly, in a first aspect, the present invention provides for use in therapy, particularly in the treatment of cancer, a compound according to one of formulae: wherein is an optional single bond; is an optional double bond; is either a hydrogen atom or a hydroxy group; p and q are independently selected from 0 to 5; and each R is independently selected from, a C 0 alkyl group, a phenyl CJ . JO alkyl group, a hydroxy group, a C 1-10 alkoxy group, a phenyl C, .10 alkoxy group, an amino group a C,_ 6 alkylamino group, a di-C 0 alkylamino group, a halogen atom, a -O- C(O)-C 1.10 alkyl group, a -C(O)-C .10 alkyl group, a formyl group, a carboxy group, a carboxyC i Q alkylester, a carboxamido group, or where p and/or q are greater than 1, any two adjacent R groups together may optionally form the group -W-CH 2 -Z- where W and Z are independently selected from O or NH; each A may be the same or different and is a group CO 2 Q wherein Q is selected from hydrogen, C j -C^ alkyl, phenyl Cι-C 10 alkyl, amino, C j -C 10 alkylamino or di-C,-C 10 alkylamino; or the two A groups together form a group -Y-X-C(O)-, wherein Y is selected from C(O) or (CH 2 )n, with n selected from 0, 1 or 2, and X is selected from CH 2 , NH or oxygen; B is a group selected from a hydrogen atom, a hydroxy group, a C, .10 alkyl group, a C 0 alkoxy group, an amino group a C μ 10 alkylamino group, a di-C μ6 alkylamino group, or a halogen atom, a -O-C(O)-C, .10 alkyl group, a -C(O)-C, .10 alkyl group, a formyl group, a carboxy group, a carboxyC, .10 alkylester, a carboxamido group; or the two B groups together form a group -CH 2 -CH 2 - or -CH=CH-, wherein one or more H atom may be replaced by a group selected from a hydroxy group, a C 0 alkyl group, a C J. J Q alkoxy group, an amino group, a C ,_ 10 alkylamino group, a d--C,_ 10 alkylamino group, or a halogen atom, a -O- C(O)-C,_ 10 alkyl group, a -C(O)-C,. 10 alkyl group, a formyl group, a carboxy group, a carboxyC^ Q alkylester, a carboxamido group; or a pharmaceutically acceptable derivative thereof. Preferably the therapeutic use is in the treatment of cancer in mammals, for example, humans, primates, livestock animals (eg. sheep, cows, horses, goats, pigs), companion animals (eg. cats, dogs), laboratory test animals (eg. mice, rats, guinea pigs, rabbits) or captured wild animals. More preferably, the therapeutic use is in the treatment of cancer in humans. Accordingly, the present invention also provides a method of treating cancer comprising the administration of an effective amount of a compound according to any one of Formulae I-IV, or a pharmaceutically acceptable derivative, to a subject in need thereof. The cancers or tumours which may be treated by the compounds, compositions and methods of the invention may simple (monoclonal, i.e., composed of a single neoplastic cell type), mixed (polyclonal, i.e., composed of more than one neoplastic cell type) or compound (i.e., composed of more than one neoplastic cell type and derived from more than one germ layer). Some examples of cancers which may be treated by the invention include breast, colon, uterus, prostate, lung, ovarian, skin, liver and stomach cancers, tumours and melanomas. As used herein, the term "Cι- 10 alkyl" when used in the terms "C, .10 alkyl group", "C 0 alkylamino group", "di-C ,. 10 alkylamino group", phenyl C , .10 , carboxy C 0 alkylester, "-C(O)-C 0 alkyl" and "-O-C(O)-C 0 alkyl" refers to an alkyl (or alkylene) group, which contains 1-10 carbon atoms, preferably 1 to 6 carbon atoms, ie. methyl, ethyl, propyl, butyl, pentyl or hexyl, and includes, where applicable, branched and cyclic alkyl groups as well as straight chain alkyl groups. Particularly preferred alkyl groups are C,_ 4 alkyl groups, particularly methyl, ethyl and propyl. A "phenyl C r C 10 alkyl" group refers to a phenyl group linked by a C,-C 10 alkylene chain. The term "C,_ 10 alkoxy" is intended to refer to C _ l0 alkyl group as defined above, when linked by an oxygen atom. The term "halogen" refers to fluorine, chlorine, bromide or iodine. As used herein the terms "phenyl", "amino", "carboxamido" and "alkyl" as used alone