WO2000004023A9 - Anhydride modified cantharidin analogues useful in the treatment of cancer - Google Patents

Abstract

Anhydride modified cantharidin analogues useful in the treatment of certain forms of cancer also methods for the screening for anti-cancer activity of these analogues and/or their ability to sensitise cancer cells to cancer treatment. The modified cantharidin analogues have structure (I) or (II), wherein R1, R2, R3 and R4 are H, aryl or alkyl; X is O, N or S; Y is O, S, NH, NR; R is alkyl or aryl; A and B are H or CH3; W and Z are CHOH or C=O. These compounds inhibit protein phosphatase.

Description

ANHYDRIDE MODIFIED CANTHARIDIN ANALOGUES USEFUL IN THE TREATMENT OF CANCER

TECHNICAL FIELD

This invention relates to compounds useful in the treatment of certain forms of

cancer; processes for producing these compounds; methods of treatment using these

compounds per se; methods of treatment using these compounds which methods also

increase the sensitivity of cancer cells to other treatments; methods of screening these

compounds for anti-cancer activity; and methods of screening these compounds for

anti-cancer activity and/or ability to sensitise cancer cells to other methods of treatment.

More particularly, the compounds are specific inhibitors of protein phosphatases 1 and

2 A.

BACKGROUND ART

Protein phosphatase inhibitors and the abrogation of cell cycle checkpoints

The regulation of protein phosphatases is integral to the control of many cell

processes, including cell growth, transformation, tumour suppression, gene transcription,

apoptosis. cellular signal transduction, as neurotransmission, muscle contraction,

glycogen synthesis, and T-cell activation. The role of protein phosphatases in many of

these processes is often mediated via alterations in the cell cycle. Cell cycle progression

is tightly regulated to ensure the integrity of the genome. During cell division it is

imperative that each stage of the cell cycle be completed before entry into the next, and

this is achieved tluough a series of checkpoints. The cell cycle can be broken down into

four phases, the first gap (G^, is followed by a phase of DNA synthesis (S-phase); this is

followed by a second gap (G ) which in turn is followed by mitosis (M) which produces

two daughter cells in G). There are two major control points in the cell cycle, one late in

SUBSTITUTE SHEET (RULE 26) G and the other at the G₂/M boundary. Passage through these control points is

controlled by a universal protein kinase, cdkl . The kinase activity of cdkl is dependant

on phosphorylation and the association with a regulatory subunit, cyclin B. The periodic

association of different cyclins with different cyclin dependent kinases (cdk) has been

shown to drive different phases of the cell cycle; thus cdk4-cyclin Dl drives cells

through mid G_b cdk2-cyclin E drives cells in late G_b cdk2-cyclin A controls entry into

S-phase and cdc2-cyclin B drives the G₂/M transition (O'Connor, 1996, 1997).

Following DNA damage induced by chemotherapy or radiation treatment these

checkpoints are responsible for halting cell cycle progression in G_j S and/or G₂ phases

(O'Connor, 1996). The cell undergoes a cell cycle arrest so that the damaged DNA can

be repaired before entry into S phase or mitosis. The phase at which the cell cycle is

halted will depend upon the type of DNA damaging agent used and the point during the

cell cycle that the damage was incurred (O'Connor, 1997). The cell cycle is controlled

and regulated by an intricate phosphorylation network (Stein et al., 1998). More

particularly, activation of cdk/cyclin complexes requires the phosphorylation of a

conserved threonine residue, which are catalysed by CAK kinase, as well as the removal

of inhibitory phosphorylations by the phosphatase cdc25. Cdc25 is only active in its

phosphorylated form. Therefore, protein phosphatase 2 A (PP2A) can inhibit the

activation of cdk/cyclin complexes by inhibiting CAK activity and by dephosphorylating

cdc25. The G,/S checkpoint is predominantly regulated by the cdk/cyclin D/E complex

that mediates its effects by phosphorylating and inactivating the tumour suppressor

protein retinoblastoma (pRb). The phosphorylation of pRb prevents it from interacting

with the S-phase transcription factor E2F. E2F controls the transcription of proteins

SUBSTITUTE SHEET (RULE 26) - j - needed for DNA synthesis and entry into S-phase including thymidylate synthase.

Accordingly, the inactivation of pRb by phosphorylation permits entry into the S-phase

and vice versa. However, protein phosphatase 1 (PPl) can dephosphorylate pRb and

inhibit the cell cycle (Durfee et al., 1993). Thus, PPl and PP2A are both negative

regulators of the cell cycle. Inhibition of PPl and PP2A would abrogate these

checkpoints and prematurely force cells through the cell cycle.

Serine/threonine phosphatases, which are responsible for protein

dephosphorylation. comprise a unique class of enzymes consisting of four primary

subclasses based on their differences in substrate specificity and environmental

requirements. Of the serine/threonine phosphatases, protein phosphatases 1 and 2 A (PPl

and PP2A, respectively) share sequence identity between both enzyme subunits (50% for

residues 23-292; 43% overall), are present in all eukaryotic cells and are together

responsible for 90% of all cellular dephosphorylation. Knowledge of structure and

subsequent correlation of binding function for both PP 1 and PP2 A would therefore

provide a vital link toward understanding the biochemical role of these enzymes. A goal

of the medicinal chemist is the development of potent and selective inhibitors of these

protein phosphatases.

The natural toxins, okadaic acid, calyculin A, microcystin-LR and tautomycin are

representative of a structurally diverse group of compounds that are all potent protein

phosphatase 1 (PPl) and 2A (PP2A) inhibitors. Okadaic acid is more specific for PP2A

(IC₅₀ InM) than PPl (IC₅₀ 60nM), while calyculin is slightly more specific for PPl (IC₅₀

0.5-1. OnM) than PP2A (IC₅₀ 2nM). All of these phosphatase inhibitors are known to

abrogate cell cycle checkpoints, particularly the G₂ checkpoint of the cell cycle and

SUBSTITUTE SHEET (RULE 26) induce cellular mitoses (Yamashita et al., 1990). Abrogation of the G₂ checkpoint means

that the cell does not have the capacity to detect DNA damage or malformation of the

genome prior to entry into mitosis. Therefore, cells which have a deficient G₂ checkpoint

are unstable, and incapable of detecting DNA damage, initiating G₂ arrest, or undergoing

DNA repair. Such cells enter the mitotic stage of the cell cycle prematurely with

malformed spindles. The abrogation is of the G₂ checkpoint in the cell cycle by okadaic

acid is mediated via the activation of cdc2/Hl kinase. the major mitotic inducer, and

results in a premature mitotic state (Yamashita et al., 1990). Although okadaic acid is

known as a tumour promoter, in some cell types, it has been shown to revert the

phenotype of oncogene-transformed cells to that of normal cells, and to inhibit neoplastic

transformation of fibroblasts (Schonthal, 1991 ).

Furthermore, okadaic acid has been shown to selectively enhance the cytotoxicity

of vinblastine and the formation of apoptotic cells, in HL60 cells which are p53 nul

(Kawamura, 1996). Interestingly, calyculin enhances irradiation killing in fibroblast cells

at doses that are non toxic when given as a single treatment. (Nakamura and Antoku,

1994). Data also shows that okadaic acid can abrogate the G,/S checkpoint of the cell

cycle. In this context, okadaic acid has been shown to overide the S-phase checkpoint

and accelerate progression of G₂-phase to induce premature mitosis (Gosh et al., 1996).

