GB2362650A - Triterpenoid derivatives - Google Patents

Abstract

The present invention relates to the use of a compound of formula I in therapy, eg. for treating a patient suffering from leukaemia, cancer or other proliferative disorder. A further embodiment relates to the use of a compound of formula I in an assay for detecting the phosphorylation state of cellular substrates. The present invention also relates to certain novel compounds of formula I and the chemical synthesis thereof. <EMI ID=1.1 HE=52 WI=61 LX=789 LY=1158 TI=CF> <PC>wherein the symbols are as defined in the specification.

Description

2362650 TIUTERPEN011) DERIVATIVES The present invention relates to the

therapeutic use and the biological activity of triterpenoid derivatives. The invention further relates to novel triterpenoid derivatives.

To date, the prior art has primarily focussed on compounds that are capable of regulating the cell cycle by virtue of inhibiting cyclin dependent kinases (CDKs). Examples of such compounds include butyrolactone 1, flavopiridol, bohemin, olomoucine, roscovitine, purvanalol and indarubicine.

There is considerable support in the literature for the hypothesis that CDKs and their regulatory proteins play a significant role in the development of human tumours. Thus, in many tumours a temporal abnormal expression or activity of CDKs has been observed, together with a major deregulation of protein inhibitors (mutations, deletions). This results in the activation of CDKs and consequently in defective regulation of the GI/S transition. Unlike normal cells, tumour cells do not arrest in GI, and since they become independent of growth factors, they pass the GI restriction point and enter the S phase very rapidly.

In contrast to the prior art, the present invention relates to compounds which are anti-proliferative, but which are believed to operate via a mechanism other than CM inhibition.

The GI/S transition of the mammalian cell cycle is tightly regulated by the retinoblastoma protein (pRb). Retinoblastoma gene mutations or deletions predispose individuals to familiar retinoblastoma and other types of cancers. The pRb protein is a docking protein, which in hypophosphorylated form has the capacity to bind and thus to inactivate Sphase transcription factors such as 2 DPA and E2F. However, following phosphorylation by GI/S cyclin-dependent kinases (CDKs) (CDK4/cyclin DI-D3, CM6/cyclin DI-D3, CM2/cyclin A), hyperphosphorylated pRb releases the transcription factors and S phase is initiated. Within the S phase, the pRb protein phosphorylation is maintained by the activity of CM2/cyclin E complexes. Thus, hyperphosphorylation of the pRb protein plays a key role in the molecular pathology of cancer cells with altered CDK activity.

The present invention relates to the use of triterpenoid compounds derived from the natural products betulin and betulinic acid (BA) as shown in formula (A).

The compounds of the present invention are referred to hereinafter as betulinines.

-,ill H R H HO H R CH20R Betulin R COOK Betulinic acid (A) With regard to their biological and therapeutic activity, the compounds disclosed herein are believed to be of specific benefit in the treatment of 20 proliferative diseases such as cancers and leukaemias.

Several of the compounds suitable for use in the present invention are already known in the art, for example those disclosed in Collect. Czech. Chem.

1 1 3 Commun. 52, 1052 (1987), Sejbal I et al. However, these disclosures do not include any indication as to possible biological activity of such compounds. A first aspect of the present invention relates to the use of a compound of formula 1, or a pharmaceutically acceptable salt thereof, in therapy.

R,, R 12 '1 H X'71 R(4 X6 xl H. R5 "' H R2 R, 1 wherein:

X' is C=Q CHOW', CHOCOR la X6 is C=O, CH2 X7 is 0 Xl is C=Q CHOCOR Ib. CHOR1b. CH-Hal R-5 and R' l, R12 are Me and wherein R' is H or lower alkyl, and Hal is Br, Cl, I, F; Rl"b are the same or different groups of W.

In a preferred aspect, the invention relates to the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for treating a patient suffering from leukaemia, cancer or other proliferative disorder.