or in the terms "alkoxy", "alkylamino", "dialkylamino", "phenylalkyl", "carboxylalkyl ester", "-C(O)-alkyl" (acyl) and "-O-C(O)-alkyl" (acyloxy) also refer to these groups where they can be optionally substituted by one or more substituents eg. "alkyl" refers to optionally substituted alkyl. Suitable optional substituents include, hydroxy, alkyl, halo, phenyl, alkoxy, amino, alkylamino, dialkyl-imino, nitro, acyl, acyloxy, carboxy, carboxy ester, carboxamido, formyl, cyano, nitro, sulphate, phosphate. Optional substimtion of "alkyl" is also intended to refer to one or more degrees of unsaturation i.e. one or more double or triple bonds, so as to form C 0 alkenyl and C 0 alkynyl groups. In a preferred embodiment of the invention, the two A groups together form a group -Y-X-C(O)-. In a preferred embodiment, X is NH or oxygen. Even more preferably, X is oxygen. In another preferred embodiment, Y is (CH 2 )n. More preferably n is 1. A particularly preferred embodiment of -Y-X-C(O)- is -CH 2 -O-C(O)- or -C(O)-O-C(O)-. In another prefered embodiment, A is the group -CO 2 Q. Examples of Q include: H, CH 3 , CH CH 3 (CH 2 ) 2 CH 3 (CH 2 ) g CH 3 (CH 2 ) 6 CH 3 (CH 2 ) 7 CH 3 (CH 2 ) 8 CH 3 CH(CH 3 ) 2 CH 2 CH(CH 3 ) 2 CH 2 CH 2 CH(CH 3 ) 2 CH 2 Ph, (CH 2 ) 2 Ph, and (CH 2 ) 3 Ph, preferably with Q being an alkyl chain of at least C 2 , or an alkylene-phenyl group of at least C 2 -phenyl. In yet another preferred embodiment, the compounds for use in the present invention contain the moiety (i): wherein (i) may be present in either the Z- or E- form. A particularly preferred form of moiety (i) is the E- form. In still another preferred embodiment, each R group is independently selected from C,_ 4 alkyl, hydroxy, C alkoxy, eg., methoxy, ethoxy, propoxy (n- and iso-), butoxy (/--, sec- and t-), benzyloxy or acetoxy or, where p and/or q is greater than 1, two adjacent R groups may together form the group -O-CH 2 -O-. A preferred value for p and q is 1, 2 or 3. The -O-CH 2 -O- group may reside on the phenyl group at the 2,3- or 3,4- positions. In a particularly preferred form, when two adjacent R groups together form the group -O-CH 2 -O-, this is substituted on the phenyl group at the 3,4- positions. In another preferred embodiment, when p and/or q are 1, 2 or 3, the R groups reside on the benzene rings at the 3-, 4- or 5- positions. Particularly preferred compounds for use in the present invention have the general Formula I as defined above: A preferred subgroup of Formual I has the structure formula F Examples of compounds of general Formula I are Compounds I-B to S as defined below: I-B Q=H I-C Q=CH 3 I-D Q=CH 2 CH 3 I-E Q=(CH 2 ) 2 CH 3 I-F Q=(CH 2 ) 3 CH 3 I-G Q = (CH 2 ) 4 CH 3 I-H Q=(CH 2 ) 5 CH 3 I-I Q=(CH 2 ) 6 CH 3 I-J Q=(CH 2 ) 7 CH 3 I-K Q= (CH 2 ) 8 CH 3 I-L Q=CH(CH 3 ) 2 I-M Q=CH 2 CH(CH 3 ) 2 I-N Q=(CH 2 ) 2 CH(CH 3 ) 2 I-O Q=CH 2 Ph I-P Q=(CH 2 ) 2 Ph I-Q Q=(CH 2 ) 3 Ph A particularly preferred compound for use in the present invention is Compound I-A which is isolated from the heartwood extracts of Taiwania cryptomeriodies Hayata. Compound I-A may be obtained from the natural source as described in the Examples. The compounds of Formula 1-B may be prepared by condensation of piperonal with a succinate such as diethyl succinate in the presence of a base such as sodium methoxide. Compounds of Formula I-C to I-Q may then be prepared by appropriate esterification of the carboxylic acid groups. It will be recognised that amidation of the carboxylic acid groups, or where appropriate the ester group, with the appropriate amine will afford the compounds where A is CO 2 NR'R" wherein R' and R" are independently selected from hydrogen, or C 0 alkyl. Methods of amidation are known to the skilled person (see for example Advanced Organic Chemistry Reactions, Mechechanisms, and Structure; 3rd Edition, Jerry March, Wiley Interscience). The compound I-R can be formed by treating I-B with a suitable dehydrating agent such as acetyl chloride. Compound I-S is also known as savinin, or Taiwanin B, and is also isolable from Taiwania cryptomerioides Hayata. The incorporation of suitable R groups