WO2010013011A1 - P53 tumor suppressor protein activating - Google Patents

Abstract

The invention provides a compound of formula (I), wherein X, L^1-4 and R^1-4 are as defined herein, with the proviso that the compound is not (i) 4-benzoyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester; (ii) 4-(2,5-difluorobenzoyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester); (iii) 4-acetyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester; or (iv) 4-(3-dimethylamino-acryloyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester, or a pharmaceutically acceptable derivative thereof.

Description

P53 TUMOR SUPPRESSOR PROTEIN ACTIVATING

INTRODUCTION

The present invention relates to compounds that have been found to activate the p53 tumour suppression protein. These compounds thus find use, for example, in the treatment of hyperproliferative diseases such as cancer.

BACKGROUND OF THE INVENTION

The P53 tumour suppressor protein is a central mediator of cellular stress response and plays a major role in preventing tumour development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell cycle arrest and apoptosis. Its importance as a tumour suppressor is reflected by its high rate of mutation in human cancer, with more than 50% of adult human tumours bearing inactivating mutations or deletions in the TP53 gene. In many cancers where p53 is wild-type, the p53 pathway may be altered by other oncogenic events. This means that the p53 response is probably defective in most cancers.

After many years of intensive research, whether p53 status influences prognosis in cancer patients is still a matter of debate. However, irrespective of whether activation of p53 plays a role in response to treatment, it is clear that many classic, and current, cancer therapeutics (radiation, DNA interacting compounds and anti-mitotics) do activate p53's activity as a transcription factor. Hence it is reasonable to think that compounds that activate p53 could lead to the identification of novel agents with therapeutic value.

No claim is made herein to the compound 4-benzoyl-3,5-dimethyl-1/-/-pyrrole-2- carboxylic acid ethyl ester, since this is a compound commercially available as part of the ChemBridge DIVERSet collection (commercially available from ChemBridge Corporation). Likewise, no claim is made herein to the compound 4-(2,5- difluorobenzoyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester. This compound is described in both WO 01/37820 and WO 01/60814 (both Sugen, Inc.) as an intermediate compound to compound 21 described in each of these publications. Likewise, no claim is made herein to the compounds 4-acetyl-3,5-dimethyl-1/-/-pyrrole-2- carboxylic acid ethyl ester or 4-(3-dimethylamino-acryloyl)-3,5-dimethyl-1/-/-pyrrole-2- carboxylic acid ethyl ester since these compounds are both described in WO 2004/083203 (Vertex Pharmaceuticals Incorporated) as intermediate compounds to a pyrrole-substituted pyrimidine described in Example 2 in this publication. BRIEF SUMMARY OF THE INVENTION

Use of a p53 activation as a biomarker in a cell-based assay has led to the identification of new p53 activating compound referred to herein as JJ78 or JJ78:1. Synthetic elaboration of JJ78:1 has led to the identification of analogues with improved efficacy in cell lines and activity in a preclinical tumour model.

Viewed from one aspect, therefore, the invention provides a compound of formula (I):

(wherein:

X is NR^X, O, S, SO or SO₂, wherein R^x is H or a C_1-6 straight-chain, branched or cyclic alkyl substituted once or twice with OH, SH, NH or NH₂; each of L¹ and L³ is independently present or absent and if present is selected from O, S, SO, SO₂ or NR^y; each of R¹ and R² is independently a straight-chain, branched or cyclic alkyl, aryl, heteroaryl, or R¹ together with R², forms a straight-chain diradical R^1"2 whereby to connect L¹ (if present) and L², and is optionally substituted with one or more substituents selected from the group comprising straight-chain, branched or cyclic alkyl, aryl, heteroaryl, halo, hydroxyl, carboxy, acyl, thioacyl, alkoxy, alkylthio, alkyl sulfoxy, alkyl sulfone, thiol, -C(=O)-, -O-, -S-, -S(O)-, -S(O)₂-, ester, amido, sulfonamido, amino, nitro and cyano;

L² is a linking moiety comprising a contiguous saturated or unsaturated hydrocarbyl diradical sequence of from 1 to 6 atoms, said linking moiety optionally comprising one or more of the following functionalities as part of the contiguous sequence: -C(=O)-, -SO₂NR^y-, -S(O)-, S(O)₂-, C(=0)N(R^y)-, -C(=O)O-, -C(=S)-,

-C(=S)O-, -C(=S)S-, -C(=O)S-, -C(=N-OH)-, -C(=N-OR^y)-, -C(=NR^y)-, -C(=N-NH₂)-, -C(=N-NHR^y)-, -C(=O)N(R^y)C(=O)-, -C(=S)N(R^y)C(=S)-, -C(=O)N(R^y)C(=S)- and -C(=N-N(R^y)₂)-;

L⁴ is absent or present and if present is a linking moiety comprising a contiguous saturated or unsaturated hydrocarbyl diradical sequence of from 1 to 6 atoms, said linking moiety optionally comprising one or more of the following functionalities as part of the contiguous sequence: -C(=O)-, -C(OH)-, -C(OR^y)-, -C(SH)-, -C(SR^y)-, -SO₂NR^y- -S(O)-, S(O)₂-, C(=O)N(R^y)-, -C(=O)O-,-C(=S)-, -C(=S)O-, -C(=S)S-,-C(=O)S-, -C(=N- OH)-, -C(=N-OR^y)-, -C(=NR^y)-, -C(=N-NH₂)-, -C(=N-NHR^y)-,-C(=O)N(R^y)C(=O)-, -C(=S)N(R^y)C(=S)-, -C(=O)N(R^y)C(=S)-, -C(=N-N(R^y)₂)-, -O-, -S-, -N(R^y)-, -C(NH₂)-, -C(NR^y ₂)-, -C(NHR^y)-, -C(NH₂)-; each of R³ and R⁴ is independently a straight-chain, branched or cyclic alkyl, aryl, heteroaryl, or R³ together with R⁴, forms a straight-chain diradical R^3"4 whereby to connect L³ (if present) and L⁴, and is optionally substituted with one or more substituents selected from the group comprising straight-chain, branched or cyclic alkyl, aryl, heteroaryl, halo, hydroxyl, carboxy, acyl, thioacyl, alkoxy, alkylthio, alkyl sulfoxy, alkyl sulfone, thiol, -C(=O)-, -O-, -S-, -S(O)-, -S(O)₂-, ester, amido, sulfonamide amino, nitro and cyano; each R^y is independently selected from a group comprising hydrogen and a straight-chain, branched or cyclic alkyl, aryl, heteroaryl, optionally substituted with one or more substituents selected from the group comprising straight-chain, branched or cyclic alkyl, aryl, heteroaryl, halo, hydroxyl, carboxy, acyl, alkoxy, alkylthio, alkyl sulfoxy, alkyl sulfone, thiol, -C(=O)-, -O-, -S-, -S(O)-, -S(O)₂-, ester, amido, sulfonamido, amino, nitro and cyano; with the proviso that the compound is not (i) 4-benzoyl-3,5-dimethyl-1H-pyrrole- 2-carboxylic acid ethyl ester; (ii) 4-(2,5-difluorobenzoyl)-3,5-dimethyl-1H-pyrrole-2- carboxylic acid ethyl ester); (iii) 4-acetyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester; or (iv) 4-(3-dimethylamino-acryloyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester, or a pharmaceutically acceptable derivative thereof.

Viewed from a further aspect the invention provides a compound of the invention, but without the provisos, together with a pharmaceutically acceptable carrier.

Viewed from a further aspect the invention provides a compound of the invention for use in medicine.

The invention also provides the use of a compound of the invention, but without the provisos, for the preparation of a medicament for use in a method or treatment or prophylaxis of a disease involving cell proliferation, in particular cancer.

The invention also provides a compound of the invention, but without the provisos, for use in a method as defined in the preceding aspect of the invention.

The invention also provides methods of treatment or prophylaxis of the human or animal body compromising administering a compound of the invention to a patient in need thereof, in particular for the treatment or prophylaxis of a disease involving cell proliferation, in particular cancer. -A-

Other aspects and embodiments of the present invention will become apparent from the discussion and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows the results of staining and treating HeLa cells grown with JJ78, with established tubulin poison vincristine (VX-680), paclitaxel (Taxol®) and an untreated cell fixing and staining for the mitotic marker P-S10-HH3 and DNA using Hoescht. The experiment in Fig. 1 demonstrates that in the presence of JJ78 cells enter mitosis (as indicated by the positive P-S10-HH3 staining). This shows that cells treated with JJ78 primarily arrest in mitosis, not in G2.

Fig. 2A shows detail of the synthesis of compounds JJ78:1-6 of the invention, reaction conditions: a) AICI₃, 2,5-difluorobenzoyl chloride, chlorobenzene (55%); b)

KOH, ethanol (70%); c) SOCI₂, DMF (cat.), CH₂CI₂; d) EtNH₂/Et0H, CH₂CI₂ (59%); e) benzyl alcohol (70%); f) DCC, Λ/-hydroxysuccinimide, CH₂CI₂ (33%); g) benzyl amine, CH₂CI₂ (70%); h) K₂CO₃, iodomethane, CH₃CN (96%).

Fig. 2B shows details of the synthesis of JJ78:17-18: reaction conditions: i) AICI₃, acid chloride, chlorobenzene.

