This invention relates to pyrazole compounds that inhibit or modulate the activity of Cyclin Dependent Kinases (CDK), Glycogen Synthase Kinases (GSK) and Aurora kinases, to the use of the compounds in the treatment or prophylaxis of disease states or conditions mediated by the kinases, and to novel compounds having kinase inhibitory or modulating activity. Also provided are pharmaceutical compositions containing the compounds and novel chemical intermediates.

Background of the Invention

Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a wide variety of signal transduction processes within the cell (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book. I and II, Academic Press, San Diego, CA). The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (e.g., Hanks, S.K., Hunter, T., FASEB ./., _, 9:576-596 (1995); Knighton, et al, Science, 253:407-414 (1991); Hiles, et al, Cell, 70:419-429 (1992); Kunz, et al, Cell, 73:585-596 (1993); Garcia-Bustos, et al, EMBO J., 13:2352-2361 (1994)).

Protein kinases may be characterized by their regulation mechanisms. These mechanisms include, for example, autophosphorylation, transphosphorylation by other kinases, protein-protein interactions, protein-lipid interactions, and protein- polynucleotide interactions. An individual protein kinase may be regulated by more than one mechanism.

Kinases regulate many different cell processes including, but not limited to, proliferation, differentiation, apoptosis, motility, transcription, translation and other signalling processes, by adding phosphate groups to target proteins. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. Phosphorylation of target proteins occurs in response to a variety of extracellular signals (hormones,

neurotransmitters, growth and differentiation factors, etc.), cell cycle events, environmental or nutritional stresses, etc. The appropriate protein kinase functions in signalling pathways to activate or inactivate (either directly or indirectly), for example, a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor. Uncontrolled signalling due to defective control of protein phosphorylation has been implicated in a number of diseases, including, for example, inflammation, cancer, allergy/asthma, disease and conditions of the immune system, disease and conditions of the central nervous system, and angiogenesis.

Cyclin Dependent Kinases

The process of eukaryotic cell division may be broadly divided into a series of sequential phases termed Gl, S, G2 and M. Correct progression through the various phases of the cell cycle has been shown to be critically dependent upon the spatial and temporal regulation of a family of proteins known as cyclin dependent • kinases (cdks) and a diverse set of their cognate protein partners termed cyclins. Cdks are cdc2 (also known as cdkl) homologous serine-threonine kinase proteins that are able to utilise ATP as a substrate in the phosphorylation of diverse polypeptides in a sequence dependent context. Cyclins are a family of proteins characterised by a homology region, containing approximately 100 amino acids, termed the "cyclin box" which is used in binding to, and defining selectivity for, specific cdk partner proteins.

Modulation of the expression levels, degradation rates, and activation levels of various cdks and cyclins throughout the cell cycle leads to the cyclical formation of a series of cdk/cyclin complexes, in which the cdks are enzymatically active. The formation of these complexes controls passage through discrete cell cycle checkpoints and thereby enables the process of cell division to continue. Failure to satisfy the pre-requisite biochemical criteria at a given cell cycle checkpoint, i.e. failure to form a required cdk/cyclin complex, can lead to cell cycle arrest and/or cellular apoptosis. Aberrant cellular proliferation, as manifested in cancer, can often be attributed to loss of correct cell cycle control. Inhibition of cdk enzymatic

activity therefore provides a means by which abnormally dividing cells can have their division arrested and/or be killed. The diversity of cdks, and cdk complexes, and their critical roles in mediating the cell cycle, provides a broad spectrum of potential therapeutic targets selected on the basis of a defined biochemical rationale.

Progression from the Gl phase to the S phase of the cell cycle is primarily regulated by cdk2, cdk3, cdk4 and cdkό via association with members of the D and E type cyclins. The D-type cyclins appear instrumental in enabling passage beyond the Gl restriction point, where as the cdk2/cyclin E complex is key to the transition from the Gl to S phase. Subsequent progression through S phase and entry into G2 is thought to require the cdk2/cyclin A complex. Both mitosis, and the G2 to M phase transition which triggers it, are regulated by complexes of cdkl and the A and B type cyclins.

During Gl phase Retinoblastoma protein (Rb), and related pocket proteins such as pl30, are substrates for cdk(2, 4, & 6)/cyclin complexes. Progression through Gl is in part facilitated by hyperphosphorylation, and thus inactivation, of Rb and pl30 by the cdk(4/6)/cyclin-D complexes. Hyperphosphorylation of Rb and pi 30 causes the release of transcription factors, such as E2F, and thus the expression of genes necessary for progression through Gl and for entry into S-phase, such as the gene for cyclin E. Expression of cyclin E facilitates formation of the cdk2/cyclin E complex which amplifies, or maintains, E2F levels via further phosphorylation of Rb. The cdk2/cyclin E complex also phosphorylates other proteins necessary for DNA replication, such as NPAT, which has been implicated in histone biosynthesis. Gl progression and the Gl/S transition are also regulated via the mitogen stimulated Myc pathway, which feeds into the cdk2/cyclin E pathway. Cdk2 is also connected to the p53 mediated DNA damage response pathway via p53 regulation of p21 levels. p21 is a protein inhibitor of cdk2/cyclin E and is thus capable of blocking, or delaying, the Gl/S transition. The cdk2/cyclin E complex may thus represent a point at which biochemical stimuli from the Rb, Myc and p53 pathways are to some degree integrated. Cdk2 and/or the cdk2/cyclin E complex therefore