A second aspect of the present invention relates to novel betulinines of structural formula la, or pharmaceutically acceptable salts thereof, 4 R,, R 12 H X7 R3 R4 X6 XX H R5 H R2 R, wherein:

]a X'is C=Q CHOW', CHOCOW' X6 is C=Q CH2 X7 is 0 X8 is C=Q CHOCOR 1b, CHOR1b. CH-Hal R2-5 and R' l, R 12 are Me and wherein R' is H or lower alkyl, and Hal is Br, Cl, 1, F; la-Ib R are the same or difFerent groups of R'.

with the proviso that when X6 is CH2, X7 is 0, W-5, W' and R 12 are Me; when X' is C=Q X 8 is not CHBr, CHOR CH2, or CHOAc; and - when X8 is C=Q X' is not CHOH or CHOAc.

As used herein, the term lower alkyl means a linear or branched chain alkyl group containing from 1 to 6 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl.

1 1 Descrintion of the Preferred Embodiments Within the options provided for the groups X1, 3&?, 111-5 and R1 1-12 of formula 1, the following options are preferred:

a-1b Preferably, R' is as defined below for the relevant group R'. X' is preferably C=Q CHOR1a or CHOCOR1a, wherein W' is preferably H and methyl respectively. Preferably, When X8 is CHOCOR1b, R1b is preferably methyl. Preferably, when X8 is CHHal, Hal is preferably Br.

Preferably, when X8 is CHOR 1b, R 1b is preferably H.

In a more preferred embodiment of the first aspect of the invention, the compounds of use are selected from those shown in Table 1 below:

Table 1 (Compounds of general structure I).

No. xl r 1 xf)C R", R11'1z 111.1 CHOAc C=0 0 C=0 Me 111.2 C=0 C=0 0 CHOAc Me 111.3 C=0 CH2 0 CHBr Me 111.4 C=0 CH2 0 CHOH Me 111.5 C=0 CH2 0 CHOAc Me 111.6 CHOH CH2 0 C=0 Me 111.7 CHOAc CH2 0 C=0 - Me In respect of the second aspect of the invention, the preferred embodiments regarding the compounds are identical to those given above for the first aspect 20 with application of the proviso of formula Ia.

The most preferred compounds of the second aspect are those in Table 1 not marked, i.e. compounds HI. 1 and 111.2.

1 6 In respect of the invention as a whole, it is preferable that the proliferative disorder is cancer or leukaemia. More preferably, the cancer or leukaemia is p53, Rb, hormone and multidrug resistance independent.

More specifically, the present invention relates to a method of treating patients suffering from cancer by administering therapeutically effective amounts of a compound of formula I or pharmaceutically acceptable salts or esters thereof.

Without wishing to be bound by theory, preliminary studies suggest that rather than influencing the activity of cyclin dependent kinases, the compounds of the present invention appear to operate via an alternative mechanism. In particular, it is believed that the betulinines of the present invention may inhibit cell proliferation and induce cancer cell death in a manner which involves mainly post-translational modifications, namely the phosphorylation, of a key regulatory protein involved in cellular proliferation. More specifically, it is believed that the betulinines of the invention effect a change in the phosphorylation state of the Rb protein. Such a mechanism may be advantageous as it is thought that the compounds of the present invention may be capable of inhibiting cell proliferation in proliferating tumour tissue, but not in healthy tissue.

Thus, in a further embodiment the present invention relates to a method of treating a cancerous or leukaemic proliferative disease through effecting a change in the pRb protein phosphorylation state by the administration of a therapeutically effective amount of a compound of formula 1, or pharmaceutically acceptable salts or esters thereof.

The compounds of the present invention are also capable of inducing apoptosis (programmed cell death) in proliferative cells. Thus, in an additional embodiment, the present invention relates to a method of inducing cell death in 7 proliferative cells comprising administering a therapeutically effective amount of a compound of formula I or pharmaceutically acceptable salts or esters thereof.

A further aspect of the present invention relates to use of betulinines of formula I as research chemicals and as compounds for clinical and/or laboratory diagnostics. More particularly, the invention relates to the use of betulinines as research chemicals for studying the phosphorylation/de-phosphorylation processes of cellular substrates, cellular proliferation, purification of target 10 molecules, and/or cell cycle studies.