Fig. 2C shows activity of selected JJ78:1 analogues shown in terms of their relative potency order with the most potent analogue assigned as 1. Fig. 3A shows selected data showing levels of p53 activation in cells as a function of compound concentration for a series of JJ78:1 analogues.

Fig. 3B shows inhibition of tubulin polymerisation in vitro by JJ78:1 derivatives. A selection of JJ78 derivatives produced via SAR studies were tested in the in vitro tubulin polymerisation assay. All compounds were tested at 10 μM (0.5% DMSO). Polymerisation curves are the average of three independent experiments.

Fig. 3C shows that JJ78:12 is more potent at inhibiting tubulin polymerisation in vitro than JJ78:1. JJ78:1 (10 μM) and increasing concentrations of JJ78:12 were tested. DMSO concentration was 0.5% (v/v) in all assays. Polymerisation curves are the average of three independent experiments. Fig. 3D plots mitotic index calculation (i.e. % of cells positive for P-S10-HH3 staining) for selected JJ78:1 derivatives in MCF7 cells.

Fig. 3E shows images of interphase and mitotic cells after treatment of MCF-7 cells with the indicated concentrations of JJ78:1 and JJ78:12 for 24 hours. Fixed cells were stained for α tubulin (light grey/white) and DNA using Hoescht dye (dark grey). Fig. 4 shows that the known tubulin depolymerising agent Nocodazole, JJ78:1 and JJ78:12 have similar effects in cells. Fig. 4A shows two-dimensional FACS analysis of SKNSH cells cultured with either active (SKNSH-pCMV) or inactive p53 (SKNSH-DNp53) in the absence or presence of the indicated compounds for 48 hours. 20 minutes before harvesting cells where pulse labelled with BrdU, harvested and processed. Treatment with all three compounds (nocodazole, JJ78:1 and JJ78:12) leads to the accumulation of cells with a 4N DNA content. Thus JJ78 (or JJ78:1 ) leads to the accumulation of cells with a 4N DNA content irrespective of their p53 status. Nocodazole, an established tubulin poison leads to a similar outcome. Cells with a 4N DNA content are likely to be either in the G2 or M (mitosis) phases of the cell cycle. Fig. 4B shows time-course analyses of p53 levels in MCF7 cells (harbouring active p53) incubated with the indicated compounds. p53 is detected with DO1 antibody. PCNA detection using PC10 antibody is used as a loading control. p53 accumulates only after long term treatment (16 hours or above) with the three compounds. Fig. 5 relates to the antitumour activity of JJ78:12. Fig. 5(A) shows cell viability, as monitored by MTT assay, of ARN8 human melanoma cells (p53 wild type) incubated with the indicated concentrations of compound for 48 hours.

Fig. 5B plots PK data for JJ78:12.

Fig. 5C shows the results of JJ78:12 in vivo. ARN8 melanoma cells were injected in the flank of SCID mice and allowed to develop into tumours. JJ78:12 (in 20% cyclodextrin) was administered by intraperitoneal injection at 30 mg/kg (n=10) and tumour growth was measured over a period of 14 days. Control animals (n=10) were treated with 20% cyclodextrin. Tumour size was measured with callipers. The left panel shows the measurements obtained from each individual tumour. Note that untreated tumours would reach sizes beyond 1 ,000 mm3 by day 15. Despite this, and in order to provide a statistical analysis, measurements performed on days 1 through 8 were averaged between groups and plotted (right panel). Error bars correspond to 95% confidence intervals. Mice receiving JJ78:12 had significantly reduced tumour growth as analysed by appropriate choice of 2-sample t-test (normal distributions) and Mann Whitney U-test (non-normal distributions). Difference between the JJ78:12 and control distributions were statistically significant on days 3 to 8 (p=0.014, 0.013, 0.004, 0.009, 0.002 and 0.001 , respectively). JJ78:12 did not induce signs of toxicity at the concentration tested. DETAILED DESCRIPTION OF THE INVENTION

The present invention arises from the recognition that the compounds described herein possess anti-tumour activity. This has been demonstrated by the studies described below where it has been shown that JJ78 affects cells primarily after the onset of mitosis and delays cell cycle progression thereafter. Moreover, additional immunofluorescence imaging of cells showed that the microtubule network in interphase appears disordered and that there are numerous aberrant mitotic figures where cells are unable to form a mitotic spindle. An in vitro assay demonstrated that JJ78 directly decreased tubulin polymerisation. Thus JJ78 has been found to be a moderate inhibitor of tubulin polymerisation in vitro (see figure 3D), appears to be effective in delaying cell cycle progression and, without wishing to be bound by theory, it is believed to be through this mechanism that JJ78 and the other compounds described herein exert an increase in p53-dependent transcriptional activity.

The compounds of and utilised in the various aspects of the present invention of formula (I) are based upon a central 5-membered ring, the ring comprises a contiguous chain of four carbon atoms attached to the heteroatom X.

The heteroatom may be nitrogen, oxygen or sulfur. Where the heteroatom is sulfur, this may be present as a sulfide, sulfoxide or sulfone (-S(0)o-₂-). Where the heteroatom X is nitrogen, this may either be substituted with a hydrogen atom or with a substituted alkyl group. The alkyl group is typically a Ci_-6 straight-chain, branched or cyclic alkyl radical substituted once or twice with OH, SH, NH or NH₂.

By alkyl is meant herein a saturated or unsaturated, but not aromatic, hydrocarbyl moiety unless the context dictates to the contrary. The alkyl groups described herein may thus have one or more sites of unsaturation, which may be constituted by carbon-carbon double bonds or carbon-carbon triple bonds. Generally, the alkyl substituents described herein will be saturated alkyl radicals unless the context dictates to the contrary.

Where an alkyl group is substituted, the substituent may be comprised within the alkyl chain. An example of a substitution would be the presence of ether (-O-), thioether (also known as sulfide), sulfoxide or sulfone moieties (-S(O)_0-2-), ester, amide or amino moieties. Alternatively, the substituent(s) may be pendant from the alkyl chain.

Examples of such substituents are halo, hydroxyl, acyl, thiol and the like.

Typically alkyl moieties will comprise from 1 to 20 carbon atoms, more usually 1 to 10 carbon atoms, for example 2 to 6 carbon atoms. In certain embodiments of the invention X is NH, O or S. In particular embodiments of the invention X is NH. Typically, R¹ and R² are independent monoradicals, that is to say they do not together from diradical R^1"2.

Where R¹ and R² are independent radicals, each may be a straight-chain, branched or cyclic alkyl, or aryl, or heteroaryl radical, each of which may be optionally substituted.

By aryl is meant herein both monocyclic aryl (i.e. phenyl) or polycyclic aryl radicals such as napthyl or anthracyl. Heteroaryl moieties or cyclic aromatic moieties comprise a heteroatom, typically O, N or S in place of a carbon atom and any hydrogen atoms attached thereto. Heteroaryl moieties may likewise be monocyclic (e.g. pyridyl, furyl, pyrrolyl and pyrimidinyl). An example of a polycyclic heteroaryl is indanyl. Typically the heteroatoms in any heteroaryl moieties present is or are oxygen or nitrogen.

In certain embodiments of the invention, the compounds of formula (I) may comprise diradical R^1'2. Where such a diradical is present, this may serve to form a 5- to 8- membered ring together with L² and L¹ (if present), and the carbon atoms to which the terminal atoms of the diradical (L¹JR^{1 2}L² is connected. Typically R^1"2 will be an alkylene diradical, that is to say the diradical formed formally by removal of a hydrogen atom from an alkyl radical. As with the monovalent radicals R¹ and R², the diradical R^1"2, if present, may be substituted with one or more substituents selected from those defined for R¹ and R².

In certain embodiments of the invention the diradical (L¹)R^{1 2}L² may be L¹- C(H)=C(H)-L², e.g. those diradicals wherein L¹ is present and in particular where L¹ is O₁S or NR^y, most typically O, and/or wherein L² is C=O or SO₂, most typically C=O. In these or other embodiments of the invention wherein R^1"2 is present, R^1"2 may be substituted. Where R^1"2 is based upon the ethylene diradical (-C(H)=C(H)-), both carbon atoms or only one carbon atom of the ethylene diradical may be substituted. Where one carbon atom is substituted, this is typically at a carbon atom adjacent to L².

A particular embodiment of the invention comprises the diradical L¹R^{1 2}L² and is of the formula -O-CH(Y)-C(=O)- wherein Y is a cyclic alkyl (e.g. cyclohexyl), aryl or heteroaryl moiety. In particular embodiments of the invention substituent Y is a phenyl radical optionally substituted at one or more of positions 2, 3, 5 and 6, for example with a substituent selected from the group comprising halo (in particular chloro or fluoro), hydroxyl, thiol, amino, alkyloxy or alkylthio.

By carboxy is meant herein the functional group CO₂, which may be in deprotonated form (CO₂ ^"). By acyl and thioacyl are meant the functional groups of formulae -C(O)-alkyl or - C(S)-alkyl respectively.