In addition, okadaic acid has been shown to significantly increase the fraction of

quiescent cells entering the S-phase via modifications in the phosphorylation state of

pRb (Lazzereschi et al., 1997). Other studies have shown that the hyperphosphoryation

state of pRb forces cells prematurely into S-phase and pRb can be kept in a

phosphorylated state via protein phosphate inhibition (Herwig and Strauss, 1997). Cells

SUBSTITUTE SHEET (RULE 26) lacking functional pRb show increased apoptosis and cytotoxicity following 5-

fluorouracil and methotrexate treatment (Herwig and Strauss, 1997). We propose that

cell death would be substantially enhanced in cells forced to enter the S-phase

prematurely (via G, checkpoint abrogation) and which were lacking key S-phase

components such as dTMP (via TS inhibition).

The okadaic acids class of compounds, with the exceptions of okadaic acid,

cantharidin (Honaken) and thyrisferyl 23 -acetate (Matszawa et. al) (being PP2A

selective) exhibit poor selectivity. Furthermore, the concentration of PPl and PP2A

inside cells is such that high concentrations of these inhibitors are required to generate a

response in vivo resulting in the loss of effectiveness of any in vitro selectivity (Wang).

Cantharidin (exo.exo-2.3-dimethyl-7-oxobicyclo[2.2.1]heptane-2,3-dicarboxylic

acid anhydride), is a major component of the Chinese blister beetles:

Mylabris phalerata or M. cichorii)(Yang; Cavill et. al). The dried body of these beetles

has been used by the Chinese as a natural remedy for the past 2000 years. Although

Western medicine decreed cantharidin to be too toxic in the early 1900's (Goldfarb et.

al) its purported aphrodisiac qualities (the active ingredient of "Spanish Fly"), and its

widespread occurrence in cattle feed still results in numerous human and livestock

poisonings (Schmitz).

Li and Casida, and previous work in this laboratory (McCluskey et. al) (and

more recently Pombo-Villar, Sodeoka) has assisted in the delineation of certain features

crucial for inhibition of PP2A by cantharidin analogues (Figure 1). However the

corresponding picture for PPl is not so clear, the majority of data refers to possible

SUBSTITUTE SHEET (RULE 26) interactions with the known crystal structures, and in some cases the inhibition values

for PPl are not reported.

Involvement of Tumour Suppressor Gene p53

The most commonly mutated gene in human cancers is the tumour suppressor

gene p53, which is abnormally expressed in more than 50% of tumours. The

development of chemotherapeutic agents which selectively target cancer cells with

mutant p53 is certainly desirable, for two main reasons. Firstly, cells that have an

abnormal p53 status are inherently resistant to conventional chemotherapy and produce

the more common, and more aggressive tumours such as colon carcinoma and non small

cell lung cancer. Secondly, a chemotherapy regime that targeted only those cells with a

mutant p53 phenotype would potentially produce fewer side effects since only the

cancer cells would be killed and not the p53 proficient normal healthy cells.

DISCLOSURE OF THE INVENTION

In relation to the discussion above, the present inventors believed that the

replacement of the ether O atom of the anhydride with N or S (as N-H and N-R, where

R = alkyl or aryl) would allow them to probe the H-bonding requirements of this region

of cantharidin analogues. Previous studies in their laboratory had shown limited

tolerance for modification of the 7-oxa position. An ability to modify these heteroatoms

is crucial to the development of selective inhibitors based on this simple skeleton.

There is not, at present, an inhibitor with either absolute specificity or high

enough selectivity which renders the inhibitor effectively specific in vivo.

It has surprisingly been found that anhydride modified cantharidin analogues,

which are the subject of this invention, may possess one or more of the properties of

SUBSTITUTE SHEET (RULE 26) being potent, selective, oxidatively stable, and cell permeable inhibitors of protein

phosphatases 1 and 2A.

Therefore, according to the first aspect of this invention there are provided cell

permeable inhibitors of protein phosphatases 1 and 2A, said inhibitors being anhydride

modified cantharidin analogues.

According to a particular embodiment of the first aspect of this invention there

are provided compounds of the formula:

wherein R, and R₂ are H, aryl or alkyl; X is O, N or S; Y is O, S, SR, NH, NR, CH₂OH,

CH₂OR; R is alkyl or aryl; A and B are H or CH₃; W and Z are CHOH or C=0 and R

and R₂ can cyclise to form a ring as follows:

wherein R₃ and R₄ are H, aryl or alkyl.

The aryl group may suitably be phenyl or naphthyl for example, and may be

attached via a carbon spacer of between 6 and 10 carbon atoms. The alkyl group may

suitably be C C^.

SUBSTITUTE SHEET (RULE 26) According to the second aspect of this invention there is provided a process for

producing anhydride modified cantharidin analogues. The process may include the

steps of:

dissolving a diene in a suitable solvent and adding to the resultant solution an

ene.

According to a third aspect of the invention there is provided a process for

producing anhydride modified cantharidin analogues, involving the step of reacting a

diene with an ene.

The process may further involve hydrogenation of the adduct of the diene and

ene and/or optionally, ring opening of the adduct.

Generally, the reaction conditions for the production of the anhydride modified

cantharidin analogues are dependent on the aromaticity of the starting diene. Suitable

reaction conditions are exemplified below.

According to a fourth aspect of this invention there is provided a method of

treating a cancer which method comprises administering to a patient in need of such

treatment, an effective amount of an anhydride modified cantharidin analogue of the

first aspect of this invention, together with a pharmaceutically acceptable carrier, diluent

and/or excipient.

The method may be carried out in conjunction with one or more further

treatments for treating the cancer.

According to a fifth aspect of this invention there is provided a method of

sensitising cancer cells to at least one method of treating cancer, which method of

sensitising comprises administering to a patient in need of such treatment, an effective

SUBSTITUTE SHEET (RULE 26) amount of an anhydride modified cantharidin analogue of the first aspect of this

invention, together with a pharmaceutically acceptable carrier, diluent and/or excipient.

According to a sixth aspect of the invention there is provided a method of

treating cancer which method comprises:

administering to a patient in need of such treatment, an effective amount of an

anhydride modified cantharidin analogue to sensitise cancer cells of the patient to one or

more cancer treatments; and utilising the one or more cancer treatments.

According to a seventh aspect of this invention there is provided a method of

screening a compound for anti-cancer activity.

According to an eighth aspect of this invention there is provided a method of

screening compounds for use in the fourth aspect of this invention, said method

comprising screening for anti-cancer activity; and screening for ability to abrogate either

the G_| or the G₂ checkpoint of the cancer cell cycle. The method may also comprise

screening for the ability of said compounds to sensitise cancer cells to one or more

cancer treatments.

The one or more cancer treatments mentioned above may be selected from

treatments involving cisplatin, irradiation, taxanes and antimetabolites.

The invention will hereinafter be described with reference to Examples and the

accompanying figures.

Brief Description of the Figures

Figure 1 is a schematic representation of the structure activity data generated for

inhibition by PP2A by cantharidin analogues;

Figure 2: New cantharidin analogues.

Figure 3: Cytotoxicity of cantharidin and the new cantharidin analogues.

SUBSTITUTE SHEET (RULE 26) Figure 4: Cell cycle analysis 12h following exposure to cantharidin, MK-2 or MK-4.