into the compounds of Formula I may be effected by replacing piperonal with an the appropriately substituted benzaldehyde. Thus compounds I-T to I-W can be prepared using the appropriate methoxy and benzyloxy substituted benzaldehyde, optionally protected by one or more protecting groups. Where appropriate, eg. when R is OH or amino based, the substiuents on the benzaldehyde may be optionally protected by one or more protecting groups which may be removed at a later stage if desired. Suitable protecting groups are known to the skilled person and examples are described in Protective Groups in Organic Synthesis by T. W. Greene & P. Wutz, 2nd Edition (1991), John Wiley and Son. Alternatively, the benzaldehyde may be substituted by one or more groups (synthons) which may at a later stage be converted into the desired R group(s) by one or more synthetic transformations. Groups which may be convertible into suitable R groups are known to the skilled person and are described in standard reference texts e.g., March (supra) and Comprehensive Organic Transformations, Richard C. Larock, V.C.M. Publishers (1989). Alternatively, the R group(s) (or the synthon therefor) may be incorporated into the molecule after condensation of a suitable benzaldehyde with the succinate, for example by electrophilic aromatic substimtion or Friedel-Crafts acylation. Imides may be formed from the appropriate anhydride by reaction with ammonia (see for example March supra). The term "pharmaceutically acceptable derivative" refers to any pharmaceutically acceptable salt, ester, solvate, hydrate or any other compound which, upon administration to the recipient is capable of providing (directly or indirectly) a compound as described herein. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic , butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. The compounds as defined by Formulae I-IV (or their pharmaceutically acceptable derivatives) are administered to the subject in need of therapeutic treatment in an effective amount. As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages lie within the range of about 0.1 ng per kg of body weight to about 10 g per kg of body weight per dosage. The dosage is preferably in the range of 1 μg to 10 g per kg of body weight per dosage. More preferably, the dosage is in the range of 1 mg to 10 g per kg of body weight per dosage. In a preferred embodiment, the dosage is in the range of 1 mg to 5 g per kg of body weight per dosage. In another preferred embodiment, the dosage is in the range of 1 mg to 2 g per kg of body weight per dosage. In yet another preferred embodiment, the dosage is in the range of 1 mg to 1 g per kg of body weight per dosage. The active ingredient may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. Thus, in yet a further aspect of the invention, the present invention provides a composition comprising a compound according to any one of Formulae I-IV together with a pharmaceutically acceptable carrier or excipient. The carrier or excipient must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parental (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked poly vinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface- active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Compositions for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter. Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient. It should be understood that in addition to the active ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. An example of a carrier for formulating the compounds for use in the present invention is 1,2-propanediol/ethanol (1 :1 v/v). The present invention also provides novel compounds according to any one of general Formulae I-IV as defined above or a pharmaceutically acceptable derivative thereof. One or more embodiments of the present invention may also provide methods, compositions, agents or compounds which have an advantage over, (or avoid a disadvantage associated with) known methods, compositions, agents or compounds used in the chemotherapeutic treatment of cancerous conditions. Such advantages may include one or more of increased therapeutic activity, reduced side effects, reduced cytotoxicity to non- cancerous cells, improved solubility or dispersibility for formulation into pharmaceutical compositions, improved stability or a more readily available means of obtaining said compound, eg. by simpler or higher yielding synthetic processes. 