Alkyloxy and alkylthio are of the formulae -O-alkyl and -S-alkyl respectively, wherein alkyl is as defined hereinbefore. An ester comprises a unit of the formula -C(O)O- and may be flanked by straight- chain, branched or cyclic alkyl, aryl or heteroaryl moieties. Thus, for example, where an alkyl group is substituted with an ester moiety, this be connected either through the oxygen atom or the carbonyl carbon atom thereof, where the ester is pendant from the alkyl; or connected to the alkyl group through both the oxygen and carbonyl carbon thereof. Similarly, amido (-(NR^y)C(O)-) and sulfonamide (-SO₂N(R^y)-) groups, wherein R^y is as hereinbefore defined, typically hydrogen, may be attached to an alkyl group, for example, by either the carbon and/or oxygen atoms (with amido groups) or sulfur and/or nitrogen atoms (with sulfonamide groups).

An amino group is of the formula N(R^y)₃ wherein each R^y is selected independently from the group R^y as hereinbefore defined. An amino group, for example, may substitute an alkyl group such that the nitrogen atom is connected to the alkyl group through a single bond. In this case, the remaining two groups R^y may either both be hydrogen, both not be hydrogen, or one be hydrogen and one not be hydrogen.

L² connects the central 5-membered ring of compounds (I) to R² (or R^1'2). Typically, L² is based upon an alkylene diradical. Typically, therefore, L² will be a substituted, unbranched, alkylene diradical comprising from one to six atoms in the sequence of atoms that leads directly from the carbon atom of the five-membered ring to which L² is connected and the R² or R^1"2 moiety to which it is attached. Thus L² may be, for example, a keto or thioketo (-C(O or S)-), sulfonomide, sulfoxide, sulfone, amide, ester, or thioester or one or more of these or other functional groups to which an alkylene radical is attached to either or both sides of these divalent functional groups.

Alternatively, L² may be based on an arylene or heteroarylene radical, or a combination of alkylene, arylene and heteroarylene radicals.

As is understood by those skilled in the art, analogously to the structural relationship between alkyl monoradicals and alkylene diradicals, arylene and heteroarylene diradicals, may be considered to be formed formally from aryl and heteroaryl monoradicals respectively by abstraction of a hydrogen atom.

In certain embodiments of the invention L² may be a keto, thioketo or sulfone moiety, and is typically a keto moiety. Where R³ and R¹ are each present as such, i.e. are not part of diradicals R^1"2 and

R^3"4 respectively, these moieties are each independently selected, in certain embodiments of the invention, from the group comprising straight-chain, branched or cyclic saturated Ci_-6 alkyl groups. Typically the moieties R¹ and R³ are alkyl groups. In certain embodiments of the invention R¹ and R³ are unsubstituted alkyl groups. Most typically, R¹ and R³ are the same, e.g. the same unsubstituted alkyl group, in particular a C_1-6 unsubstituted alkyl group.

In certain embodiments of the invention L⁴ is absent, for example where R⁴ is a substituted alkyl group, e.g. a C_1-6 alkyl group terminally substituted with hydroxyl and optionally substituted with one or two alkyl substituents.

Analogously to the way in which R¹ and R² may together form a diradical R^1"2, R³ and R⁴ may also together form a diradical R^3"4 as hereinbefore defined. R^3"4, where present, may be considered to form together with L⁴, if present, and, if present L³, a diradical (L³JR³^(L⁴) whereby to form a 5- to 8-membered ring together with the carbon atoms to which the terminal atoms of diradical (L³)R^3"4(L⁴) is connected. In certain embodiments of the invention this 5- to 8-membered ring is an aromatic or heteroaromatic ring. In certain embodiments of the invention the resultant 5- to 8- membered ring, whether aromatic, heteroaromatic or otherwise, may be substituted with one or more substituents selected from those defined for R³ and R⁴.

Typically L⁴ is present.

It will be appreciated, therefore, that compounds of formula (I) may comprise one or more rings fused to the central X-containing 5-membered ring. Thus, where no diradicals are R^1"2 and R^3"4 are present, the central motif in the compound will be the 5- membered monocyclic ring depicted figuratively in the structure of the compounds of formula (I). If one of R¹ -R² or R^3"4 is present, at least a bicycle will be present. The possibility of additional rings fused to either R¹-R² and R³-R⁴ is contemplated by this invention leading to the possibility of polycyclic compounds comprising three or more fused rings even where one of R^1"2 and R^3"4 is absent. Typically, however, the diradicals

R^1"2 and R^3"4, whilst they may be substituted, are not substituted by way of a ring fused to them.

It will be appreciated that where R^1"2 and R^3"4 are each present, the compounds of formula (I) will comprise (at least) a central fused tricyclic system.

The compounds of the present invention may be used for the treatment and/or prophylaxis of conditions and diseases involving abnormal cell death associated with abnormalities with the p53 protein, its function and/or the p53 pathway.

In particular, diseases involving abnormal proliferation of cells are treatable with the compounds recited herein. Examples of such diseases include cancers, hyperproliferation disorders (including warts, psoriasis, inflammatory bowel disease), rheumatoid/autoimmune conditions, sickle cell anemia, thalasemias and the like.

In certain embodiments of the invention, the compounds are used for the treatment or prophylaxis of cancer. Examples of cancers which may be treated by the active compounds include, but are not limited to, a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non- small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g. exocrine pancreatic carcinoma, stomach, cervix, thyroid, prostate, or skin, for example squamous cell carcinoma; a hematopoietic tumour of lymphoid lineage, for example leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma; a hematopoietic tumor of myeloid lineage, for example acute and chronic myelogenous leukemias, myelodysplastic syndrome, or promyelocytic leukemia; thyroid follicular cancer; a tumour of mesenchymal origin, for example fibrosarcoma or habdomyosarcoma; a tumor of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma pigmentoum; keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma. Examples of other therapeutic agents that may be administered together

(whether concurrently or at different time intervals) with the compounds of the formula (I) include but are not limited to topoisomerase inhibitors, alkylating agents, antimetabolites, DNA binders and microtubule inhibitors (tubulin target agents), such as cisplatin, cyclophosphamide, doxorubicin, irinotecan, fludarabine, 5FU, taxanes or mitomycin C. For the case of active compounds combined with other therapies the two or more treatments may be given in individually varying dose schedules and via different routes.

The combination of the agents listed above with a compound of the present invention would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner.

Where the compound of the formula (I) is administered in combination therapy with one, two, three, four or more, preferably one or two, preferably one other therapeutic agents, the compounds can be administered simultaneously or sequentially. When administered sequentially, they can be administered at closely spaced intervals (for example over a period of 5-10 minutes) or at longer intervals (for example 1 , 2, 3, 4 or more hours apart, or even longer period apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).

The compounds of the invention may also be administered in conjunction with non-chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery and controlled diets.

The patient is typically an animal, e.g. a mammal, especially a human. By a therapeutically or prophylactically effective amount is meant one capable of achieving the desired response, and will be adjudged, typically, by a medical practitioner. The amount required will depend upon one or more of at least the active compound(s) concerned, the patient, the condition it is desired to treat or prevent and the formulation of order of from 1 μg to 1 g of compound per kg of body weight of the patient being treated.

Different dosing regiments may likewise be administered, again typically at the discretion of the medical practitioner. As alluded to hereinafter the low toxicity of the compounds of the invention, allow for at least daily administration although regimes where the compound(s) is (or are) administered more infrequently, e.g. every other day, weekly or fortnightly, for example, are also embraced by the present invention.

By treatment is meant herein at least an amelioration of a condition suffered by a patient; the treatment need not be curative (i.e. resulting in obviation of the condition). Analogously references herein to prevention or prophylaxis herein do not indicate or require complete prevention of a condition; its manifestation may instead be reduced or delayed via prophylaxis or prevention according to the present invention.

For use according to the present invention, the compounds or physiologically acceptable salt, solvate, ester or other physiologically acceptable functional derivative thereof described herein may be presented as a pharmaceutical formulation, comprising the compound or physiologically acceptable salt, ester or other physiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic and/or prophylactic ingredients. Any carrier(s) are acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release-controlling matrix, or is coated with a suitable release-controlling film. Such formulations may be particularly convenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.

Injectible preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Altematively, an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.

As one possibility such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self- propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent.

Suitable liquid propellants include propane and the chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.

Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.

As a further possibility an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.

Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.

It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. Formulations suitable for topical formulation may be provided for example as gels, creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either directly spread upon the surface of the wound or ulcer or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated. Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and then applied to the site to be treated.

Therapeutic formulations for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus particularly suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water. Liquid concentrates of this invention suitably contain the compound or a derivative or salt thereof and may optionally include a veterinarily acceptable water- miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals.

The present invention will now be described with reference to the following non- limiting examples.

Investigation of induction of cell cycle arrest at mitosis through p53 activation assay

A cell-based p53-dependent reporter was conducted using T22-RGC-ΔFos-lacZ murine cells expressing β-galactosidase under the control of a p53-dependent promoter. Compounds were assayed in each plate at 10 μM as described (Lain et a/., Cancer Cell 2008, vol 13 pp.454-463). SKNSH cells with either active (SKNSH-pCMV) or inactive p53 (SKNSH-DNp53) were cultured in the absence or presence of the lead compounds at 10 μM for 48 hours. 20 minutes before harvesting the cells had BrdU added to the media. Cells were then harvested and processed for two-dimensional FACS analysis.

For mitotic index calculations HeLa cells were grown in the presence of the indicated compound for 24 hours, fixed and stained for the mitotic marker P-S10-HH3 and DNA using Hoescht.