The present invention therefore further relates to the use of a compound of formula 1 in the manufacture of a medicament for use in the treatment of a proliferative disease.

As used herein the phrase "manufacture of a medicamenC includes the use of a compound of formula 1 directly as the medicament in addition to its use in a screening programme for flirther anti-proliferative agents or in any stage of the manufacture of such a medicament.

Such a screening programme may for example include an assay for determining the phosphorylation state of cellular substrates and determining whether a candidate substance is capable of mimicking the activity of a betulinine of formula 1.

Thus, in a further embodiment, the invention relates to the use of a compound of formula I or a pharmaceutically acceptable salt, crystal form, complex, hydrate, or hydrolysable ester thereof, in an assay for determining the phosphorylation state of cellular substrates, and optionally in the identification of candidate 30 compounds that act in a similar manner.

8 Preferably, the cellular substrate, the phosphorylation state of which is being assayed is Rb protein.

Such assays may be carded out by incubating a betulinin either alone or together with a candidate substance with a relevant cell line and assessing the phosphorylation profile the Rb protein over a period of time. If a candidate substance is present it's effect on the activity of the control betulinin will be evident by running the corresponding controls (betulinin alone and candidate alone). Further information on such assays including appropriate cell lines,reagents and Rb antibodies is given below.

Rb phosphorylation assay; Since Rb protein contains multiple phosphorylation sites for CDKs, its phosphorylated form has molecular weight about 110 kDa, while the molecular weight of hypophosphorylated protein is only 105 kDa. This small difference in molecular weight is enough to separate both forms by conventional SDS-PAGE electrophoresis.

CEM cells may are cultured in Dulbeco's modified essential medium with 4. 5 g dextroselI, 10% of foetal calf serum, 2 mM glutarnine, 100 Unil penicillin and 100 ptg/ml streptomycin with/without below indicated concentrations of betulinin. At selected time points, cells axe harvested, washed in ice cold Hank's balanced salt solution and solubilized on ice using the SDS-PAGE sample buffer containing protease and phosphatase inhibitors (10 ig/ml of leupeptin, 10 4g/ml of aprotinin, 10 ggImI of soybean trypsin inhibitor, 100 4mol of benzamide, 1 mM of sodium vanadate, 1 niM of NaF, 1 mM of phenylphosphate) and boiled immediately.

9 Total cellular proteins (100 Ag/well) are separated on SDS-PAGE electrophoresis, blotted on polyvinyldifluoride membranes and total Rb protein, including proteolytic fragment(s) detected using a pRb monoclonal antibody (Oncogene, Germany, Rb(Ab-5), CaO OP66 Rev 02-Sept-96 EB, Clone LM95. 1) and visualized by chemiluminiscence (ECL-Westem Blotting Systen-4 Amersham). Details of the Western blot technique are described in Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K (Eds): Short Protocols in Molecular Biology, 2nd edition, John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore, 1992, page 10-33 - 10-35.

The compounds of the present invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.

Pharmaceutically acceptable salts of the product of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 6-6, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrobalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of I to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutarnic acid; with benzoic acid; or with organic sulfonic acids, such as (CI-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methaneor ptoluene sulfanic acid.

Esters are formed either using organic acids or alcohol s/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid. with saturated or 5 unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cl-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).

In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers and tautomers of compounds of formula I or Ia. The man skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.

The invention furthermore relates to the compounds of, or of use, in the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation from the solvents used in the synthetic preparation of such compounds.

The invention further includes the compounds of, or of use, in the present invention in prodrug form. Such prodrugs are generally compounds of formula I or la wherein one or more appropriate groups have been modified such that the modification is reversed upon administration to a human or mammalian subject.

Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include esters (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.

The present invention also encompasses pharmaceutical compositions comprising the compounds of the invention. In this regard, and in particular for human therapy, even though the compounds of the present invention (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.