Figure 5: Cell cycle analysis 18h after 6Gy of radiation and 12h after exposure to cantharidin, MK-2 or MK-4. Figure 6 (a-c): Combination index versus fraction affected: HCT116 colon cells in simultaneous combination with cisplatin and MK-4.

Figure 7 (a-b): Combination index versus fraction affected: HT29 colon cells in simultaneous combination with cisplatin and MK-4.

Figure 8 (a-c): Combination index versus fraction affected: HCT116 colon cells in simultaneous combination with taxotere and MK-4

Figure 9 (a-c): Combination index versus fraction affected: HT29 colon cells in

simultaneous combination with taxotere and MK-4.

Best and other Modes for Carrying Out the Invention

As mentioned above, the reaction conditions for producing anhydride modified

cantharidin analogues encompassed by the present invention generally depend on the

aromaticity of the starting diene. This is illustrated by a description of examples of the

methods wherein the starting materials are furan (Method 1 below): thiophene (Method

2 below); and pyrrole (Method 3 below).

Method 1 : Furan as the starting diene

A solution of furan (5 equivalents) is dissolved in a suitable solvent (about 5 times

the volume of furan, the solvent can be for example, ether (for room temperature

reactions); or benzene or xylene (the latter two for reactions at 80 and 130°C

respectively). To this solution is added one equivalent of the ene. The reaction is then

heated (or stirred at room temperature), typically for 24 hours (2 days in the case of the

room temperature reaction). Upon cooling (or standing at room temperature) a precipitate

forms and is collected by vacuum filtration. The adduct is then purified by

SUBSTITUTE SHEET (RULE 26) recrystalisation from for example, chloroform or ethanol. In the case of the furan +

maleic anhydride compound care is exercised to minimise heating as this causes a reto-

Diels-Alder reaction yielding only the starting materials.

Method 2: Thiophene as the starting diene

Thiophene (l .Olόg, 0.012 mol) and maleic anhydride (0.558.0.006 mol) are

mixed at room temperature in 2.5 mL of distilled dichloromethane. This mixture is then

placed inside a high pressure reactor. They are compressed to a pressure of 17kbar at

40°C for a period of 71 hours, after which the pressure is released and the product

purified by chromatography.

Method 3 : Pyrrole as the starting diene

To [Os (NH₃)₅OsO₂ CF₃ )] (CF₃SO₃)₂ , (0.3511 g, 0.4 mmol) and activated

magnesium (0.151 1 g), pyrrole (0.45 mL, 0.6 mmol), DME (1 mL) and DMAc (0.3 mL)

are added in that order. The mixture is stirred for 1 hour, the temperature gradually

rising to 40°C and then dropping. The brown slurry is filtered through a thin pad of

celite, and the cake washed with DME in small portions (4 x 2 mL). The filtrate is

added to dichloromethane (15 mL). Vigorous stirring results in the formation of yellow

coloured precipitate which is collected by vacuum filtration, followed by an ether wash

(2 x 2.5 mL). The product is dried under a stream of nitrogen yielding a yellow-tan

solid (0.343g, 84%). To this pyrrole complex is added maleimide (0.05g, 0.515 mmol)

(or any other "ene", eg maleic anhydride, dimethyl maleate, etc) in acetonitrile. The

mixture is allowed to stir at room temperature for 60 min. after which the solvent is

removed by vacuum, yielding the exo isomer (0.359g, 64%). The crude material is

purified by ion-exchange column (Sephadex-CM C-25, 2 x 10 cm), using NaCl as the

SUBSTITUTE SHEET (RULE 26) mobile phase. The complexes are precipitated by the addition of a saturated sodium tetraphenylborate solution.

The types of cancer which are amenable to treatment by these compounds

include those types of cancer which are inherently resistant to conventional

chemotherapy. Typically, these types of cancer are represented by the more common

and more aggressive tumour types such as, but not limited to, colon cancer and non

small-cell lung cancer.

The compounds of this invention are suitably administered intravenously,

although other modes of administration are possible. Pharmaceutically acceptable

diluents, adjuvants, carriers and/or excipients may be used in conjunction with the

compounds of this invention.

Suitable such pharmaceutically acceptable substances are those within the

knowledge of the skilled person and include compounds, materials and compositions

deemed appropriate.

Actual dosage levels of the compounds of the invention may be varied so as to

obtain an amount of the active ingredient which is effective to achieve the desired

response for a particular patient, composition and mode of administration.

The dosage level can be readily determined by the physician in accordance with

conventional practices and will depend upon a variety of factors including the activity of

the particular compound of the invention to the administered, the route of

administration, the time of administration, the rate of excretion of the particular

compound employed, the age, sex, weight, condition, general health and prior medical

history of the patient being treated, and like factors well known in the medical arts.

SUBSTITUTE SHEET (RULE 26) The compounds of this invention may also sensitise cancer cells to other

methods of treatment. For example, typically these methods include irradiation and

treatment with platinum anti-cancer agents, for example cisplatin.

In addition, sensitisation may also be brought about by, for example the use of

the plant alkaloids vinblastine and vincristine, both of which interfere with tubulin and

the formation of the mitotic spindle, as well as taxanes and antimetabolites, including 5-

fluorouracil, methotrexate and antifolates.

In particular, the compounds of this invention sensitise those cells with deficient

p53 activity.

When screening for anti-cancer activity as contemplated by the invention,

various cancer cell lines may be chosen. These are typically both haematopoietic and

solid tumour cell lines with varying p53 status and include: L1210 (murine leukaemia,

p53 wildtype), HL60 (human leukaemia, p53 nul), A2780 (human ovarian carcinoma,

p53 wildtype), ADDP (cisplatin resistant A2780 cells, p53 mutant), SW480 (human

colon carcinoma, p53 mutant), WiDr (human colon carcinoma, p53 mutant), HT29

(human colon carcinoma, p53 mutant), HCT116 (human colon carcinoma, p53

wildtype) and 143B (human osteosarcoma, p53 mutant).

In addition to the methods for screening for anti-cancer activity, the following

procedures may be suitably used in the remainder of the screening process. For

example, when screening for the ability to abrogate the G_j and/or the G₂ checkpoint of

the cancer cell cycle, the following are suitably used:

SUBSTITUTE SHEET (RULE 26) Cell cycle method

The cells are fixed in 70% ethanol and stored at - 20°C until analysis is

performed (1-2 weeks). After fixing, the cells are pelleted and incubated in PBS

containing propidium iodide (40mg/ml) and RNase A (200 mg/ml) for at least 30 min at

room temperature. The samples (2 X 10 events) are analysed using a Becton Dickson

FACScan, fluorescence is collected in fluorescence detector 2 (FL2), filter 575/30 nm

band pass. Cell cycle distribution is assessed using Cell Quest software (Becton

Dickson).

Those protein phosphatase inhibitors which show abrogation of either the Gj or

G₂ checkpoint will then be exploited in combination studies with either radiation

exposure or chemotherapy drugs incubation. The MTT (3-[4,5-dimethylthiazol-2-

yl]2,5-diphenyl-tetrazolium bromide) assay is used to determine whether a synergistic,

antagonistic or additive effect is induced. The Median Effect method is adopted to

mathematically determine the optimal combination index of the treatments chosen

(Chou and Talalay, 1984). This method has been extensively used to investigate the

cytotoxicity of various drug combinations including cisplatin and D1694 (Ackland et al

1996; 1998). A combination index value less than 1 indicates synergism, a value equal

to 1 indicates additivity and a value greater than one indicates antagonism.