Treatment of cells with two established tubulin poisons (paclitaxel and vincristine) under our standard conditions also caused an induction of the p53-dependent reporter in cells (unpublished data). Exactly how tubulin poisons lead to the induction of p53- dependent transcription is not established.

JJ78 appears to accumulate cells in the G2/M phase of the cell cycle. Accumulation of cells in G2/M was observed in cells with functional p53 as well as in cells where p53 function is abolished by overexpression of a dominant negative form of p53. Fig. 4A shows an example of the two-dimensional FACS plots obtained with compound JJ78. It is important to note that cells with inactivated p53 could still replicate their DNA (as determined by BrdU incorporation) acquiring a DNA content higher than 4N whereas cells with active p53 are precluded from entering a new DNA synthesis phase (Fig. 4A). This observation further highlights p53's role in preventing polyploidy (Giono and Manfredi, 2006, J Cell Physiol. 209, 13-20). To narrow down the mechanism of action of JJ78 an analysis of mitotic index was carried out. Aurora B phosphorylates histone H3 at serine 10 upon entry into mitosis (Gurley et al., 1978, Eur. J. Biochem. 84, 1-15; Hsu et al., 2000, Ce// 102, 279- 291 ). Compounds that arrest cells after the onset of mitosis (like paclitaxel) give rise to the accumulation of cells that are positive for phospho-serine H3, whereas compounds that prevent cells from entering mitosis (like VX-680, an inhibitor of Aurora kinases) abolish H3 phosphorylation at serine 10 (Harrington et al., 2004, Nat Med. 10, 262-267). An example of this assay using HeLa cells treated with compound JJ78 is shown in Fig. 1B. JJ78 gave rise to an increase in H3 phosphorylation at serine 10, indicating that it allows entry into mitosis and delay cell cycle progression at this stage. HeLa cells are defective for p53 function due to the expression of the human papillomavirus E6 oncoprotein (Finzer et al., 2002, Cancer Lett. 188, 15-24), which suggested that p53 induction is not likely to be responsible for the mitotic arrest induced by the compounds. In order to confirm this hypothesis, we carried out the same analysis using SKNSH- pCMV cells (with active p53) and SKNSH cells expressing a dominant negative p53 form (SKNSH-DNp53) (Smart et al., 1999, Oncogene 18, 7378-7386). Treatment of both cell types with J J78 led to an increase in H3-phosphoserine 10 positive mitotic cells demonstrating that it induces a failure in mitotic progression independently of p53 function. Cells with inactive p53 showed a higher mitotic index probably reflecting their faster progression through the cell cycle.

JJ78 directly impairs tubulin polymerisation

Further immunofluorescence imaging of cells treated with JJ78 showed that the microtubule network in interphase appears disordered and that there are numerous aberrant mitotic figures where cells are unable to form a mitotic spindle. Given that agents that delay mitotic progression can bind to and perturb tubulin polymerisation we used an in vitro assay to test whether the compounds promote the stabilisation of tubulin polymers (like paclitaxel) or destabilise tubulin polymers (like vinca alkaloids or nocodazole). This in vitro assay indicated that JJ78 directly decreases tubulin polymerisation as the polymerisation curve shows a slow nucleation and growth phase. This led to the conclusion that JJ78 directly targets tubulin and, like vinca alkaloids and nocodazole, prevents its polymerisation.

JJ78, a moderate inhibitor of tubulin polymerisation in vitro (Fig. 3BJ seemed to be highly effective in delaying cell cycle progression and caused a significant increase in p53-dependent transcriptional activity at 0.5 μM (Fig.3A). More importantly, JJ78's chemical tractability made it a good candidate for optimisation studies. Synthesis and Structure Activity Relationship studies on JJ78

A sample of JJ78 (subsequently referred to as JJ78:1 ) was initially obtained from a commercial source. To further confirm bioactivity, chemical structure and purity, larger quantities of JJ78:1 were synthesised by Friedel-Crafts acylation of ethyl 3,5-dimethyl- 1 H-pyrrole-2-carboxylate (1) with 2,5-difluorobenzoyl chloride in the presence of aluminium trichloride in line with literature precedent (Eur. J. Med. Chem., 1993 28, 481- 498) (Fig. 2A). The structure of JJ78:1 was also confirmed by small molecule X-ray crystallographic analysis.

Inspection of the chemical structure of JJ78:1 immediately raised questions about the role of the ester functional group. Saponification of JJ78:1 with potassium hydroxide provided the corresponding carboxylic acid JJ78:2 in high yield (Fig. 2A). The acid chloride formed on treatment of JJ78:2 with thionyl chloride was then converted to the corresponding ethylamide JJ78:3 and benzyl ester JJ78:4 derivatives using standard protocols. Acid JJ78:2 was also converted to the corresponding benzylamide JJ78:5 via the activated ester 2. The biological activity of all the derivatives of JJ78:1 discussed here was assessed using the p53-reporter assay described above. The reasons for selecting this assay included its robust and relatively easy protocol, its ability to readout out biological activity in a cellular environment (as opposed to in vitro) and the fact that it provided a semi-quantitative measure for analysing the effects of compounds on the tubulin network in cells in contrast to the use of immunofluorescence-based approaches the results from which are viewed as qualitative. When tested in the p53 reporter assay, carboxylic acid JJ78:2, possibly due to low membrane permeability, showed no activity, implying that either the biological target was intracellular and hence inaccessible to JJ78:2 or alternatively that the ethyl ester makes a significant contribution to the binding of JJ78:1 to tubulin. The analysis of compounds JJ78:4 and JJ78:5 added further weight to the possibility that a relatively small group is advantageous at position 2 of JJ78:1 (Fig. 2A for numbering system). Bulkier groups (present in JJ78:4 and JJ78:5) were less well tolerated at the 2-position. Evidence that the pyrrole NH functionality or alternative hydrogen bond donor was advantageous for the activity of JJ78:1 was gained from analysis of the NMe-analogue, JJ78:6 (Fig. 2A), synthesised in good yield by reaction of JJ78:1 with iodomethane in the presence of a weak base. JJ78:6 was shown to be inactive implying either binding site constriction in this region or the possible advantageousness of a hydrogen bond donor.

One of the few remaining sites for potential modification in JJ78:1 was the 4- aroyl-position and it was therefore decided to explore the effect of modification at this position whilst retaining the other functionality. The trisubstituted pyrrole 1 (Fig. 3A) was shown to exhibit no biological activity highlighting the importance of the 4-substituent. Acylation of 1 was carried out in parallel employing a modified version of the protocol used to prepare JJ78:1. Analogues JJ78:7-11 were prepared in good yield with high levels of purity using this protocol. The relative activity of these analogues is shown in Fig. 2C. A sufficient spread of potencies was observed across the series to allow a meaningful analysis of the data. A clear trend emerged suggesting an unfavourable steric effect from 4'-substitution. The exception to this trend is JJ78:7, which despite having a large 4'-chloro substituent in addition to the 3'-chloro, exhibited the highest potency of the series. This observation led to the intriguing possibility that an analogue bearing a single 3'-chloro substituent might be significantly more active.

This analogue, ethyl 4-(3-chlorobenzoyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (JJ78:12), was synthesised as part of an analogue expansion series to further investigate SAR. This third compound set also included JJ78:13, a derivative of JJ78:1 in which the bridging ketone had been reduced to a methylene group with triethylsilane {Eur. J. Med. Chem., 1993 28, 481-498) and JJ78:14, an analogue bearing a 2'- methoxybenzoyl group in the 4-position of the dimethylpyrrole ring (Fig. 2C). A byproduct was isolated during synthesis of JJ78:14, in which the 2'-methoxy group had been cleaved giving the corresponding phenol JJ78:15, under the reaction conditions. A further aroyl analogue JJ78:16 was synthesised by S_NAr displacement of the 2'-fluoro group present in JJ78:1 by morpholine. This reaction occurred in preference to the anticipated conversion of the ethyl ester to the corresponding morpholinoamide. In addition to these derivatives, a final pair of analogues was prepared with the goal of exploring the importance of the 3- and 5-methyl substituents in JJ78:1. Ethyl pyrrole-2- carboxylate (3) (Fig. 2B) was treated with two aroyl chlorides to give JJ78:17 and JJ78:18 respectively. X-ray crystallographic analysis of both of these analogues confirmed that variation in the position of acylation occurred as a function of the acylating agent. For example, 2,5-difluorobenzoyl chloride reacted to give, as the major product, JJ78:17, the result of acylation at the 4-position of the pyrrole providing a direct analogue of JJ78:1. However, the use of 3-chlorobenzoyl chloride led predominantly to reaction at the 5-position providing JJ78:18 under analogous conditions. It has been previously reported that the proportion of 4- and 5-acyl regioisomers formed in this system is dependent on reaction conditions, catalyst and acylating reagent (Murakami, Heterocycles, 1988, 27, 8, 1855-1860; and Murakami, Chem. Pharm. Bull. 44(1 ) 48-54 (1996)). Reduction of the ketone functionality in JJ78:1 to a methylene group in JJ78:13 caused an increase in concentration of compound required to induce maximal p53- dependent reporter stimulation and the amplitude of the induction was also reduced (Figs. 2C and 2D). This supports a view that the carbonyl functionality or other surrogate is advantageous for maximal biological activity. Interestingly the X-ray structural analysis of JJ78:1 suggests that, in the solid state at least, the carbonyl group in JJ78:1 is nearly co-planar with the pyrrole ring (torsional angle C(3)-C(4)-C(10)-O(IO) = -21.6°) and that the aromatic ring is significantly distorted out of the plane of the pyrrole ring (torsional angle C(4)-C(10)-C(11)-O(16) = -60.1°). More rotational flexibility in this region of the molecule would be expected following reduction of the carbonyl group, potentially consistent with a decrease in activity resulting from an increased entropic energy cost on binding of JJ78:13 to its target compared with JJ78:1.