Thus, the present invention also relates to pharmaceutical compositions comprising betulinines or pharmaceutically acceptable salts or esters thereof, together with at least one pharmaceutically acceptable excipient, diluent or carrier.

By way of example, in the pharmaceutical compositions of the present invention, the compounds of the invention may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilising agent(s). Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook t 12 of Pharmaceutical Excipients, 2d Edition, (1994), Edited by A Wade and PJ Weller.

The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.

For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 250 ing and more preferably from 10-100 ing, of active ingredient per dose.

Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between I and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers, and preservatives as may be required.

Injectable forms may contain between 10 - 1000 nig, preferably between 10 - 250 ing, of active ingredient per dose.

13 Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.

A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

in an exemplary embodiment, one or more doses of 10 to 150 mglday will be administered to the patient for the treatment of malignancy.

The invention further relates to methods of chemical synthesis of the above described compounds.

In one embodiment, the invention relates to a process for preparing a compound of formula 1, as defined above, wherein X6 is C=Q R, R12 H 0 R3 R4 X8 H R5 xl 7 H R2 R, R, R12 H 0 R3 R4 0 X8 H RS xl H R2 R, lc 1b 14 comprising oxidising a compound of formula Ib to a compound of formula lc.

In a preferred embodiment the compound of formula lb is oxidised to a compound of formula lc by treating with chromium trioxide.

The preparation of the compounds of the present invention will be discussed in greater detail below, with specific reference to the preferred embodiments. The man skilled in the relevant art would be able to prepare other compounds of the invention by selection of the appropriate reagents, The following scheme illustrates the synthesis of compounds of formula 1 where W-Rs, R", R 12 are methyls, X' is oxygen, X6 is C=Q X1,8 are CHOAc or C=O.

or X8 = C=O, X' = CHOAc X8 = CHOAc, X' = C=0 Conditions: a, oxidation (e.g. with chromium trioxide).

- 1 - 0 X X, X8 = C=O, X' = CHOAc or X8 = CHOAc, X' = C=0 This invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAWLES General 1 1 The chemical shift values (6-scale, ppm) and coupling constants Q, Hz) in the 'H and 13 C NMR spectra were obtained using a Varian UNITY-INOVA 400 FT spectrometer (H at 400 MHz and 13C at 100.6 MHz) in deuterochloroform with tetramethylsilane (for 'H NMR data 3 = 0 ppm) as an internal standard. For the 13C NMR data 3(CDC13)=77.00 ppm. The value for a multiplet, either defined (doublet (d), triplet (t), quartet (q), septet (sept.) or not (m) at the approximate mid point is given unless a range is quoted (s=singlet, b=broad)).

Electron impact mass spectra (E1MS) were measured on an INCOS 50 instrument. Ionising electron energy 75 eV, ion source temperature 150 'C.

EBIS was used to determine molecular weights, M' corresponding to the molecular ion.

Ether is diethylether. TBF and dioxane were dried over sodium. Acetic acid was purified before use by chromium trioxide treatment and distillation. Reactions were run at room temperature unless otherwise stated. The reaction progress was monitored by thin layer chromatography (TLQ on silicagel 60 G (Merck, detection by spraying with 10 % sulphuric acid and heating). The work-up procedure involves dilution with specified solvent (otherwise the organic reaction solvent), extraction with water and then brine or sodium hydrogencarbonate, drying over anhydrous magnesium sulphate, and evaporation under vacuum to give a residue.

EXAWLE I 2cc-acetgxy-3-oxo-18(x-oleanan-28.190-olid Chromium trioxide (2 g; 20 mmol) was added to a solution of 3-oxo-190, 280epoxy- I 8oc-oleanan-2-yl acetate [Sejbal J., Minot J., Vystrcil A.: Collect. Czech. Chem. Commun. 52, 1052 (1987)] (1 g; 2 mmol) in acetic acid (15 ml). The mixture was stirred for 3 hours at a higher temperature. The resulting 16 precipitate was filtered off and washed with acetic acid and methanol. The yield of white crystalline solid was 0.3 g (30%), m.p. 315 - 317 'C, [(XID +47' (c 0.29. CH03), The 13 C NMR spectrum of the title compound is as follows: 209.2, 179. 7, 170.1, 85.9, 71.8, 57.4, 50.9, 48.7, 46.7, 46.2, 46.1, 40.7, 40.1, 38.2, 35.9, 33.6, 33.4, 32.3, 31.9, 28.7, 27.8, 26.3, 25.5, 24,7, 23.9, 21.2, 21.0, 20.8, 18.9, 17.0, 15.7, 13.6.