Cytotoxicity assay

When screening for the ability to sensitise cancer cells to conventional

chemotherapy and irradiation, the following methods are suitably used:

Cells in a subconfluent phase are transferred to 96- well microtitre plates. LI 210

cells are plated at a density of 1000 cells/well in lOOμl medium, while all other cell

SUBSTITUTE SHEET (RULE 26) lines are plated at a density of 2000-25000 cells/well. The cells are left for 24h prior to

treatment to ensure exponential growth has been achieved, 24h after plating (day 0),

lOOμl of phosphatase inhibitor is added to each well, control wells received lOOμl of

medium only. Drug exposure time is 72h (day 3). The effect of phosphatase inhibition

is tested in triplicate over a concentration range of 1 x 10^"3M - 1 x 10^"8 M. Growth

inhibitory effects are evaluated using the MTT assay and absorbance read at 540 nm.

The IC₅₀ is the drug concentration at which cell growth is 50% inhibited based on the

difference of optical density on day 0 and day 3 of drug exposure. Cytotoxicity is

evaluated using a spectrophotometric assay which determines the percentage of cell

growth following exposure of the cells to various concentrations of the phosphatase

inhibitors for a period of 72 hours. The subsequent dose response curve is used to

calculate IC_5ϋ values (the drug concentration at which cell growth is 50% inhibited).

Most drug discovery has focused on the development of new single agents.

However, in light of the success of combination chemotherapy it is increasingly

apparent that successful anticancer treatment of the future will be based upon the

discovery of agents which are synergistic in their action. In view of this, the cytotoxicity

of phosphatase inhibitors in combination with either radiation, cisplatin, taxanes,

antimetabolites or plant alkaloids is examined. As indicated above, calyculin which by

itself is not cytotoxic, enhances irradiation induced cell death. Similarly abrogation of

the G₂ checkpoint by either, caffeine or UCN-01, also enhances the cytotoxicity of γ

irradiation in cells with mutant p53 (CA46 and HT-29 cells) (Powell et al., 1995;

Russell et al.. 1995: Wang et al., 1996). DNA damage induced by irradiation causes

both a G, and G₂ cell cycle arrest. In p53 mutant cells, the G_j checkpoint is absent.

SUBSTITUTE SHEET (RULE 26) However, following irradiation the cells will still arrest in the G₂ phase, and potentially

repair the damage. P53 mutant cells are generally more resistant to conventional

chemotherapy and produce more aggressive tumours. Therefore, in p53 deficient cells,

DNA damage that is not detected by the G, checkpoint will be picked up by the G₂

checkpoint. If the cells are deficient in both of these checkpoints then it is believed that

the cells will be unable to initiate repair mechanisms and will be more unstable and

increasingly susceptible to cell death induced by DNA damage.

Cisplatin is another commonly used anticancer treatment which binds to DNA

and produces DNA crosslinks and strand breaks. Cisplatin is particularly useful in the

treatment of testicular carcinoma, small cell carcinoma of the lung, bladder cancer, and

ovarian cancer. Repair of cisplatin induced DNA damage is mediated via nucleotide

excision repair which is coordinated by p53 activation of Gadd45 (Smith et al., 1994).

In this context, it has been suggested that cells that are p53 mutant are more sensitive to

cisplatin treatment (Hawkins et al., 1996). A number of researchers have investigated

this proposal in p53 mutant cell lines and in p53 mutant tumours, with mixed results.

While it is apparent that cisplatin is more cytotoxic in cells lines that are deficient in p53

(induced via papillomavirus) compared to the p53 proficient cells (Hawkins et al.,

1996), it is harder to test this hypothesis in tumours and in cisplatin resistant cells as

they may have several undefined mutations in their genome which would confound such

studies (Herod et al., 1996). Nevertheless, the G₂ abrogator UCN-01 (7-

hydroxystaurosporine, a protein kinase inhibitor) has been shown to markedly enhanced

the cell-killing activity of cisplatin in MCF-7 cells defective for p53 function (Wang et

al., 1996).

SUBSTITUTE SHEET (RULE 26) The development of chemotherapeutic agents which selectively target p53

mutant cells is desirable since 50% of tumours have either a mutated or deleted p53

gene. Many of these p53 deficient cells and tumours are inherently resistant to

conventional chemotherapy and represent the common more aggressive tumour types

such as colon cancer, and non-small cell lung cancer. Thymidylate synthase (TS)

inhibitors are another class of commonly used anticancer agents. TS catalyses a critical

step in the pathway of DNA synthesis by converting dUMP to dTMP by methylation

using the co-substrate N5,N10-methylene tetrahydrofolate (CH₂-THF) as a methyl

donor. This step is the only de novo source of dTMP, which is subsequently metabolised

to dTTP exclusively for incorporation into DNA during synthesis and repair (Jackman &

Calvert, 1995). Thus, TS is a key regulatory enzyme during the S-phase of the cell cycle.

Lack of dTTP results in DNA damage and ultimately cell death, but the process(es) by

which cell death occurs is not clear. TS inhibitors such as fluorouracil, raltitrexed, and

LY231514 play a pivotal role in anticancer treatment and are often the first line

treatment of many cancers (Peters & Ackland, 1996). We propose that the TS inhibitor

Thymitaq (Zarix, Ltd) be used in combination with cantharidin analogues. Thymitaq is a

direct and specific TS inhibitor which does not require active transport into the cell nor

does it require intracellular activation for its action.

The following examples are not to be construed as limiting on the scope of the

invention as indicated above.

SUBSTITUTE SHEET (RULE 26) Example 1

Chemistry

Anhydride modified cantharidin analogues were synthesised by a variety of

modified literature procedures, as set out in schemes 1 and 2. These modifications are

embodied in the three methods, which depend on the aromaticity of the starting dienes,

set out above. The dimethyl ester (3), which was prepared by the application of high

pressure, 17kbar, 40°C, 61 hours, as shown in scheme 3.

Scheme 1. a. Furammaleic anhydride (5:1). diethylether. 2d. RT, 96%; b. H₂ / 10% Pd-

C/ EtOH; c. p-TosOH, MeOH, chromatography; d. H₂ / 10% Pd-C/ Acetone; e. NaBH₄

then HC1.

SUBSTITUTE SHEET (RULE 26) Scheme 2. Reagents and Conditions: f. Fura maleimide (5:1), diethyl ether, 7d, in

dark, 75%, exo product; g. FuramMaleimide (5: 1), diethylether, sealed tube 12h, 90°C,

66%,endo product.

Scheme 3. Reagents and Conditions: h.

Furan:dimethylmaleate (2:1), CH₂C1₂ ,17 Kbar, 40°C. 61 h, 56%.

Example 2

Development of potent, selective, oxidativelv stable, and cell permeable inhibitors of

protein Phosphatases 1 and 2A.

Crude natural product extracts have yielded isopalinurin and a series of

cantharidin analogues have been synthesised. In this context, the present inventors have

developed the simple cantharidin analogue which is PPl selective (IC₅₀ = 50mM, with

0% inhibition of PP2A at concentrations >1000mM) representing the first small

molecule to exhibit selectivity for PP 1. Results have indicated that a series of simple

synthetic modification of the cantharidin skeleton also allows the synthesis of a PP2A

selective compound (see Figure 1 ).