The S_NAr reaction product JJ78:16 showed good cellular activity being equipotent with JJ78:7 (Fig. 3A). JJ78:16 was, however, less potent than the 3'-chloro containing analogue JJ78:12 which showed a significant increase in potency compared to both JJ78:7 and JJ78:1 enabling p53 transcriptional activity induction to be detectable at a final concentration of JJ78:12 of only 10 nM in the cell-based assay (not shown). A final concentration of above 100 nM of JJ78:1 was required to give a detectable level of p53 activation, hence JJ78:12 is at least 10-fold more active in cells than JJ78:1. Interestingly, the oxime containing analogue JJ78:19 of JJ78:12 was active but less active at increasing p53 dependent transcription than JJ78:12. The 3,5-unsubstituted pyrrole JJ78:17, a direct analogue of JJ78:1 , showed no evidence of bioactivity. This somewhat surprising result may reflect a change in the degree of rotational freedom associated with the aromatic ring of the 4-substituent in JJ78:17 compared with JJ78:1 , possibly resulting in a greater entropic cost on binding of JJ78:17 to its biological target. The 2,5-disubstituted compound JJ78:18 which bears a 3'-chlorobenzoyl group was also found to be inactive (Figs 2C and 3A) adding to the view that there is a very well-defined pocket available for the binding of the active analogues.

Validation of JJ78:12 as a tubulin poison As a whole, the efficacy of particular JJ78 analogues in the tubulin biochemical assay correlated directly with their level of p53 activation in cells (Figs. 2C, 3A and 3B). This strongly supports that tubulin is the primary target of JJ78:1 and JJ78:12 in cells and that their inhibitory effect on tubulin polymerisation is related to an increase in p53 activity. It is worth noting however, that although in vitro tubulin polymerisation inhibition assays showed that JJ78:12 is significantly more potent than JJ78:1 (Fig. 3C), the increase in potency in this biochemical assay was more than 5-fold but below 10-fold, and therefore less marked than the improvement in the p53 activation cell-based assays. This difference between biochemical and cell-based assays could be due to a variety of reasons including improved cell permeability and intracellular stability properties of JJ78:12 over JJ78:1. It is unlikely that JJ78:12's elevated efficiency in the p53 reporter assay is due to an additional mode of action of JJ78:12 on the p53 pathway because JJ78:12 is also highly effective at increasing the mitotic index (Fig. 3D). Furthermore, the increase in potency of JJ78:12 at inducing p53 transcription was matched by the increased potency at causing microtubule disordering in cells (Fig. 3E). This was especially noticeable in mitotic cells as the mitotic spindle could not form in the presence of 50 nM JJ78:12.

Further strengthening that the effects on cells of JJ78:1 and its more potent derivative JJ78:12 occur through their ability to depolymerise tubulin, nocodazole, JJ78:1 and JJ78:12 induce the same type of cell cycle patterns (Fig. 4A). Another feature shared by these three compounds is that their effect on p53 levels requires that compounds are present in cell cultures for more than 8 hours (Fig. 4B). Instead, compounds that act like the nutlins to decrease p53 degradation by directly inhibiting mdm2 (Vassilev et al., 2004, Science 303, 844-848), can increase p53 levels within 30 minutes of incubation (not shown). This delay in p53 induction suggests that p53 responds to tubulin poisons at a particular stage of the cell cycle. Only when a sufficient number of cells accumulate at this stage it becomes possible to observe an effect on p53 levels in the total cell population.

JJ78 derivative JJ78:12 shows anti-tumour activity in mice xenografts Further evidence of the increased potency of JJ78:12 over the initial hit compound JJ78:1 was demonstrated by cell viability assays. The cytotoxicity of JJ78:1 and the optimised derivative JJ78:12 on the growth of human ARN8 melanoma cells was assessed by 48h MTT assays (Fig. 5A). JJ78:1 Gl₅₀ equals 223 nM, whereas for JJ78:12 Gl₅₀ equals 63 nM. Given the ability of JJ78:12 to kill tumour cells at two digit nanomolar concentrations we tested its ability to reduce tumour growth in vivo. In spite of the fact that JJ78:12 showed a relatively short half life in vivo (Fig. 5B), mice receiving JJ78:12 at 30 mg/kg as a single agent showed a reduction in ARN8 tumour growth (Fig. 5C) without any noticeable toxic effect on the animals.

It is important to note that we have been able to administer a therapeutically active amount of JJ78:12 on a daily basis. Materials and Methods Cell lines

MCF-7, HeLa and ARN8 cells were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle medium supplemented with 10% FCS and 50 μg/ml gentamycin. SKNSH cell lines (CMVNeo and DNp53) and T22- RGC-ΔFos-LacZ were also cultured in this manner but the media was supplemented with 1 mg/ml G418 (Gibco). H 1299 cells were obtained from the ATCC and grown in RPMI-1640 supplemented with 10 % FCS and 50 μg/ml gentamycin.

T22-RGC-ΔFos-LacZ p53 Activation Assay

T22-RGC-ΔFos-LacZ cells were subcultured at 1 x 1O⁴ CeIIs per well in a 96 well plates 24 hours prior to compound treatment. Compounds were added to the cells for 24 hours at a DMSO concentration of 1 %. After treatment the medium was removed and the cells were lysed using 50 μl of 1 x Reporter Lysis Buffer (Promega). Plates were gently shaken for 2 hours to complete the lysis of the cells. After 2 hours 150 μl of CPRG reaction mix (97.5 % v/v phosphate buffer pH7.5 0.1 M, 1.95 % v/v CPRG (Roche) 4mg/ml, 0.52 % v/v 0.1 M magnesium chloride / 4.5 M β-mercaptoethanol) were added and sampes were incubated at 37 ⁰C. Absorbance at 570 nm was measured at 24h hours. Activation ratios were calculated as the absorbance of treated wells minus the background, divided by the absorbance of untreated wells minus the background. Background absorbance was determined by reading the absorbance of wells containing all reagents for the development of the signal but without any cells present.

FACS analysis Cells were seeded at 1 x 10⁵ cells per well in 6 well plates. 24 hours compounds were added and left for 48 hours. Bromodeoxyuridine (BrdU), supplied by Sigma, was added to the cells 30 minutes before harvesting to a final concentration of 30 μM. Cells were harvested using trypsin, spun down and resuspended in 1 ml of PBS and then fixed adding 3 ml of ice-cold ethanol for 1 hour. The cells were treated with a warmed 1 mg/ml pepsin solution (Sigma) in 3OmM HCI pH 1.5, for 30 minutes at 37°C. Samples were centrifuged and cell pellets were incubated in 1 ml of 2M HCI for 20 minutes at RT before being washed in PBS and then antibody buffer (0.5 % w/v BSA, 0.5 % v/v Tween-20 in PBS). Samples were incubated with a mouse monoclonal antibody to BrdU (Becton Dickinson) in antibody buffer for 1 hour at RT. This was followed by extensive washing using PBS before the addition of a secondary anti-mouse IgG FITC conjugated antibody (Sigma) and incubated for 30 minutes at RT in the dark. Samples were washed with PBS and centrifuged. Pellets were resuspended in 0.5 ml of propidium iodide (25 μg/ml) diluted in PBS. Samples were analysed using a Becton Dickinson FACScan operated by the CELLQuest software.

Immunofluorescence staining and imaging of cells

Cells were seeded into 2 well glass slide chambers (Nunc) at 5 x 10⁴ cells per chamber, incubated for 24 hours and treated as specified. Cells were washed in DPBS (Gibco) before being submerged in ice-cold 1 :1 v/v mix of methanol and acetone for 8 minutes. Fixed cells were then washed in DPBS supplemented with 0.1 % v/v Tween- 20 (PBST) 4 times for 5 minutes each. Cells were then blocked using 5 % w/v nonfat milk in PBST. Primary and secondary antibodies were diluted in this solution and all washes were carried out in PBST. After blocking, cells were incubated with primary antibodies for 1 hour at room temperature before being washed 4 times for 5 minutes. Cells were then incubated with the corresponding species type secondary fluorescently labelled antibodies (Alexa Fluor 488 and 594 from Molecular Probes, Invitrogen) in a zero light environment for 45 minutes at room temperature. Cells were again washed twice for 5 minutes before being submerged in Hoescht 33258 DNA stain (Sigma) for 1 minute at 40 μg/ml. Once removed from Hoescht stain glass coverslips were mounted onto the cells using Hydromount (National Diagnostic) supplemented with 2.5 % Dabco (1 ,4-Diazabicyclo-[2.2.2.]octane)- supplied by Sigma. Samples were visualised using an Axiovert 200 M microscope from Zeiss powered by the Volocity software from Improvision. Images were captured using a Hamamatsu Orca ER camera and the light source was provided by the Exfo X-cite 120 light source.