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 which fall within the spirit and scope. 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 or features. The invention will now be further described with reference to the following non-limiting Examples. EXAMPLES GENERAL METHODS AND MATERIALS Preparation of Compound I- A (Taiwanin A) The heartwood of Taiwania cryptomerioides Hayata was extracted with methanol and subsequently partitioned between n-hexane/water (1:1). The n-hexane soluble material was fractionated by sequential column chromatography of silica-gel. The active component, Compound I-A, as determined by Η nmr spectroscopy and X-ray crystallography, was eluted at the fraction of n-hexane/EtOAc (95:5) and was further purified by reverse phase high performance liquid chromatography to homogeneity. For bioassay, the compounds were dissolved in ethanol and were filtered through 0.25 μm filter (Millipore). Extracts of Compound I-A are maintained at pH 7.0 or above. Cell Culture Stock cultures of human hepatoma cells Hep 3B, Hep A2, HuH-7 and normal human skin fibroblast were maintained in DMEM medium supplemented with 10% fetal calf serum and antibiotics (100 units/ml of penicillin and streptomycin) in a humidified atmosphere containing 5% CO and 95% air at 37°C. Stock cultures of human non-small lung cancer cell line H1299 and human stomach cancer cell line SCM-1 were maintained in RPMI-1 medium supplemented with 10% fetal calf serum and antibiotics (100 units/ml of penicillin and streptomycin) in a humidified atomsophere containing 5% CO 2 and 95% air at 37 °C. The cultures were passaged by trypsinization for every 4 day. For experimental purposes, cells were plated either in 24-well plates at 1 x 10 5 cells/well or in 100 mm culture dishes at 15 x^O cells/dish in DMEM medium containing 10% fetal calf serum. Determination of Cell Growth Human cells were seeded in 24-well plates at a density of 10 6 cells/well in DMEM(RPM11640) medium containing 10% fetal calf serum. After 24h incubation, cells were washed three times with phosphate buffered saline (pH 7.0) and treated with various concentration of drags in serum-free DMEM for 2, 4, 6 days. The medium was changed every two days. The viable cells in each well were determined by trypan blue exclusion and haemocytometer counting. Determination DNA Content by Flow Cytometeric Analysis Cells grew on DMEM containing 10% fetal calf serum. After 24 h attachment, the medium was changed to serum free DMEM alone or plus drugs for indicated time points. The harvested cells were counted, stained with propidium iodide and followed by flowcytometry analysis. Preparation of Cell Extract Quiescent cultures of cells were washed twice with PBS, lysed at 4°C in 700 ml of a solution containing 50 mM Hepes, pH 7.4, 4 mM EDTA, 2 mM EGTA, 1% Triton X-100, 50 mM PMSF, 20 mg/m. leupeptine, 100 mM NaVO4 (lysis buffer), centrifuged at 55,000 rpm for 30 min. The ly sates were stored at -20 °C. Protein concentration in the ly sates was determined using brabufal. Western Blotting Treatment of quiescent culture of cells with drugs, cell lysis, and immunoprecipitation were performed as described above. After SDS-PAGE, proteins were transferred to nitrocellulose membranes. Membranes were blocked using 4% non-fat dried milk in TBS Blocking solution, and incubated for 2 h with suitable antibodies as indicated in TBS (25 mM Tris/HCl, pH 7.4, 125 mM NaCl) containing 4% non-fat dried milk. The nitrocellulose membranes were washed three times in blocking solution, followed by a 1 h incubation with horseradish peroxidase-conjugated rabbit - anti-mouse IgG (Amersham Corp.) diluted 1 :5000 in 4% blocking solution. The Immunoblot were then washed five times in blocking solution before developing using the ECL chemiluminescence kit (Amersham