The present inventors have previously demonstrated that a facile ring opening of

an anhydride is crucial to inhibition of PP2 A. This is not possible with c (previous

studies with the 7-0, and this analogue indicated considerable hydrolytic stability of the

maleimide link). It is also interesting to note that endothal thioanhydride is three fold

more potent than cantharidin, with the S atom being an important factor. It is thus

SUBSTITUTE SHEET (RULE 26) envisaged that the 7-S group presents itself to the active sites metals and the N-H of the

maleimide occupies the hydrogen bond cavity normally reserved for the 7-0 substituent

cantharidin.

Structure of cantharidin and selective analogues

(a) (b)

(c)

(a) Shows structure of cantharidin;

(b) Shows PP 1 selective analogue; and

(c) Shows PP2A selective analogue. In the case of panel (c) IC₅₀ ~ 25mM.

On the basis of these results and previous experience in our laboratory (synthesis

and molecular modelling of cantharidin inhibitors at PPl and PP2A), we have designed a

series of analogues which are more active and selective, whilst retaining the desirable

properties of stability and cell permeability.

The synthetic pathways to these analogues are shown in schemes 1-3. Each

scheme allows for modification of the basic skeleton, and in some cases the insertion of

beneficial feature that were present in the more complex natural toxin(s) (eg okadaic

acid, calyculin, microcystin, etc). The inclusion of these features is designed to provide

enhanced selectivity and potency.

SUBSTITUTE SHEET (RULE 26)

Example 3

Synthetic development of a series of PPl and PP2A analogues of cantharidin.

(i) Diels-Alder addition (maleic anhydride) and subsequent manipulations of X;

(ii) Diels-Alder addition (substituted maleic anhydrides), introduction and manipulation

of Z (Z = hydrophobic tail; eg long chain nitrile: cf Calyculin A, long chain terminating

in a spiro acetal: cf Tautomycm. Okadaic acid; long chain terminating in an aromatic

ring: cf Adda in Microcystin-LR; (iii) stereospecific ring opening of the anhydride

allowing further manipulations of the newly released functional groups (see scheme 2).

In this instance we have developed synthetic protocols in our laboratory that

allow the facile assembly of these analogues. Biological evaluation and molecular

modelling of the most active molecules will allow compounds to be evaluated.

Additional modification to the basic structure can be obtained as exemplified

below.

SUBSTITUTE SHEET (RULE 26)

X = CH₂, S, O

Example 4

A specific example of one class of cantharidin analogue that shows promise as a

selective inhibitor of protein phosphatases 1 and 2A.

Example 5

Stereospecific route towards 7-azabicvclo [2.2.11 heptanes

We have shown that the introduction of the bridgehead nitrogen improves the

potency, selectivity and stability of similar analogues, the above pathway has been

developed to further improve the bio-activity of these analogues. The synthetic routes

alluded to herein may allow the rapid assembly of the target molecules.

Those agents which meet the requirements of being stable, specific, potent, and

membrane permeable protein phosphatase inhibitors are screened for their anti-cancer

activity.

SUBSTITUTE SHEET (RULE 26) Example 6

Biochemistry

All synthesised compounds were tested for their ability to inhibit protein

phosphatases 1 and 2A. Initial investigations were carried out at 100 mM. Promising

analogues were then assayed in triplicate for estimation of IC₅₀ values.

Protein phosphatase 1 and 2A were partially purified from chicken skeletal

muscle essentially as described by Cohen Protein phosphatase activity was measured at

37°C in 50 mM Tris-HCl buffer (pH 7.4). 0.1 mM EDTA, 5 mM caffeine, 0.1% 2-

mercaptoethanol and 1 mg/ml bovine serum albumin using 30 mg [^J P] -phosphorylase

as substrate. The total assay volume was 30 ml. The assay conditions were restricted to

20% dephosphorylation to ensure linearity and inhibition of protein phosphatase activity

was determined by including cantharidin or its analogues at the required concentrations

in the reaction buffer. Reactions were terminated by the addition of 0.1 ml ice cold 20%

trichloroacetic acid. Precipitated protein was pelleted by centrifugation and the

radioactivity in the supernatant measured by liquid scintillation counting. Data is

expressed as the percentage inhibition with respect to a control (absence of a competing

compound) incubation.

Example 7

Screening various PPl and PP2A inhibitors for anti-cancer activity

(a) Cytotoxicity of protein phosphatase inhibition:

Those PPl and PP2A inhibitors which fulfil the requirements detailed above

were tested in various cancer cell lines. The cells lines chosen for study included both

haematopoietic and solid tumour cell lines with varying p53 status and include:

SUBSTITUTE SHEET (RULE 26) L1210 (murine leukaemia, p53 wildtype),

HL60 (human leukaemia, p53 nul),

A2780 (human ovarian carcinoma, p53 wildtype),

ADDP (cisplatin resistant A2780 cells, p53 mutant),

SW480 (human colon carcinoma, p53 mutant),

WiDr (human colon carcinoma, p53 mutant).

HT29 (human colon carcinoma, p53 mutant)

HCT1 16 (human colon carcinoma, p53 wildtype)

143B (human osteosarcoma, p53 mutant)

Anti-cancer screening of the protein phosphatase inhibitors is assessed using the

MTT assay. This assay determines cell viability by the ability of mitochondrial

dehydrogenase to produce formazan crystals from 3-(4,5-dimethylthiazol-2-yl) -2, 5-

diphenyltetrazolium bromide. The viable cell number/well is directly proportional to the

production of formazan, which following solubilization, can be measured

spectrophotometrically (540nm). This technique is also used by the National Cancer

Institute to screen for new anticancer agents.

As described herein a number of cantharidin analogues have been synthesised

and tested for their anticancer activity in nine cancer cell lines using the MTT assay after

72 h exposure. These new analogues are shown in Figure 2 and have been designated

MK-1 through to MK-9. The cytotoxicity (IC₅₀) of these cantharidin analogues is shown

in Table 1 and Figure 3. In summary, the MK-1 analogue did not show any significant

cytotoxicity in any of the cell lines tested (IC₅₀ >1000μM). Only marginal cytotoxicity

across all cell lines tested was observed for MK-3 (IC₅₀ 247 to >1000μM), MK-7 (IC₅₀

SUBSTITUTE SHEET (RULE 26) Table 1

ICso values of tumour cell lines after 72 h continuous exposure to cantharidin and cantharidin analogues.

Tumour Cell p53 IC-50 (mean + SE) after 72h continuous exposure (μM) type line status Cantharidin MK-1 MK-2 MK-3 MK-4 MK-5 MK-7 MK-8 MK-9

Murine Leukaemia L1210 wt 18 ± >1000 185 ±51 647 ± 132 680 ± 97 >1000 367 ± 37 337 ± 19 192 ±56

Human Leukaemia HL60 nul 13 ± >1000 177 ±3 247 ± 55 393 ± 103 323 ± 13 293 ±7 297 ±3 133 ±9

Human Ovarian A2780 wt ± >1000 157 ±9 317 ± 17 333 ± 55 567 ± 109 357 ± 102 313 ±61 187 ±9

Human Ovarian ADDP mt 12 ± 0.8 >1000 183 ±17 >1000 275 ± 56 260 ± 40 210 ±18 208 ± 19 233 ± 23

Human Osteosarcoma 143B mt 10.2 ±1.2 >1000 248 ± 29 665 ± 225 450 ± 50 >1000 327 ± 67 385 ± 43 223 ± 44

Human Colon HCT116 wt 12 ± >1000 160 ±10 >1000 78 +7 143 ± 23 180 ±20 173 ±22 107 ± 12

Human Colon HT29 mt 6.4 ± 0.7 >1000 183 ±20 530 ± 112 14 ± 0.3 28 ± 1 297 ± 58 373 ± 54 205 ± 13

Human Colon WiDr mt 6.1 ± 0.5 >1000 198 ± 53 620 ± 31 15±3 31 ± 10 320 ± 20 367 ± 44 190 ± 35

Human Colon SW480 mt 17.5 ± >1000 155 ±9 444 ± 27 88 ±5 247 ± 14 333 ± 22 353 ± 20 147 ± 14

_|-

CΛ wt = wildtype, mt = mutant.