Via this general procedure the following compound was prepared:

3 P-acetoxy-2-oxo- 1 8(x-oleanan-28,190-olide EXANTLE: 2 BIOLOGICAL ACTIVITY OF BETULE\TWS In vitro pAotoxic activity of betulinines on tumor cell lines One of the parameters used as the basis for colorimetric assays is the metabolic activity of viable cells. For example, a microtiter assay which uses the tetrazolium salt MTT is now widely used to quantitate cell proliferation and cytotoxicity [Hajdikh K Nfihdl V, Nfinarik J, Fiber E, afirovi K WeigI E, Antilek P.: Cytotechnology, 1996, 19, 243-245]. For instance, this assay is used in drug screening programs and in chemosensitivity testing. Because tetrazolium salts are cleaved only by metabolically active cells, these assays exclusively detect viable cells. In the case of the MTT assay, yellow soluble tetrazolium salt is reduced to a coloured water-insoluble formazan salt. After it is solubilized, the formazan formed can easily and rapidly be quantified in a conventional ELISA plate reader at 570 nin (maximum absorbancy). The quantity of reduced formazan corresponds to the number of vital cells in the culture.

17 Human T-lymphoblastic leukaemia cell line CEM was used for routine screening of these compounds. To prove a common mechanism of action, selected compounds which showed activity in a screening assay were tested in a panel of celi lines (Table 2). These lines were from different species and of different histogenetic origin and they possess various alterations in cell cycle regulatory proteins and hormone dependence status (Table 2). The cells were maintained in Nunc/Corning 80 cm plastic tissue culture flasks and cultured in cell culture medium (DNEM with 5 g/1 glucose, 2mM glutamine, 100 U/ml penicillin, 100 gglml streptomycin, 10% foetal calf scrum and sodium bicarbonate). Individual compounds were dissolved in 10% dimethylsulfoxide/saline, pH 8.0.

The cell suspensions that were prepared and diluted according to the particular cell type and the expected target cell density (2.500-30.000 cells per well based on cell growth characteristics) were added by pipette (800) into 96/well microtiter plates. Inoculates were allowed a pre-incubation period of 24 hours at 37C and 5% C02 for stabilisation. Four-fold dilutions of the intended test concentration were added at time zero in 20gl aliquots to the microtiter plate wells. Usually, test compounds were evaluated at six 4-fold dilutions. In routine testing, the highest well concentration was 250 pM, but it may differ, depending on the agent, All drug concentrations were examined in duplicate. Incubations of cells with the test compounds lasted for 72 hours at 37'C, in 5% C02 atmosphere and 1001/o humidity. At the end of the incubation period, the cells were assayed by using the NfTT assay. Ten microliters of the MTT stock solution were pipetted into each well and incubated further for 1-4 hours. After this incubation period, formazan was solubilized by the addition of 1000well of 10% SDS in water (pH=5.5) followed by further incubation at 37'C overnight. The optical density (OD) was measured at 540nm with the Labsystem iEMS Reader NIF(UK). The tumour cell survival (TCS) was calculated using the following equitation: TCS=(ODdug exposed well / Mean 18 OD,.t,,>1,,,,.ns) x 100%. The WS5o value, the drug concentration lethal to 50% of the tumour cells, was calculated from the obtained dose response curves.

To evaluate the anti-cancer activity of betulinines, their cytotoxic activity against CEM cell line was examined using the screening assay. Potent compounds were further tested against a panel of cell lines of different histogenetic and species origin (Table 2).