Corp.). DNA Laddering Test Cells were treated with various concentrations of drugs or serum free DMEM alone (control), for 48 hours. The DNA of harvested dying cells was isolated and fractionated on a 2% agarose gel. Immunofluoresent and Hoechst Staining Cells were treated with serum-free DMEM alone (control), plus drugs for 48 hours and harvested to be permeated by acetone followed by fixing with 3% formaldehyde in phosphate buffered saline (PBS). Nuclei of the fixed cells were incubated with first antibody in 10% BSA phosphate buffered saline for 1 hour in covered glass, washed with PBS buffer three times, followed by a second antibodies incubation and then stained with Hoechst dye 33258 (1 μg/ml) for immunofluoresent counting and photograph. In vivo Test The six weeks old male Balb/c-nu/nu nude mice were obtained from national animal center which is charged by the National Science Coucil, Taipei. The obtained nude mice were stabilized in the animal room for two weeks. Hep3B cells which would transform into tumour, at 1 x 10 7 cells/mouse were then injected subcutaneously (S-C) into nude mice. After 5 days of Hep3B cells implanted into nude mice, various dose of drugs or saline (control) were given by I.P., three times/week for continuous three weeks. Those experimental nude mice were kept for another three weeks then the animal was sacrificed and the weight of the tumour measured. The tumour size of the animal were measured every week. Example 1 Dose response curves of Compound I-A with human cell lines (Hep G2/A2, Hep 3B/T2, SCM-1 (stomach), HuH-7 (liver), H1299 (lung) and fibroblast (human normal skin)) are depicted in Figures l(a)-(c). Dosages were administered at concentrations of 0-8 μM and cell numbers were determined against a control (no Compound I-A) after 2 days (b) and 6 days (c). Strong cytotoxicity to human cancer cells is exhibited, however, the cytotoxicity is much weaker in human normal skin fibroblast cells. Example 2 The cytotoxicity of Compound I-A was compared against that of the known anti- cancer drugs Taxol and Colchicine. Each compound was administered at concentrations of 0-7.5 μM and cells were counted at 2,4,6 and 8 days. Figures 2 and 3 depict the dose response curves of Compound I-A, Taxol and Colchicine administered in serum-free media for Hep G2/A2 and Hep 3B/T2 cell lines respectively. Figures 4 and 5 depict the dose response curves of Compound I-A, Taxol and Colchicine administered in culmre medium (with sera) for Hep G2/A2 and Hep 3B/T2 cell lines respectively. Example 3 The cytoxicity of each of Compound I-A, Taxol and Colchicine on non-cancerous cell (normal human skin fibroblasts) was compared. The active compounds were administered at concentrations of 0-2.5 μM and cell survival was monitored at 2,4 and 6 days. Figure 6 shows that Compound I-A was, generally less cytotoxic to normal non- cancerous cells than Taxol or Colchicine. Example 4 The reversibility of the cytotoxic effect an HepG2/A2 and Hep 3B/T2 cell lines of Compound I-A, Taxol and Colchicine administered at concentrations of 0-0.55 μM, was evaluated by removal of the drag after 2 days of incubation and monitoring the cell survival at 4, 6 and 8 days. The active compound was administered in serum-free and culmre media. The results are depicted in Figures 7-10. Example 5 Comparative flow cytometric analysis was conducted on Compound I-A, Taxol and Colchicine in Hep G2/A2 and Hep 3B/T2 cell lines. The dosages of Compound I-A, Taxol and Colchicine were 2.5 μM, 1.1 μM and 2.5 μM respectively. Analysis was conducted at 6, 24, 48 and 72 hours, with the experiments conducted in both serum-free (SF) and culture media (CM). The results are shown below in Tables 1 and 2 and graphically depicted in Figures 11 and 12 and indicate that Compound II-A, Taxol and Colchicine all arrest the cell cycle at the G2/M phase. Table 1 Serum-Free Hep GA/A2 Table 1 continued Serum-Free Hep 3B/T2 Table 2 Culmre-Media Hep G2/A2 Table 2 continued Cultore Media 3B/T2 Example 6 Cell death by apoptosis is illustrated by DNA fragmentation, Micronucleation Staining, Nuclear Mitotic Apparatus Protein (NuMA) analysis. For the DNA fragmentation analysis, HepG2/A2 and Hep 3B/T2 cell lines were culmred in serum free and culmre medium. Compound I-A (Taiwanin A) was administered at 0.25 μM and 2.5 μM and compared against Taxol (1.1 μM). Analysis was performed 48 hours after the addition of the drugs. The results are illustrated in Figures 13 and 14. Staining (Hoechst dye) of Hep G2/A2 and Hep 3B/T2 cell lines treated with Compound I-A (Taiwanin A) (2.5 μM) and Taxol (1.1 μM) after 48 h showed micronucleation in the cell lines treated with Compound IA and Taxol (Figures 15 and 16). For NuMA analysis, Hep G2/A2 and Hep 3B/T2 cells were treated with Compound I-A (2.5 μM), Taxol (1.1 μM) and Colchicine (2.5 μM) (serum-free media). After 48 hours, cells were collected and SDS-PAGE analysed. Markers: M=240 kDa, 1= 220 kDa and R= 180 kDa (Figure 17). Immunofluoresence staining of α-tubulin on Hep G2/A2 cell lines treated with Compound I-A (2.5 μM) indicated that Compound I-A can depolymerize microtubules during cell division. Example 7 In vivo experiments in nude mice were carried out as described above. Compound I-A was administered at 2.5 mg/kg, 500 μg/kg and 100 μg/kg and weight and volume of the mmours was recorded at the first administration of the drag and every 5 days for 3 weeks. Taxol (10 mg/kg) was monitored for comparision. The results are depicted in Figures 18-21 which indicated a reduction of transformed tamour of Hep 3B/T2 cells in both size and weight in a Compound I-A dosage dependent manner. Example 8 EC 50 values of Compound I-A, C, E, F, G, H, J, K, R and S in on Hep G2/A2 and Hep 3B/T2 cell lines and are shown below in Table 3. Table 3 Example 9 The effects of Compound I-A on microtubule polymerization were analyzed by immunoblotting with anti-α-tubulin antibody, monitoring of absorbance at 350nm. HepG2/A2 cells were left untreated (Ctl) or were treated with 1.5 μM Taxol or 2.5 μM Taiwanin-A for 24 hours. Cells were collected and processed for quantitation of tubulin polymerization. The soluble (S) and polymerized (P) fractions were analyzed by immunoblotting with anti-α-tubulin antibody. MAP-depleted tubulin were purified from porcine brain. Each reaction mixture (0.15 ml) contained lmg/ml of tubulin, 0.1M MES bufer (0.1 M MES, lmM EGTA, lmM MgCl 2 , pH6.8) and 10 μM taxol or 2.5 μM Compound I-A in the presence of GTP at 37 °C. Microtubule polymerization was monitored by change of absorbance at 350nm. The effect of Compound I-A on tubulin polymerization. HeLa cells were left untreated (lane 1), or treated with 2.5 μM Compound I-A (Lane 2), 50 nM taxol (Lane 3), 0.7 μM nocodazole (Lane 4), or 5 μM colchicine (Lane 5) for 24 hours. The polymerized fractions were analyzed by SDS-PAGE, and immunoblot was performed with anti-α tubulin monoclonal antibodies. The results are depicted in Figures 22-24. Example 10 Preparation of Compounds IB-R The synthesis of compounds IB-R was performed according to the procedures of Swoboda G. et al, 3. Chem. Soc , C. 161-162 (1967). Stobbe condensation of piperonal with diethyl succinate in the presence of sodium methoxide furnished α,α'-dipeperonlylidenesuccinic acid I-B. I-B was dehydrated with acetyl chloride to give α,α'-dipipero-nylidenesuccinic anhydride (I-R). Compound I-B was dissolved in each of alcohols (CH 3 OH, EtOH or PrOH), and was treated with concentrated H 2 SO 4 under reflux for 6h. The reaction products are dimethyl, diethyl, and dipropyl α,α'-di-peperonylidenesuccinate (I-C to E), respectively. K 2 CO 3 and I-B were dissolved in DMSO solution, and were heated at 50° for lh, then n-BuBr was added to the mixtare as it descended to room temperatare. The reaction mixtare was stirred overnight. After purification, the product, I-F was isolated. Compounds I-G to J were synthesized in a manner analogous to compounds I-C to E. A solution of I-B and CH 3 ONa in CH 3 OH were stirred 1 h, and then benzyl bromide was added to afford I-O. Compound I-B was dissolved in CH 2 C1 2 and was treated with SOC for 3h under reflux. To the solution, 2-phenylethanol or 3-phenylpropan-l-ol was added, and the mixtare was stirred under room