180-367μM) and MK-8 (IC₅₀ 173-385μM). Greater cytotoxicity was observed with MK-

2 (IC₅₀ 157-248μM) and MK-9 (IC₅₀ 107-233μM) which was also consistent across the

nine cell lines. The greatest cytotoxicity was observed with the MK-4 and MK-5

analogues, however, the magnitude of this response was cell line dependent. In this

context, MK-4 and MK-5 were selectively more cytotoxic in the human colon cancer

cell lines (IC₅₀ 14-88μM; 28-247μM) compared with leukaemia (IC₅₀ 393-680μM; 323-

>1000μM) ovarian (IC₅₀ 275-333μM; 260-567μM), and osteosarcoma (IC₅₀ 450μM;

>1000μM) cells respectively.

(b) Abrogation of cell cycle checkpoints:

The ability of the protein phosphatase inhibitors to abrogate the G, or G₂

checkpoint of the cell cycle may be determined by cell cycle analysis using flow

cytometry. Briefly, asynchronous cell cultures are harvested 18h after 6Gy irradiation

and/or 12h incubation with the protein phosphatase inhibitor. Depending upon the p53

status of the cell line, radiation treatment alone will induce arrest in either G, and/or G₂

phase of the cell cycle.

Data shown in Table 2 and Figure 4 show the cell cycle response of LI 210,

HL60, HT29 and HCTl 16 cells to cantharidin and the new cantharidin analogues MK-2

and MK-4 after 12h exposure. In summary, cantharidin and MK-2 produced a similar

response and induced G₂ arrest in all four cell lines tested. MK-4 also induced G₂ arrest

but only in L1210, HL60 and HCTl 16 cells. In HT29 cells, MK-2 induced G, cell cycle

arrest. The magnitude of the cell cycle arrest induced by these drugs directly correlated

with their cytotoxicity in the respective cell lines. The ability of the parent compound

cantharidin to inhibit cell growth is also shown (IC₅₀ 6.1-18μM). The cytotoxicity of the

SUBSTITUTE SHEET (RULE 26) Cell Cycle Analysis Table 2

Cell Cycle Distribution (percentage of total) of tumour cell lines 12h after cantharidin or cantharidin analogue treatment.

Method : Flow Cytometry of Propidium Iodide stained cells.

Agent L1210 cells HL60 cells HCTΪ 16 cells HT29 cells μM sub G, G, S G₂+M sub G, G, S sub G, G, S G₂+M sub G, G, S G₂+M

Cantharidin 0 0.5 47.4 34.3 19.4 1.9 45.5 25.8 28.2 6.5 43.3 14.6 36.4 11.1 45.3 8.0 36.0

1 0.5 45.8 33.7 21.6 1.5 44.0 26.1 29.7 2.2 39.9 17.2 41.9 9.0 46.2 7.8 37.4

5 0.6 46.5 32.6 21.9 1.7 41.4 27.7 30.6 2.9 39.9 16.8 41.8 4.0 47.4 9.3 39.8

10 0.5 49.1 33.0 18.9 1.7 41.8 27.5 30.4 G₂ arrest 6.2 38.0 14.9 42.0 2.8 42.7 14.6 40.3 G₂ arrest

50 1.9 22.0 27.8 50.6 G₂ arrest 19.3 16.2 31.6 34.7 Cell Death 11.1 25.1 17.8 48.1 G₂ arrest 15.1 46.0 14.7 26.0 Cell Death

MK-2 0 0.4 40.1 28.4 32.1 2.1 45.6 21.0 32.6 4.7 44.2 13.7 36.8 6.0 46.4 9.3 37.5

^ 50 0.3 42.7 26.2 31.7 1.8 44.1 23.8 31.4 1.3 47.2 13.8 37.4 9.4 45.3 7.6 37.1 NJ

100 0.6 45.2 22.4 32.4 1.8 43.3 23.6 32.4 1.7 47.2 16.0 34.5 3.6 49.8 8.2 37.6 tn 250 2.4 46.7 14.3 36.7 3.2 37.7 23.8 36.4 G₂ arrest 1.4 52.8 11.1 34.3 4.2 41.4 11.2 42.5 G₂ arrest

CΛ 500 3.9 26.3 10.1 60.0 G₂ arrest 18.8 17.8 21.6 43.1 Cell death 2.5 39.4 11.3 46.5 G₂ arrest 5.2 44.5 15.4 33.6 S-phase

MK-4 0 0.8 42.0 26.9 31.7 2.3 49.9 21.6 27.4 4.1 44.0 12.5 39.4 5.5 45.7 7.4 41.4

50 0.5 42.0 26.9 32.0 1.9 44.7 22.3 32.3 4.5 43.9 11.4 40.7 4.7 51.4 12.3 31.6

100 0.4 43.2 25.4 32.5 2.5 45.3 22.6 30.6 2.0 41.4 13.6 44.1 6.0 52.3 12.5 29.4

250 0.5 45.7 24.6 30.5 6.0 40.0 23.0 32.0 3.9 36.2 14.1 46.9 7.0 53.2 11.9 27.6

500 1.1 47.5 18.6 33.9 Slight Δ 6.1 27.8 22.8 44.4 G₂ arrest 9.6 29.0 15.7 46.5 G₂ arrest 3.4 53.7 14.1 29.1 G, arrest

cantharidin is greater than for its analogues. Interestingly, cantharidin also showed slight

selectivity towards the colon cancer cells.

If the protein phosphatase inhibitor abrogates the G₂ checkpoint then the cells

will not arrest in the G₂ phase of the cell cycle and the cells will continue through the

cell cycle and accumulate in the Gi phase of the cell cycle only. Similarily if the protein

phosphatase inhibitors abrogates the G, checkpoint then the cells will not arrest in the G_j

phase of the cell cycle and accumulate in the G₂ phase of the cell cycle only. Cell cycle

analysis using propidium iodide labelling of DNA has been used extensively in our

laboratory to assess the effect of specific anticancer agents that induce S-phase cell cycle

arrest and apoptotic cell death (Sakoff, Ackland and Stewart, 1998). Experiments were

performed on a Becton Dickinson FACScan and using Cell Quest software.

Data shown in Table 3 and Figure 5 show the cell cycle response of LI 210,

HL60, HT29 and HCTl 16 cells. The cells were treated with 6Gy of radiation and then

treated with cantharidin 6h later. The ability to abrogate cell cycle arrest was assessed

12h after the addition of the drugs. Cantharidin and MK-2 both abrogated radiation

induced G, arrest in all cell lines. MK-4 also abrogated G, arrest in L1210, HL60 and

HCTl 16 cells. In HT29 cells, MK-4 induced abrogation of the G₂ checkpoint. It is

important to note that the exposure of HT29 cells to MK-4 induced the greatest

cytotoxicity (IC₅₀ 14μM) as determined by the MTT assay. Not surprisingly, the ability

to abrogate the G₂ checkpoint was more lethal than the ability to abrogate the G_!

checkpoint.