Fluorescence based In vitro Tubulin Polymerisation Assays

In vitro tubulin polymerisation assays were carried out following manufacturer's instructions (Cytoskelton). This involved firstly reconstituting purified bovine neuronal tubulin to a final concentration of 10 mg/ml in 'Buffer 1' (80 mM Piperazine-N,N'-bis[2- ethanesulfonic acid] sequisodium salt; 2.0 mM Magnesium chloride; 0.5 mM Ethylene glycol-bis(b-aminuteso-ethyl ether) N,N,N',N'-tetra-acetic acid, pH6.9 10 μM fluorescent reporter) supplemented with GTP (10 mM). This tubulin was then further diluted to 2 mg/ml in a reaction buffer containing Buffer 1 and a Tubulin Glycerol Buffer (same as Buffer 1 with 60 % glycerol v/v and no fluorescent reporter) to produce a 20 % glycerol concentration. GTP was added to this mix to a final concentration of 1 mM. Compounds were then added (the final concentration of DMSO was 0.5 % v/v in all samples). Polymerisation was monitored by measuring fluorescence (Ex. 355 nm Em. 460 nm) every minute for 60 minutes using a plate reader heated to 37°C (Molecular Devices SPECTRA MAX GEMINI XS driven by SoftMax Pro 4.6). Data was exported to Microsoft Excel for analysis where the data was normalised by subtracting the fluorescence reading at zero minutes from each point.

Chemical synthesis

Ethyl 4-(2\5'-Difluoro-benzoyl)-3,5-dimethyl-1 H-pyrrole-2-carboxylate (J J78: 1 )

Aluminium chloride (0.88 g, 6.6 mmol) and 2,5-difluorobenzoyl chloride (1.13 g, 6.4 mmol) were stirred together at ambient temperature in chlorobenzene (10 mL) for 5 minutes before cooling to 0⁰C. A solution of ethyl 3,5-dimethylpyrrole-2-carboxylate (3.0 mmol, 0.50 g) in chlorobenzene (5 mL) was then added and the mixture was stirred at 0⁰C for 90 minutes, then for 2.5 hours at ambient temperature. The reaction mixture was heated to 80⁰C for 2 hours then cooled back to ambient temperature. The resulting solution was poured onto ice-water (50 mL) and was extracted with dichloromethane. The organic phase was washed with 1 M aqueous NaOH solution (1 x 3OmL), saturated brine (1 x 3OmL) and dried over MgSO₄. Concentration in vacuo provided a residue that was further purified by flash silica chromatography eluting with dichloromethane to give the desired compound JJ78:1 as a white solid (0.51 g, 55%): m.p. 137-138⁰C; ¹H NMR (300 MHz, CDCI₃) δ ppm 1.38 (3 H, t, J=7.2 Hz), 2.28 (3 H, s), 2.31 (3 H, s), 4.34 (2 H, q, J=7.0 Hz), 7.01 - 7.23 (3 H, m), 9.15 (1 H, br. s.); MS (ES+) m/z 330 [M+Na]⁺; HRMS calc'd for Ci₆H₁₅F₂NO₃Na 330.0918, found 330.0912; CHN analysis (C₁₆H₁₅NO₃F₂ O-IH₂O) requires: C, 62.17; H, 4.96; N, 4.53; Found: C, 62.02; H, 4.82; N, 4.46. A sample of JJ78:1 was recrystallised from chloroform to give needle-like crystals that were suitable for X-ray crystallographic analysis.

General procedure for the parallel synthesis of JJ78:1 analogues

Aluminium chloride (147 mg, 1.1 mmol) was added to chlorobenzene (2 mL) in each of the parallel reactor tubes. The appropriate aroyl chloride was then added with stirring under argon atmosphere for 5 minutes. The parallel reactor was then cooled to - 3°C in an ice-brine bath. To each reaction tube was added a solution of the pyrrole 1 (0.5 mmol) in chlorobenzene (1 mL) and the reaction stirred for 90 minutes at -3°C. The reaction was subsequently stirred at ambient temperature for 1 hour followed by heating to 80⁰C for 2 hours. Each reaction was then submitted to an aqueous work up as described in the synthesis of JJ78:1. Desired products JJ78:7-12 and JJ78:14-15 were isolated (24-82% yield) following purification by flash silica chromatography and subsequent re-crystallisation.

Ethyl 4-(3',4'-dichloro-benzoyl)-3,5-dimethyl-1 W-pyrrole-2-carboxylate (J J78:7) Synthesised according to general procedure from 1 and 3,4-dichlorobenzoyl chloride. Yellow crystals, 41 mg (24%): ¹H NMR (300 MHz, CDCI₃) δ ppm 1.38 (3 H, t, J=7.1 Hz), 2.22 (3 H, s), 2.28 (3 H, s), 4.35 (2 H, q, J=7.1 Hz), 7.54 (2 H, m), 7.80 (1 H, s), 9.52 (1 H, br. s); ¹³C NMR (75 MHz, CDCI₃) δ ppm 12.46, 13.60, 14.47, 60.59, 118.81 , 122.42, 128.27, 128.87, 130.58, 131.14, 132.95, 136.57, 137.37, 139.93, 161.78, 191.18.

Ethyl 4-(4'-chloro-benzoyl)-3,5-dimethyl-1 W-pyrrole-2-carboxylate (J J78:8)

Synthesised according to general procedure from 1 and p-chlorobenzoyl chloride. 80 mg (52%): ¹H NMR (300 MHz, CDCI₃) δ ppm 1.39 (3 H, t, J=7.1 Hz), 2.24 (3 H, s), 2.28 (3 H, s), 4.36 (2 H, q, J=7.1 Hz), 7.57 (4 H, dd, J=8.4, 75.8 Hz), 9.60 (1 H, br. s); ¹³C NMR (75 MHz, CDCI₃) δ ppm 12.40, 13.52, 14.47, 60.50, 118.63, 122.86,

128.72, 128.98, 130.65, 137.06, 138.53, 138.61 , 161.85, 192.58.

Ethyl 4-(4'-methyl-benzoyl)-3,5-dimethyl-1 W-pyrrole-2-carboxylate (J J78:9) Synthesised according to general procedure from 1 and p-methylbenzoyl chloride. Beige crystals, 73 mg (51%): ¹H NMR (300 MHz, CDCI₃) δ ppm 1.36 (3 H, t, J=7.1 Hz), 2.23 (3 H, s), 2.25 (3 H, s), 2.41 (3 H, s), 4.33 (2 H, q, J=7.1 Hz), 7.44 (4 H, dd, J=8.0, 121.2 Hz), 9.38 (1 H, br. s); ¹³C NMR (75 MHz, CDCI₃) δ ppm 12.33, 13.46, 14.51 , 21.68, 30.99, 60.37, 118.38, 123.44, 129.08, 129.49, 135.53, 137.54, 142.97, 161.87, 193.69.

Ethyl 4-(4'-methoxy-benzoyl)-3,5-dimethyl-1W-pyrrole-2-carboxylate (JJ78:10)

Synthesised according to general procedure from 1 and p-methoxybenzoyl chloride. Off white solid, 88 mg (58%): ¹H NMR (300 MHz₁ CDCI₃) δ ppm 1.37 (3 H, t, J=7.1 Hz), 2.24 (3 H, s), 2.25 (3 H, s), 3.87 (3 H, s), 4.33 (2 H, q, J=IA Hz), 7.33 (4 H, dd, J=7.8, 243.7 Hz), 9.30 (1 H, br. s).

Ethyl 4-benzoyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (JJ78:11)

Synthesised according to general procedure from 1 and benzoyl chloride. White needles, 95 mg (70%): ¹H NMR (300 MHz, CDCI₃) δ ppm 1.36 (3 H, t, J=7.1 Hz), 2.23 (3

H₁ s), 2.24 (3 H, s), 4.33 (2 H, q, J=7.1 Hz), 7.51 (3 H, m), 7.72 (2 H, m), 9.52 (1 H, br. s); ¹³C NMR (75 MHz, CDCI₃) δ ppm 12.29, 13.46, 14.43, 60.37, 118.43, 123.14, 128.33, 129.14, 132.12, 136.98, 140.27, 161.88, 193.94.

Ethyl 4-(3'-chloro-benzoyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (JJ78:12) Synthesised according to general procedure from 1 and m-chlorobenzoyl chloride. White solid, 64 mg (42%): m.p. 137-138°C; ¹H NMR (300 MHz, CDCI₃) δ ppm 1.39 (3 H, t, .7=7.2 Hz), 2.23 (3 H, s), 2.27 (3 H, s), 4.35 (2 H, q, J=7.2 Hz), 7.35 - 7.45 (1 H, m), 7.48 - 7.56 (1 H, m), 7.60 (1 H, d, J=U Hz), 7.66 - 7.74 (1 H, m), 9.13 (1 H, br. s); MS (ES+) m/z 328 [M+Na]⁺, (ES-) m/z 304 [M-1]^"; HRMS calc'd for C₁₆H₁₆CINO₃Na 328.0716, found 328.0714; CHN analysis (C₁₆H₁₆CINO₃) requires: C, 62.85; H, 5.27; N, 5.48; Found: C, 62.78; H, 5.16; N, 4.46.