temperatare for 2 h to produce I-P and I-Q, respectively. PHYSICAL AND SPECTROSCOPIC DATA FOR COMPOUNDS IB-R Compound 1-B 1H NMR (CD 3 OD) 65.84 (2H,S), 6.64 (1H, d, J=7.7Hz), 6.94 (1H, dd, J=7.7, 1.6Hz), 7.08 (IH, d, J= 1.6Hz), 7.45 (1H.S) IR v -^cm " ^ 3413, 1665, 1499, 1454, 1041, 931, 815 mp 223 (dec), EIMS= 382 Compound 1-C 1H NMR(CDC1 3 ) δ 3.90 (3H,S), 5.91 (2H,S), 6.91 (IH, d, J=8.6Hz), 6.98 (IH, dd, J=8.6, 1.3Hz), 700 (IH, d, J= 1.3Hz), 7.79 (1H.S) IR v ^cm "1 1711, 1653, 1610, 1505, 1486, 1228, 1041, 671 mp 189-200°C, HRMS= 410.1013 Compound 1-D 1H NMR (CDC1 3 ) δ 1.13 (3H, t, J=7.1Hz), 4.15 (2H, q, J=7.1 Hz), 5.91 (2H, S), 6.71 (IH, d, J = 8.6Hz), 6.98 (IH, dd, J=8.6, 1.2Hz), 7.00 (IH, d, J = 1.2Hz), 7.77 (1H,S) IR v m^cm '1 1739, 1600, 1500, 1249, 1015, 919. mp 118-120°C, HRMS= 438.1314 Compound 1-E 1H NMR (CDCI 3 ) δ 0.81 (3H, t, J=7.3Hz), 1.53 (2H, quin, J=7.3Hz), 4.05 (2H, t, J=7.3Hz), 5.91 (2H,S), 6.71 (IH, d, J=8.2Hz), 6.99 (IH, d, J= 8.2Hz), 7.00 and 772 (each IH, S) IR v ^-cm '1 1705, 1596, 1492, 1228, 1041, 839, 809. Amorphous, HRMS= 466.1632 Compound 1-F 1H NMR (CDCI 3 ) 50.81 (3H, t, J=7.9Hz), 1.24 (2H, m), 1.48 (2H, m), 4.09 (2H, t, J=6.5Hz), 5.91 (2H, S), 6.71 (IH, d, J=8.6Hz), 6.99 (IH, dd, J=8.6, 1.2Hz), 7.01 (IH, d, J= 1.2Hz), 7.71 (1H,S) IR v maχ cm "1 = = 1712, 1600, 1513, 1493, 1222, 1036, 939 Amorphous, HRMS= 494.1936 Compound 1-G 1H NMR (CDCI 3 ) δθ.81 (3H, t, J=8.0Hz), 1.11-1.20 (4H, m), 1.52 (2H, quin, J-7.0Hz), 4.06(2H, m), 5.91 (2H,S) 6.71 (IH, d, J=7.3Hz), 7.01 (IH, dd, J=7.3, 1.5Hz), 7.02 (IH, d, J= 1.5Hz), 7,71 (IH, S) IR v maxCm-^ 1706, 1600, 1493, 1228, 1043, 930, 819 Amorphous , HRMS = 522.2249 Compound 1-H 1H HMR (CDCI 3 ) δθ.82 (3H, t, J=68Hz), 1.17 (6H, brs), 1.50 (2H, m), 4.07 (2H, m), 5.91 (2H, S), 6.71 (IH, d, J=8.6Hz), 6.98 (IH, d, J=8.6Hz), 7.01 (1H,S), 7.77 (1H,S) IR v π- a xcm " ^ 1906, 1594, 1489, 1225, 1038, 931 Amorphous, HRMS= 550.2593 Compound 1-1 1H NMR (CDCI 3 ) δθ.84 (3H, t, J=7.2Hz), 1.17 (8H, brs), 1.48 (2H, m), 5.90 (2H,S), 6.71 (IH, d, J=7.9Hz), 6.99 (IH, dd, J=7.9, 1.8Hz), 9.01 (IH, d, J= 1.8Hz), 7.77 (1H,S) IR v maxcm "1 1706, 1600, 1493, 1228, 1043, 930 Amorphous, EIMS 598 Compound 1-J 1H NMR (CDCI 3 ) δθ.88 (3H, t, J=6.2Hz), 1.19 (lDH.brs), 1.52 (2H, m), 4.07 (2H, m), 5.91 (2H, S), 6.91 (IH, d, J=8.8Hz), 6.99 (IH, d, J=8.8Hz), 7.01 (IH, S), 7.71 (1H,S) IR v -naxCin ^ 1712, 1606, 1493, 1228, 1036, 930. Amorphous, HRMS= 606.3191 Compound 1-K 1H (CDCI 3 ) δθ.85 (3H, t, J=6.5Hz), 117 (12H, brs), 1.52 (2H, m), 4.07 (2H, m), 5.91 (2H, S), 6.71 (IH, d, J-8.0Hz), 6.98 (IH, dd, J=8.0, 1.4Hz), 7.01 (IH, d, J=1.4Hz), 9.76 (1H,S) IR v 1593, 1093, 1228, 1089, 1036, 930, 811 Amorphous, HRMS 634.3525 Compound 1-L 1H NMR (CDC1 3 ) δl.03, 1.16 (each 3H, d, J=6.2Hz), 5.02 (IH, sept, J= 6.2Hz), 5.91 (2H,S), 6.71 (IH, d, J= 8.6 Hz), 6.97 (IH, dd, J=8.6, 1.2Hz), 6.99 (IH, d, J = 1.7 Hz), 7.73 (IH, S) IR v 1600, 1493, 1228, 1107, 1036 Amorphous, HRMS 466.1651 Compound 1-M 1H NMR (CDCI 3 ) δθ.79 (6H, d, J=6.7Hz), 1.82 (IH, m), 3.87 (2H, d, J=6.6Hz), 5.91 (2H,S), 6.70 (2H, d, J=8.7Hz), 7.00 (IH, dd, J = 8.9, 1.1Hz), 7.02 (IH, d, J= l.lHz), 7.78 (1H,S) IR v -naxcm "1 1706, 1600, 1493, 1228, 1038, 930 Amorphous, HRMS= 494.1935 Compound 1-N 1H NMR (CDCI 3 ) δθ.79, 0.82 (each 3H, d, J=6.1Hz), 1.39 (IH, m), 1.52 (2H, m), 4.11 (2H, t, J- 6.4Hz), 5.91 (IH, d, J = 8.6Hz), 6.99 (IH, d, J=8.6, 1.2Hz), 7.01 (lh, d, J-1.2Hz), 7.71 (IH, S) IR 1706, 1619, 1493, 1228, 1076, 1036, 930 Amorphous, HRMS= 520.2081 Compound l-O 1H NMR (CDCI 3 ) δ5.01, 5.15 (each IH, d, J= 12.6Hz), 5.90 (2H,S), 6.68 (IH, d, J = 8.6Hz), 6.97 (IH, dd, J=8.6, 1.3Hz), 6.98 (IH, d, J= 1.3Hz), 7.12 (2H, m), 7.21 (3H, m), 7.83 (1H,S) IR v m a xCm '1 1712, 1600, 1493, 1222, 1043, 930 Amorphous, HRMS= 562.1635 Compound 1-P 1 H NMR (CDC1 3 ) δ2.83, 4.31 (each 2H, t, J=6.9 Hz), 7.01 (IH, d, J = 1.5Hz), 7.05-7.20 (5H, m), 7.84 (1H,S) IR v π- a xcm -1 1712, 1600, 1228, 1036, 930 Amorphous HRMS 486.1326 Compound 1-0 1H NMR (CDC1 3 ) δl.82 (2H, m), 2.50 (2H, t, j=7.6Hz), 4.12 (2H, m), 5.93 (2H,S), 6.71 (IH, d, J = 8.8Hz), 7.01 (IH, d, J=8.8Hz), 7. 33 (1H,S), 7.10-7.20 (5H, m), 7.88 (IH, S) IR v ---axCrn- 1 1712, 1600, 1493, 1235, 1026, 930 Amorphous, EIMS 618 (7%) Compound 1-R 1H NMR (CD 3 COCD 3 ) δ5.94 (2H,S), 6.31 (IH, d, J= 1.6Hz), 6.61 (IH, d, J=8.1Hz), 6.94 (IH, dd, J = 8.1, 1.6Hz), 7.74 (1H,S) IR v ^cm^lδOδ, 1756, 1600, 1582, 1499, 1288, 1034, 925, 809 mp 210-213°C, HRMS364.0591