SUBSTITUTE SHEET (RULE 26) Table

Checkpoint Abrogation ^g

Cell Cycle Distribution (percentage of total) of tumour cell lines 18h after 6Gy of radiation and 12h after cantharidin or cantharidin analogue treatment.

Method : Flow Cytometry of Propidium Iodide stained cells.

Agent L1210 cells HL60 cells HCT116 cells HT29 cells μM sub G, G, S G₂+M sub G G, S G,+M sub G G, S G₂+M sub G G, S G₂+M

Cantharidin 0 1.6 25.3 35.8 38.8 6.6 5.3 3.2 85.3 4.9 26.8 8.7 60.1 5.9 40.7 9.2 44.7

1 1.6 27.2 25.6 37.5 6.2 5.2 3.0 85.8 4.2 26.1 13.8 58.2 16.6 35.2 10.6 38.2

5 2.4 25.8 31.9 41.5 4.4 5.7 3.5 86.8 4.0 23.6 10.4 63.3 5.3 38.6 10.6 46.3

10 3.4 24.9 29.4 43.7 5.3 5.6 4.3 85.1 4.2 25.7 9.4 62.2 6.4 21.1 12.3 60.8

50 4.9 4.1 15.6 77.4 4.3 10.2 11.3 64.9 Cell Death 12.0 12.2 15.6 63.3 G, abrogation 14.7 23.0 20.7 43.1

MK-2 0 1.6 16.4 31.1 52.1 7.0 5.9 2.2 85.1 3.7 30.8 7.9 57.2 17.3 31.8 8.7 41.3

?α 50 4.0 19.0 27.8 50.1 5.9 6.1 2.8 85.5 3.3 32.8 6.3 57.3 10.3 35.4 8.8 44.7 c 100 3.5 18.4 23.0 55.8 5.5 6.1 3.3 85.4 3.1 29.9 7.2 59.6 3.5 40.6 9.0 45.9 tn 250 6.9 11.2 10.0 71.9 8.1 5.4 2.8 83.9 6.2 23.9 4.3 65.4 2.7 24.9 12.3 59.4

CΛ 500 5.4 3.4 2.9 88.4 1.8 4.4 4.1 80.0 Cell Death 6.4 15.4 4.9 73.0 G, abrogation 8.8 24.1 20.4 45.2

MK-4 0 1.9 20.2 29.7 50.0 8.7 5.7 2.0 83.9 10.3 31.4 6.2 52.1 7.0 35.1 9.3 48.4

50 1.8 21.2 28.5 50.3 8.9 6.2 2.8 82.3 6.3 26.7 5.8 61.3 6.8 28.9 16.4 48.4

100 2.4 22.0 27.4 49.7 9.8 6.2 3.4 80.8 3.3 18.4 9.7 69.3 6.3 33.3 17.2 43.3

250 3.1 21.2 24.6 52.7 9.3 5.8 3.1 82.2 8.2 16.3 8.2 67.8 10.3 35.2 17.3 36.5

500 5.0 18.2 16.0 61.8 G, abrogation 11.6 5.2 5.4 78.2 Cell Death 14.9 13.1 10.6 61.9 G, abrogation 3.9 39.4 19.2 37.7 G2 abrogation

(c) Combination studies:

The cell lines listed above are exposed continuously to cisplatin and the

phosphatase inhibitor in various drug ratio combinations for 72h and then assayed for

cytotoxicity. Similarly, the cells are exposed to 8 Gy of radiation and incubated with the

phosphatase inhibitor and assessed for cytotoxicity at 72 h.

Data shown in Figures 6-9 shows the results of combination studies utilising the

Median Effect Method in HT29 and HCTl 16 human colon cells. This method tests the

cytotoxicity of various drug combinations from which a combination index can be

calculated. A value of greater than one indicated antagonism, a value equal to 1 indicates

additivity, while a value less than one indicates synergism. The HT29 and HCTl 16 cell

lines were chosen as they have differing p53 status and they represent the tumour types

that responded the greatest to cantharidin and its analogues.

The data show that the simultaneous combination of cisplatin and MK-4 in both

HCTl 16 and HT29 cells was additive and not synergistic using drug molar ratios of 1 : 1 ,

10:1 and 1 : 10. An additive response indicated that the drugs were mediating their effects

via two separate biochemical pathways. The simultaneous combination of taxotere and

MK-4 in HT29 cells was also additive using drug molar ratios of 1 : 10, 1 : 100, 1 :1000

(Taxotere: MK-4). However, this drug combination of taxotere and MK-4 induced a

synergistic response in HCTl 16 cells. A synergistic response indicates that the two

drugs were interacting in such a way as to enhance the overall cytotoxic response and to

induce "more than the additive¹' response of each individual agent. Consequently, the

addition of subtoxic levels of MK-4 clearly enhanced the cytotoxicity of taxotere.

SUBSTITUTE SHEET (RULE 26) Example 8

Results and Discussion

Anhydrides and simple analogues were synthesised according to literature

procedures (Eggelte et. al; 1973), and then subjected to a PPl and PP2A bio-assay (see

biochemistry) to determine their ability to inhibit these enzymes. The results of initial

screening at 100 mMs are shown in Table 4, along with IC₅₀ values in some instances.

Table 4 The inhibition of protein phosphatase 1 and 2A by anhydride modified

cantharidin analogues.

SUBSTITUTE SHEET (RULE 26)

Of the compounds listed in Table 4, only 1 and 2 show any significant inhibition

of PP2A. at 97% and 95% respectively (with little selectivity apparent for either

enzyme). Interestingly the bioisoseteric replacement of the anhydride oxygen atom of 1

results in a complete loss of inhibition. Indeed no modification of the cyclic anhydride,

is tolerated, and consequently results in no inhibition of PP2 A.

Previously we have shown that analog 2 undergoes a rapid conversion to the

dicarboxylic acid under assay conditions. We thus examined the stability of the non-

SUBSTITUTE SHEET (RULE 26) active analogues (in Table 4) and found that they were stable under assay conditions

showing no decomposition, in fact 5 can be synthesised via the Diels-Alder reaction in water (Eggelte et al; 1973).

In all instances, the corresponding dicarboxylic acid derivatives display lower

inhibitory values at PP2A (Tables 5 and 6). Even though the anhydrides undergo a facile

ring opening to the dicarboxylic acids, the original conformation presented at the active

site must also play a role in determining the overall level of inhibition. Consequently,

we believe that the conformation of anhydride carbonyl groups is more favourable for

inhibition (essentially only one conformation presented at the active site), than that of the

dicarboxylic acid (four possible minimum energy conformations, data not shown).

Table 5 Effects of anhydride to dicarboxylic acid on the inhibition of PP2A

SUBSTITUTE SHEET (RULE 26)

In an attempt to determine the feasibility of anhydride opening via nucleophilic

attack from Tyr272, we conducted a series of model experiments in which 2 was allowed

to stand in a chloroform solution of phenol. This mixture was examined periodically by

Η NMR spectroscopy and showed the growth of a new species over a period of time (ca

10 days). Further analysis indicated the presence of a phenolate ester of norcantharidin

(scheme 4). Consequently, a metal assisted or nucleophilic attack under physiological

conditions represents a possible mode of assisted ring opening with the anhydride held in

a favourable conformation within the active site. In turn the resultant diacid rapidly

binds in a more favourable manner.