Ethyl 4-(2',5'-dif luoro-benzyl)-3,5-dimethyl-1 H-pyrrole-2-carboxylate (J J78: 13) JJ78:1 (0.5 mmol, 0.15g) was treated with triethylsilane (0.32 ml_, 2 mmol) in trifluoroacetic acid (0.35 mL) in a sealed reaction tube heated to 70⁰C for 6 hours. The reaction mixture was concentrated in vacuo, redissolved in Et₂O (50 mL) and washed with 1 M aqueous NaOH (1 x 3OmL) and saturated brine (1 x 3OmL). The organic layer was then concentrated in vacuo to give a residue that was purified by flash silica chromatography eluting with dichloromethane. The resulting solid was recrystallisation from methanol to give the desired product JJ78:13 as an off-white solid (64 mg, 44%): ¹H NMR (300 MHz, CDCI₃) δ ppm 1.37 (3 H, t, J=7.1 Hz), 2.20 (3 H, s), 2.21 (3 H, s), 3.74 (2 H, s), 4.32 (2 H, q, J=7.0 Hz), 6.59 (1 H, ddd, J=3.2, 6.0, 9.0, Hz), 6.76 - 6.88 (1 H, m), 6.97 (1 H, td, J=4.6, 9.0 Hz), 9.00 (1 H, br. s); MS (Cl+) m/z 294 [M+1]⁺; HRMS calc'd for C₁₆H₁₇F₂NO₂ 294.1306, found 294.1302.

Ethyl 4-(2'-methoxy-benzoyl)-3,5-dimethyl-1 H-pyrrole-2-carboxylate (JJ78:14) and ethyl 4-(2'-hydroxy-benzoyl)-3,5-dimethyl-1 H-pyrrole-2-carboxylate (J J78: 15)

Synthesised according to general procedure from 1 and o-methoxybenzoyl chloride. Beige solid, 123 mg (82%).JJ78:14 was isolated as a by-product. Off-white solid, 5 mg. JJ78:14: ¹H NMR (300 MHz, CDCI₃) δ ppm 1.35 (3 H, t, J=7.1 Hz), 2.21 (3

H, s), 2.25 (3 H, s), 3.78 (3 H, s), 4.31 (2 H, q, J=7.1 Hz), 6.99 (2 H, m), 7.26 (1 H, dd,

J=1.8, 7.4 Hz), 7.40 (1 H, ddd, J=1.8, 7.4, 8.3 Hz), 8.99 (1 H, br. s); ¹³C NMR (75 MHz,

CDCI₃) δ ppm 11.76, 13.94, 14.41 , 55.70, 60.31 , 111.34, 118.10, 120.66, 123.49, 128.61 , 130.35, 131.28, 131.98, 139.01 , 156.61 , 161.86, 192.46; MS (ES+) m/z 324

[M+Na]⁺; HRMS calc'd for C₁₇H₁₉NO₄Na 324.1212, found 324.1208. JJ78:15: ¹H NMR (75 MHz, CDCI₃) δ ppm 1.38 (3 H, t, J=7.1 Hz), 2.27 (3 H, s), 2.29 (3 H, s), 4.35 (2 H, q, J=7.1 Hz), 6.86 (1 H, m), 7.03 (1 H, dd, J=OJ, 8.3 Hz), 7.48 (2 H, m), 9.13 (1 H₁ br. s), 12.21 (1 H, s); MS (ES+) m/z 310 [M+Na]⁺; HRMS calc'd for Ci₆H₁₇NO₄Na 310.1055, found 310.1052.

Ethyl 4-(5'-fluoro-2'-morpholin-4-yl-benzoyl)-3,5-dimethyl-1W-pyrrole-2-carboxylate (JJ78:16)

A mixture of JJ78:1 (100 mg, 0.33 mmol), morpholine (4 ml_) and water (0.2 mL) was heated to 150⁰C in a sealed tube under microwave irradiation for 2 hours. The reaction mixture was then concentrated in vacuo and the resulting residue partitioned between EtOAc (30 mL) and water (20 mL). The aqueous layer was acidified to pH 2 with dilute HCI and extracted with EtOAc (3 x 30 mL). The combined organic layers were then washed with saturated brine (1 x 50 mL), dried (MgSO₄) and concentrated in vacuo. The resulting solid was purified by recrystallisation from ethanol to give the desired compound JJ78:16 as an off-white solid (85 mg, 69%): ¹H NMR (300 MHz, CDCI₃) δ ppm 1.37 (3 H, t, J=7.2 Hz), 2.17 (3 H, s), 2.21 (3 H, s), 2.83 - 2.96 (4 H, m), 3.39 - 3.51 (4 H, m), 4.33 (2 H, q, J=7.0 Hz), 6.98 (1 H, d, J=8.7 Hz), 7.04 - 7.18 (2 H, m), 9.37 (1 H, br. s); ¹³C NMR (75 MHz, CDCI₃) δ ppm 11.58, 13.69, 53.05, 60.47, 66.84, 116.04 (d, J=23.4 Hz), 117.64 (d, J=22.1 Hz), 118.15, 119.84 (d, J=7.7 Hz), 122.68, 130.08, 137.69, 138.56, 146.35, 158.66 (d, J=243.7 Hz), 161.72, 192.77; MS (ES+) m/z 397 [M+Na]⁺; HRMS calc'd for C₂₀H₂₃N₂O₄NaF 397.1540, found 397.1522.

In vivo efficacy of compounds on the growth of xenograft tumours

Female SCID mice (Harlan) were injected subcutaneously with 1 x 10⁶ ARN8 cells (or SKNSHpCMV) suspended in 1 :1 v/v solution of DMEM containing resuspended cells: matrigel (BD Biosciences). ARN8 cells had been harvested when approximately

60-70 % confluent. Tumours were allowed to establish and reach a size of approximately 10 mm³. Mice were treated with 30 mg/kg JJ78:12 in 20% cyclodextrin everyday by intraperitoneal injection. Control animals were treated with the vehicle solution containing cyclodextrin 20 % w/v and DMSO 10 % v/v. Tumour diameters were measured using calipers and volumes were calculated using the equation

V=D4/3[(d1+d2)/4]³. The appropriate measure of centrality (mean or median) of tumour sizes was calculated for each time point, as well as the corresponding 95 % confidence intervals. Comparison of control and drug treated tumour size distributions were made by statistical tests. A Two Sample T-test or a Mann-Whitney U-test was appropriately chosen depending on whether the data was normally or non-normally distributed. Normality of data was determined by an Anderson-Darling test. An alpha-level of 0.05 was considered appropriate for determination of statistical significance. All animal experiments were in compliance with the United Kingdom Coordinating Committee on Cancer Research.

Pharmacokinetic studies of JJ78:12 in mice

Three mice were dosed by i.p. injection at 30 mg/kg with a 10 mM JJ78:12 solution in 5 % DMSO and 20 % cyclodextrine. 10 μl of blood were collected directly from the tail vein at 5, 15, 30, 60, 120, 240, 360 and 480 minutes, mixed with 10 μl of heparin solution (15 IU/ml) in a1.5 ml tube and kept on ice until storage at -80 ^°C. To each blood sample, after thawing at room temperature, 20 μl of in ternal standard (gemcitabine 10 μg/ml in methanol) and 80 μl of methanol were added, before being vortexed for 1 minute. Subsequently, 80 μl of sulfosalicylic acid (10 %) were added and samples vortexed for 1 minute, followed by a 5 minute sonication. The samples were then centrifuged at 2500 g for 10 min. Supernatants were transferred to vials and analysed by LC Q-TOF. In parallel, a calibration curve of JJ78:16 was carried out by spiking a fixed amount in blood (0 - 2500 ng/ml), followed by the extraction procedure outlined above. The percentage of extraction of JJ78:12 from the blood sample was equal to 62.5 %. LC Q-TOF analysis was carried out using a Waters 2695 HPLC coupled to a Q-TOF Micro mass (MicroMass, UK) using the positive electrospray ionisation (ESI) mode. Samples were analysed on a Luna Phenyl-Hexyl (150 mm x 3 mm, 5μm particle size, Phenomenex). The injection volume was 100 μl. The following elution program was used: a temperature of 20^°C, a flow rate of 0.2 ml/min, and an isocratic mobile phase composed of 5 % water, 90 % methanol containing 0.1 % formic acid and 5 % acetonitrile containing 0.1 % formic acid. The capillary voltage was adjusted to 4000 V, sample cone to 31 V, the cone to 50 L/h, the desolvation temperature to 300 ⁰C with a nitrogen flow to 400 L/h, and the collision energy to 28.0 V. The transitions monitored were 306.17>139.03, 264.10>112.08 for JJ78:12 and gemcitabine respectively. MS-MS data were processed using the QuanLynx quantify option of MassLynx software, version 4.1 , from Micromass. Pharmacokinetic parameters were calculated using the WinNonLin software, version 4.1. Non- compartmental and two-compartment models were used to predict the different pharmacokinetic parameters, AUC_bι_OOd. CL, t_1/2 and C_max.