Table 2 Cytotoxic activity of the most effective betulinines against a panel of different (non)rnalignant cell lines.

Compound (TCS5o[gM]) Cell Line Description Betulinic Acid 111.3

B16 mouse melanoma 36 2.1 B16F mouse melanoma, metastatic 4.6 4.7 SW620 human colon cancer, metastasis 250 1.2 U87MG human glioblastoma 250 5.1 HepG2 human hepatocellular carcinoma 3.6 1.7 A549 human lung adenocarcinoma 236 1.0 MCF-7 human breast cancer, estrogen dependent, 194 2.3 p53+/+, Rb +/+ U20S human osteosarcoma, p53+/-, Rb +/- 250 1.5 Saos2 human rhabdomyosarcoma, p53-/-, Rb-/- 250 1.8 BT549 human breast cancer, p53mut/mut 250 2.0 MDA-MB-238 human breast cancer, estrogen 195 1.4 independent. p53mutImut LNCaP human prostate cancer, androgen 244 1.1 dependent DU145 human prostate cancer, androgen 241 0.8 independent, Rb-/ HT-29 human colon cancer 250 1.6 OVCAR-3 human ovarian cancer 164 1.0 19 Caco-2 human colon cancer 20 3.0 MEL-3 human melanoma 2.7 1.3 Lymphocytes human normal lymphocytes 250 13 NlH3T3 mouse immortalised fibroblasts 250 7.2 K562-CdA human promyelocytic leukemia, 250 0.3 cladrubin resistant K562-GEM human promyelocytic leukemia, 101 0.9 gemcitabin resistant K562-ARA-C human promyelocytic leukemia, 250 0.6 cytarabin resistant Table 2 continued Compound (TCSw[pM]) Cell Line Description

Betulinic, Acid 111.3 K562-FLUD human promyelocytic leukemia, 250 0.4 fludarabin resistant CEM human T-lymphoblastic leukemia 250 1.0 CEM-DNR human T-lymphoblastic leukemia, 250 0.6 VC2 daunorubicin resistant CEM-DNR human T-lymphoblastic leukemia, 250 1.1 bulk daunorubicin resistant CEM-VCR human T-lymphoblastic leukemia, 19 3.3 1/F3 vincristin resistant CEM-VCR human T-lymphoblasbc leukemia, 24 2.9 3/D5 vincristin resistant CEM-VCR human T-lymphoblastic leukemia, 69 2.5 bulk vincristin resistant In contrast to betulinic acid, which is reported to be an agent selective for neuroectodermal derived tumours, there is no significant difference in the sensitivity of betulinines to tumours of different histogenetic origin.

The compounds are effective in submicromolar or low micromolarconcentrations. However, the non-malignant cells, e.g. NIH3T3 fibroblasts and normal human lymphocytes, tolerated substantially higher doses of betulinines than the tumour cells suggesting a favourable therapeutic index.

Notably, the effectiveness of betulinines was found to be identical in cell lines bearing various mutations or deletions in cell cycle associated proteins (Table 2). This indicates that these substances should be equally effective in tumours with various alterations of tumour suppresser genes, namely p53, Rb, etc.

1 21 Furthermore, betulinines were shown to be equally effective in drug resistant cell lines as on their maternal counterparts, thereby suggesting that classical mechanisms of multidrug resistance apparently do not apply to these compounds. This particular characteristic should be of significant therapeutic 5 benefit to chemotherapy resistant cancer patients.

Finally, the cytotoxic activity of betulinines is independent of the hormonal status of cancer cells, so the compounds should be equally effective in treatment of hormone dependent and independent cancers.

Those skilled in the art will recognise, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

Claims (21)

2-2CLAIN4S Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in therapy, R,, R12 H X7 R3 4 X6 X8 H R5 xl H R2 R, wherein:

1 la X' is C=Q CHOR ' CHOCOR1a X6 is C=Q CH2 X7 is 0 X8 is C=Q CHOCOR 1b, CHOR1b, CH_Hal R-5 and R' 1, R 12 are Me and wherein R' is H or lower alkyl, and Hal is Br, Cl, 1, F; R la-Ib are the same or different groups of R'.