Claims: We Claim: 1. A method of treating cancer comprising administration, to a subject in need thereof, of an effective amount of a compound according to one of formulae: wherein is an optional single bond; is an optional double bond; is either a hydrogen atom or a hydroxy group; p and q are independently selected from 0 to 5; and each R is independently selected from, a C l 0 alkyl group, a phenyl C j.10 alkyl group, a hydroxy group, a C 0 alkoxy group, a phenyl C lΛ0 alkoxy group, an amino group a C μ6 alkylamino group, a di-C 0 alkylamino group, a halogen atom, a -O- C(O)-C j.10 alkyl group, a -C(O)-C .10 alkyl group, a formyl group, a carboxy group, a carboxyC 0 alkylester, a carboxamido group, or where p and/or q are greater than 1, any two adjacent R groups together may optionally form the group -W-CH 2 -Z- where W and Z are independently selected from O or NH; each A may be the same or different is a group CO 2 Q wherein Q is selected from hydrogen, CJ-CJ Q alkyl, phenyl C[ -G^ alkyl, amino, CJ-CJ Q alkylamino or di-C r C 10 alkylamino; or the two A groups together form a group -Y-X-C(O)-, wherein Y is selected from C(O) or (CH 2 )n, with n selected from 0, 1 or 2, and X is selected from CH 2 , NH or oxygen; B is a group selected from a hydrogen atom, a hydroxy group, a CJ.J Q alkyl group, a C 0 alkoxy group, an amino group a C 0 alkylamino group, a di-C^ g alkylamino group, or a halogen atom, a -O-C(O)-C 0 alkyl group, a -C(O)-C,_ 10 alkyl group, a formyl group, a carboxy group, a carboxyC 0 alkylester, a carboxamido group; or the two B groups together form a group -CH 2 -CH 2 - or -CH=CH-, wherein one or more H atom may be replaced by a group selected from a hydroxy group, a C 0 alkyl group, a C 0 alkoxy group, an amino group, a C ,. 10 alkylamino group, a di-C j.jQ alkylamino group, a halogen atom, a -O- C(O)-C 0 alkyl group, a -C(O)-C,. 10 alkyl group, a formyl group, a carboxy group, a carboxyC l 0 alkylester, a carboxamido group; or a pharmaceutically acceptable derivative thereof. 2. A method according to claim 1 wherein the compound is a compound of Formula I or II. 3. A method according to claim 2 wherein the compound is a compound of Formula I. 4. A method according to claim 3 wherein the compound is a compound of Formula F . 5 A method according to claim 1 wherein two A groups together form a group, -C(O)- O-C(O)-, -CH 2 -O-C(O)-, or the A groups are each CO 2 Q. 6. A method according to claim 1 wherein when p and q are independently 1, 2 or 3. 7. A method according to claim 1 wherein each R is independently selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy, benzyloxy or acetoxy, or when p and/or q is 2 or 3, two adjacent R groups form a group -O-CH 2 -O-. 8. A method according to claim 4 wherein the compound is a compound of formula I-A: A method according to claim 4 wherein the compound is a compound of Formula: wherein Q is selected from one of CH 2 CH 3 (CH 2 ) 2 CH 3 (CH 2 ) 3 CH 3 (CH 2 ) 4 CH 3 (CH 2 ) j CH 3 (CH 2 ) 6 CH (CH 2 ) CH 3 (CH 2 ) g CH 3 CH(CH ) 2 CH 2 CH(CH ) 2 CH 2 CH 2 CH(CH 3 ) 2 CH 2 Ph, (CH 2 ) 2 Ph, or (CH 2 ) 3 Ph. 10. A method according to claim 1 wherein the compound of any one of Formulae I-IV contains the moiety (i): 11. A composition comprising a compound as described in any one of claims 1 to 10 together with a pharmaceutically acceptable carrier or excipient. 12. A novel compound according to any one of Formulae I-IV or a pharmaceutically acceptable derivative thereof.

Inventors: CHOU CHEN KUNG, KUO YUEH HSIUNG, YEH SHEAU FARN, CHOU, CHEN, KUNG, KUO, YUEH, HSIUNG, YEH, SHEAU, FARN
Applicants: CHOU CHEN KUNG, HUGHES E JOHN L, KUO YUEH HSIUNG, YEH SHEAU FARN, CHOU, CHEN, KUNG, HUGHES, E., JOHN, L., KUO, YUEH, HSIUNG, YEH, SHEAU, FARN
Application year: 1999
Main classification: C07D307
IPC codes: C07C59, C07C69, C07D317