Scheme 4

SUBSTITUTE SHEET (RULE 26) Table 6 Inhibition of PPl and PP2A by selected cantharidin analogues

The results presented herein indicate that cantharidin analogues, via anhydride

opening are more potent inhibitors of PP2 A. Analogues in which the anhydride moiety

has been modified preventing a facile ring opening (except where otherwise indicated)

are extremely poor inhibitors of PP2A (Tables 5 and 6).

However, the most interesting result reported herein (see table 4) is the selective

inhibition of PPl by the dimethyl ester (3). Simple diesterification of 2 has completely

reversed the previously reported PP2A selectivity (ca 10 fold) of norcantharidin for

PP2A to yield selective small synthetic molecule for the inhibition of either PPl or

SUBSTITUTE SHEET (RULE 26) PP2A. Again this suggests that presentation of a diacid moiety to the active site is

crucial for the inhibition of PP2 A. No such restrictions are apparent with the limited structure activity data for PPl.

A synthetic inhibitor such as 3 represents a significant advance on the currently

widespread inhibitors of PPl and PP2A.

In conclusion, the present inventors have demonstrated that a facile ring opening

of the anhydride moiety is relevant for inhibition at PP2A. Also, that modification of the

dicarboxylic acid moiety gives rise to a PPl selective compound.

The above describes some embodiments of the present invention. Modifications

obvious to those skilled in the art can be made without departing from the scope of this

invention.

Industrial Applicability

It should be clear that the present invention will find light applicability,

especially in the medical and veterinary fields.

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SUBSTITUTE SHEET (RULE 26)

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS :-

1. A cell permeable inhibitor of protein phosphatase, said inhibitor being an anhydride modified cantharidin analogue, ϊ

2. An inhibitor according to claim 1, wherein the phosphatase is phosphatase 1 and/or phosphatase 2A.

3. An inhibitor according to claim 1 or 2 wherein the anhydride modified cantharidin analogue is oxidatively stable.

4. A compound of the formula:

wherein R, and R₂ are H. aryl or alkyl; X is O, N or S; Y is O, S, SR, NH, NR, CH₂OH,

CH₂OR; R is alkyl or aryl; A and B are H or CH₃; W and Z are CHOH or C=0 and R,

and R₂ can cyclise to form a ring as follows:

wherein R₃ and R are H, aryl or alkyl.

5. A compound according to claim 3, wherein the aryl group is phenyl or naphthyl

and wherein the aryl group is attached via a carbon spacer of between 6 and 10 carbon

atoms.

6. A compound according to claim 3 or claim 4, wherein the alkyl group is C,-C₁₀.

SUBSTITUTE SHEET (RULE 26)

7. A process for producing anhydride modified cantharidin analogues for use in the

treatment of cancer or for the sensitising cancer cells to one or more cancer treatments

comprising the step of reacting a diene with an ene.

8. A process according to claim 7 further comprising hydrogenation of the adduct

5 of the diene and the ene.

9. A process according to claim 7 or 8 further comprising ring opening of the

adduct of the diene and the ene.

10. A process for producing anhydride modified cantharidin analogues, said process

including the steps of:

o dissolving a diene in a suitable solvent and adding to the resultant solution an

ene.

11. A process for producing anhydride modified cantharidin analogue, said process

including the steps of:

dissolving a furan in a suitable solvent and adding to the resultant solution an

5 ene;

incubating the solution at a temperature and for a time sufficient to form a

precipitate; and

collecting the precipitate and recrystalising the analogue.

12. A process for producing anhydride modified cantharidin analogue, said process

0 including the steps of:

mixing thiophene and maleic anhydride at room temperature in a suitable

solvent;

SUBSTITUTE SHEET (RULE 26) compressing the mixture at a temperature and pressure sufficient to facilitate a reaction to take place; and

purifying the analogue.

13. A method of treating cancer which method comprises administering to a patient

in need of such treatment, an effective amount of an inhibitor according to any one of

claims 1 to 3 or a compound according to any one of claims 4 to 6, together with a

pharmaceutically acceptable carrier, diluent and/or excipient.

14. A method according to claim 13, wherein the cancer is inherently resistant to

conventional chemotherapy.

15. A method according to claim 13 or claim 14, wherein the cancer is colon cancer

or non small-cell lung cancer.

16. A method according to any one of claims 13 to 15, wherein the inhibitor or the

compound is administered intravenously.

17. A method of sensitising cancer cells to at least one method of treating cancer,

which method of sensitising comprises administering to a patient in need of such

treatment, an effective amount of an inhibitor according to any one of claims 1 to 3 or a

compound according to any one of claims 4 to 6, together with a pharmaceutically

acceptable carrier, diluent and/or excipient.

18. A method according to claim 17, wherein the at least one cancer treatment is

selected from treatments involving irradiation and anti-cancer agents.

19. A method according to claim 17 or claim 18, wherein the cells have deficient

p53 activity.

SUBSTITUTE SHEET (RULE 26)

20. A method of treating cancer which method comprises:

administering to a patient in need of such treatment, an effective amount of an

anhydride modified cantharidin analogue of any one of claims 1 to 3 or a compound

according to any one of claims 4 to 6 to sensitise cells of the patient to one or more

cancer treatments; and

utilising the one or more cancer treatments.

21. A method of screening compounds for use in sensitising cancer cells to at least

one method of treating cancer, and comprising:

screening for anti-cancer activity; and

screening for ability to abrogate either the G_t or the G₂ checkpoint of the cancer

cell cycle.

22. A method according to claim 21 further comprising the step of screening for the

ability of the compounds of sensitise cancer cells to one or more cancer treatments.

23. A method according to claims 21 or 22 wherein the one or more cancer

treatments are selected from treatments involving cisplatin, irradiation, taxanes and

antimetabolites.

24. A method according to any one or claims 21 to 23 wherein the screening is

conducted on haematopoietic cells or solid tumour cells, having varying p53 activity.

25. A method according to claim 24, wherein the cells are selected form the group

consisting of L1210 (murine leukaemia, p53 wildtype), HL60 (human leukaemia, p53

nul), A2780 (human ovarian carcinoma, p53 wildtype), ADDP (cisplatin resistant

A2780 cells, p53 mutant), SW480 (human colon carcinoma, p53 mutant), WiDr (human

SUBSTITUTE SHEET (RULE 26) colon carcinoma, p53 mutant), HT29 (human colon carcinoma, p53 mutant), HCTl 16

(human colon carcinoma. p53 wildtype) and 143B (human osteosarcoma, p53 mutant).

26. A compound selected from a group comprising compounds (a) to (k) below:

(a) (b)

(c) ( )

(e) (f)

(g) (h)

(i) 0)

SUBSTITUTE SHEET (RULE 26) (k) (1)

(m) (n)

27. Use of an inhibitor according to claim 1 or claim 2, or a compound according to

any one of claims 3 to 5 for the manufacture of a medicament for the treatment of

cancer.

28. Use according to claim 27, wherein the cancer is colon cancer or non small-cell

lung cancer.

29. Use according to claim 27 or claim 28, wherein the medicament is administered

intravenously.

SUBSTITUTE SHEET (RULE 26)