2. Use of a compound according to claim 1 wherein R' is as defined below for the relevant group Rl"b; la X' is C=Q CHOW' or CHOCOR, wherein W' is H and methyl respectively; X8 is CHOCOR1b, wherein R 1b is methyl; X8 is CH-Hal, wherein Hal is Br; X8 is CHOR1b, wherein R 1b is H.

23

3. Use of a compound according to claims 1 or 2 wherein the compound of formula I is selected from the following table:

No. Xl)C X' 1)C C", R""z 111.1 CHOAc C=0 0 C=0 e 111.2 C=0 C=0 0 CHOAc Me 111.3 C=0 CH2 0 CHBr Me 111.

4 C=0 CH2 0 CHOH Me 111.5 C=0 CH2 0 CHOAc Me 111.6 CHOH CH2 0 C=0 Me 111.7 CHOAc CH2 0 C=0 Me Use of a compound according to claims 1 to 3 for treating a proliferative disorder.

5. Use of a compound according to claim 4 wherein the proliferative disorder is cancer.

6.

7.

8.

9.

10.

Use of a compound according to claims 4 or 5 wherein the proliferative disorder is leukaemia.

Use of a compound according to claims 4 to 6 wherein the proliferative disorder is multidrug resistant.

Use of a compound according to claims 4 to 7 wherein the proliferative disorder is independent of p53.

Use of a compound according to claims 4 to 8 wherein the proliferative disorder is independent of Rb.

Use of a compound according to claims 4 to 9 wherein the proliferative disorder is independent of hormones.

24

11. Use of a compound according to any preceding claim wherein the compound of formula 1, or pharmaceutically acceptable salt thereof, effects a change in the Rb protein phosphorylation state.

12. Use of a compound according to any preceding claim for inducing programmed cell death in proliferative cells.

13. Use of a compound of formula 1, or a pharmaceutically acceptable salt thereof, in the preparation of medicament for use in the treatment of a proliferative disorder as defined in any of claims 4 to 10.

14. Use according to claim 13 comprising use of a compound of formula I in an assay for determining the phosphorylation state of cellular substrates.

15. Use according to claim 14 wherein said assay is capable of identifying candidate compounds that influence substrate phosphorylation.

16. Use according to claim 14 or 15 wherein the cellular substrate is Rb protein.

17, A compound of structural formula Ia, or a pharmaceutically acceptable salt thereof, R,, R12 H X7 R3 4 X6 H Rr, R2/ R, wherein:

X1 is C=O, CHORia, CHOCOW' X6 is C=O, CH2 X7 is 0)e is C=O, CHOCOR1b, CHOR1b, CH-Hal la R-5 and R", R12 are Me and wherein R' is H or lower alkyl, and Hal is Br, Cl, 1, F; Rla-1b are the same or different groups of W; with the proviso that when X6 is CH2, X is 0, W-5, W' and R 12 are Me; - when X' is C=Q Xg is not CHBr, CHOR CH2, or CHOAc; and - when X8 is C=Q X' is not CHOH or CHOAc.

18. A compound according to claim 17 wherein R' is as defined below for the la-Ib relevant R XI is C=O, CHOR1a or CHOCOR1a, wherein W' is H and methyl respectively; X8 is CHOCOR1b, wherein Wb is methyl; 26 X' is CH-Hal, wherein Hal. is Br; X8 is CHOR1b. wherein R Ib is H.

19. A compound according to claims 17 or 18 selected from the table of claim 3.

20. A process for preparing a compound of formula I, as defined in claim 1, wherein X6 is C=Q R, R12 H 0 t4 R3 FR 4 H X8 H R5 xl i H R, R, R, R, 4 AHO R3 R4 X8 "-, H R5 0 / 7 - xl -:

- H R2 R, Ib IC comprising oxidising a compound of formula Ib to a compound of formula Ic.

21. A process according to claim 20 wherein the compound of formula Ib is oxidised to a compound of formula lc by treating with chromium trioxide.