Claims

1. A compound of formula (I):

wherein:

X is NR^X, O, S, SO or SO₂, wherein R^x is H or a Ci_-6 straight-chain, branched or cyclic alkyl substituted once or twice with OH, SH, NH or NH₂;

each of L¹ and L³ is independently present or absent and if present is selected from O, S, SO, SO₂or NR^y;

each of R¹ and R² is independently a straight-chain, branched or cyclic alkyl, aryl, heteroaryl, or R¹ together with R², forms a straight-chain diradical R^1"2 whereby to connect L¹ (if present) and L², and is optionally substituted with one or more substituents selected from the group comprising straight-chain, branched or cyclic alkyl, aryl, heteroaryl, halo, hydroxyl, carboxy, acyl, thioacyl, alkoxy, alkylthio, alkyl sulfoxy, alkyl sulfone, thiol, -C(=O)-, -O-, -S-, -S(O)-, -S(O)₂-, ester, amido, sulfonamido, amino, nitro and cyano;

L² is a linking moiety comprising a contiguous saturated or unsaturated hydrocarbyl diradical sequence of from 1 to 6 atoms, said linking moiety optionally comprising one or more of the following functionalities as part of the contiguous sequence: -C(=O)-, -SO₂NR^y-, -S(O)-, S(O)₂-, C(=O)N(R^y)-, -C(=0)0-, -C(=S)-, -C(=S)O-, -C(=S)S-, -C(=O)S-, -C(=N-OH)-, -C(=N-OR^y)-, -C(=NR^y)-, -C(=N-NH₂)-, -C(=N-NHR^y)-, -C(=O)N(R^y)C(=O)-, -C(=S)N(R^y)C(=S)-, -C(=O)N(R^y)C(=S)- and -C(=N-N(R^y)₂)-;

L⁴ is absent or present and if present is a linking moiety comprising a contiguous saturated or unsaturated hydrocarbyl diradical sequence of from 1 to 6 atoms, said linking moiety optionally comprising one or more of the following functionalities as part of the contiguous sequence: -C(=0)-, -C(OH)-, -C(0R^y)-, -C(SH)-, -C(SR^y)-, -SO₂NR^y- -S(O)-, S(O)₂-, C(=O)N(R^y-C(=O)O-,-C(=S)-, -C(=S)O-, -C(=S)S-,-C(=O)S-, -C(=N-OH)-, -C(=N-OR^y)-, -C(=NR^y)-, -C(=N-NH₂)-, -C(=N-NHR^y)-,-C(=O)N(R^y)C(=O)-, -C(=S)N(R^Y)C(=S)-, -C(=O)N(R^Y)C(=S)- -C(=N-N(R^y)₂)-, -O-, -S-, -N(R^y)-, -C(NH₂)-, -C(NR^y ₂)-, -C(NHR^y)-, -C(NH₂)-;

each of R³ and R⁴ is independently a straight-chain, branched or cyclic alkyl, aryl, heteroaryl, or R³ together with R⁴, forms a straight-chain diradical R^3"4 whereby to connect L³ (if present) and L⁴, and is optionally substituted with one or more substituents selected from the group comprising straight-chain, branched or cyclic alkyl, aryl, heteroaryl, halo, hydroxyl, carboxy, acyl, thioacyl, alkoxy, alkylthio, alkyl sulfoxy, alkyl sulfone, thiol, -C(=O)-, -O-, -S-, -S(O)-, -S(O)₂-, ester, amido, sulfonamide amino, nitro and cyano;

each R^y is independently selected from a group comprising hydrogen and a straight- chain, branched or cyclic alkyl, aryl, heteroaryl, optionally substituted with one or more substituents selected from the group comprising straight-chain, branched or cyclic alkyl, aryl, heteroaryl, halo, hydroxyl, carboxy, acyl, alkoxy, alkylthio, alkyl sulfoxy, alkyl sulfone, thiol, -C(=O)-, -O-, -S-, -S(O)-, -S(O)₂-, ester, amido, sulfonamido, amino, nitro and cyano;

with the proviso that the compound is not

(i) 4-benzoyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester;

(ii) 4-(2,5-difluorobenzoyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester;

(iii) 4-acetyl-3,5-dimethyl-1/-/-pyrrole-2-carboxylic acid ethyl ester; or (iv) 4-(3-dimethylamino-acryloyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester,

or a pharmaceutically acceptable derivative thereof.

2. The compound of claim 1 wherein X is NH, O, S, SO or SO₂.

3. The compound of claim 1 wherein X is NH.

4. The compound of any one of claims 1 to 3 wherein L¹ is absent.

5. The compound of any one of claims 1 to 3 wherein L³ is absent.

6. The compound of any one of any one preceding claim wherein L² comprises one or more of the following functionalities as part of the contiguous sequence: -C(=O)-, -SO₂NR^y-, -S(O)-, S(O)₂-, C(=O)N(R^y)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -C(=O)S-, -C(=N-OH)-, -C(=N-OR^y)-, -C(=NR^y)-, -C(=N-NH₂)-, -C(=N-NHR^y)-, -C(=O)N(R^y)C(=O)- and -C(=N-N(R^y)₂)-.

7. The compound of any one of any one preceding claim wherein L² is -C(=O)-, -S(O)-, S(O)₂- or -C(=S)-.

8. The compound of claim 7 wherein L² is -C(=O)- or -S(O)₂-.

9. The compound of claim 8 wherein L² is -C(=O)-.

10. The compound of any one preceding claim wherein R² is a cyclic alkyl, aryl, heteroaryl group.

11. The compound of claim 10 wherein R² is an aryl or heteroaryl group.

12. The compound of claim 11 wherein R² is a monocyclic aryl or heteroaryl group.

13. The compound of claim 12 wherein R² is a phenyl group.

14. The compound of claim 13 wherein the phenyl group is substituted once or more with substituents selected independently from halo, hydroxyl, alkoxy, alkylthio, alkyl sulfoxy, alkyl sulfone, thiol, ester, amido, sulfonamido, amino, nitro and cyano.

15. The compound of claim 14 wherein the phenyl group is substituted at one or more of positions 2, 3, 5 and 6, position 1 being the point of connectivity to L².

16. The compound of claim 14 or claim 15 wherein the phenyl group is substituted with halo, hydroxyl, thiol, amino, alkoxy or alkylthio.

17. The compound of claim 16 wherein halo is chloro or fluoro.

18. The compound of claim 17 wherein halo is fluoro.

19. The compound of claim 17 wherein halo is chloro.

20. The compound of any one of claims 14 to 19 wherein amino is morpholino.

21. The compound of any one of claims 14 to 20 wherein the phenyl group is substituted at position 2.

22. The compound of any one of claims 14 to 20 wherein the phenyl group is substituted at position 3.

23. The compound of any one of claims 14 to 20 wherein the phenyl group is substituted at position 5.

24. The compound of any one of claims 14 to 20 wherein the phenyl group is substituted at position 6.

25. The compound of any one of claims 1 to 9 wherein said diradical R^1'2 is present, and together with L² and, if present, L¹, forms a diradical (L¹ JR^{1 2}L² whereby to form a 5- to 8-membered ring together with the carbon atoms to which the terminal atoms of diradical (L¹JR^{1 2}L² is connected.

26. The compound of claim 25 wherein diradical (L¹JR^{1 2}L² is substituted with a cyclic alkyl, aryl, heteroaryl group as defined in any one of claims 10 to 24.

27. The compound of any one preceding claim wherein said diradical R^3"4 is present, and together with L⁴, if present, and, if present, L³, forms a diradical (L³)R^{3 4}(L⁴) whereby to form a 5- to 8-membered ring together with the carbon atoms to which the terminal atoms of diradical (L³JR³^(L⁴J iS connected.

28. The compound of claim 27 wherein diradical (L³)R^3'4(L⁴)forms a 6-membered aromatic or heteroaromatic ring together with the carbon atoms to which the terminal atoms of diradical (L³JR³^(L⁴J iS connected.

29. The compound of any one of claims 1 to 26 wherein L⁴R⁴ is -C(=O)OR⁴, -C(=S)OR\ -C(=O)SR⁴ or -C(=S)SR.

30. The compound of claim 29 wherein L⁴R⁴ is -C(O)OR⁴.

31. The compound of claim 29 or claim 30 wherein R⁴ is a C_1-6, e.g. C_2-4, alkyl.

32. The compound of any one of claims 29 to 31 wherein R⁴ is saturated.

33. The compound of any one of claims 29 to 32 wherein R⁴ is unsubstituted.

34. The compound of any one of claims 1 to 24 or 29 to 33 wherein R¹ and R³ are each selected from the group comprising straight-chain, branched or cyclic saturated

C_1-6 alkyl.

35. The compound of claim 34 wherein said alkyl is unsubstituted.

36. The compound of claim 34 or claim 35 wherein R¹ and R³ are the same.

37. A composition comprising a compound as defined is any one of claims 1 to 36, but without the provisos, together with a pharmaceutically acceptable carrier.

38. A compound as defined is any one of claims 1 to 36, but without the provisos, for use in medicine.

39. Use of a compound according to any one of claims 1 to 36, but without the provisos, for the preparation of a medicament for use in a method or treatment or prophylaxis of a disease involving cell proliferation, in particular cancer.

40. A compound according to any one of claims 1 to 36, but without the provisos, for use in a method as defined in claim 39.

41. A method of treatment or prophylaxis of a disease involving cell proliferation, in particular cancer, said method comprising administering a therapeutically or prophylactically useful amount of a compound according to any one of claims 1 to 36, but without the provisos, to a subject in need thereof.