represent good targets for therapeutics designed at arresting, or recovering control of, the cell cycle in aberrantly dividing cells.

The exact role of cdk3 in the cell cycle is not clear. As yet no cognate cyclin partner has been identified, but a dominant negative form of cdk3 delayed cells in Gl , thereby suggesting that cdk3 has a role in regulating the Gl/S transition.

Although most cdks have been implicated in regulation of the cell cycle there is evidence that certain members of the cdk family are involved in other biochemical processes. This is exemplified by cdk5 which is necessary for correct neuronal development and which has also been implicated in the phosphorylation of several neuronal proteins such as Tau, NUDE-I, synapsinl, DARPP32 and the

Munclδ/SyntaxinlA complex. Neuronal cdk5 is conventionally activated by binding to the p35/p39 proteins. Cdk5 activity can, however, be deregulated by the binding of p25, a truncated version of p35. Conversion of p35 to p25, and subsequent deregulation of cdk5 activity, can be induced by ischemia, excitotoxicity, and β-amyloid peptide. Consequently p25 has been implicated in the pathogenesis of neurodegenerative diseases, such as Alzheimer's, and is therefore of interest as a target for therapeutics directed against these diseases.

Cdk7 is a nuclear protein that has cdc2 CAK activity and binds to cyclin H. Cdk7 has been identified as component of the TFIIH transcriptional complex which has RNA polymerase II C-terminal domain (CTD) activity. This has been associated with the regulation of HIV-I transcription via a Tat-mediated biochemical pathway. Cdk8 binds cyclin C and has been implicated in the phosphorylation of the CTD of RNA polymerase II. Similarly the cdk9/cyclin-Tl complex (P-TEFb complex) has been implicated in elongation control of RNA polymerase II. PTEF -b is also required for activation of transcription of the HIV-I genome by the viral transactivator Tat through its interaction with cyclin Tl . Cdk7, cdk8 ₅ cdk9 and the P-TEFb complex are therefore potential targets for anti-viral therapeutics.

At a molecular level mediation of cdk/cyclin complex activity requires a series of stimulatory and inhibitory phosphorylation, or dephosphorylation, events. Cdk

phosphorylation is performed by a group of cdk activating kinases (CAKs) and/or kinases such as weel, Mytl and Mikl . Dephosphorylation is performed by phosphatases such as cdc25(a & c), pp2a, or KAP.

Cdk/cyclin complex activity may be further regulated by two families of endogenous cellular proteinaceous inhibitors: the Kip/Cip family, or the INK family. The INK proteins specifically bind cdk4 and cdkό. pl6 ^mk4 (also known as MTSl) is a potential tumour suppressor gene that is mutated, or deleted, in a large number of primary cancers. The Kip/Cip family contains proteins such as _p21 c ^ipi, w ^afi _{; p27} κ ^ipi _{and As} discussed previously p21 is induced by p53 and is able to inactivate the cdk2/cyclin(E/A) and cdk4/cyclin(Dl/D2/D3) complexes. Atypically low levels of p27 expression have been observed in breast, colon and prostate cancers. Conversely over expression of cyclin E in solid tumours has been shown to correlate with poor patient prognosis. Over expression of cyclin Dl has been associated with oesophageal, breast, squamous, and non-small cell lung carcinomas.

The pivotal roles of cdks, and their associated proteins, in co-ordinating and driving the cell cycle in proliferating cells have been outlined above. Some of the biochemical pathways in which cdks play a key role have also been described. The development of monotherapies for the treatment of proliferative disorders, such as cancers, using therapeutics targeted generically at cdks, or at specific cdks, is therefore potentially highly desirable. Cdk inhibitors could conceivably also be used to treat other conditions such as viral infections, autoimmune diseases and neuro-degenerative diseases, amongst others. Cdk targeted therapeutics may also provide clinical benefits in the treatment of the previously described diseases when used in combination therapy with either existing, or new, therapeutic agents. Cdk targeted anticancer therapies could potentially have advantages over many current antitumour agents as they would not directly interact with DNA and should therefore reduce the risk of secondary tumour development.

Diffuse Large B-cell Lymphomas ( ^" DLBCD

Cell cycle progression is regulated by the combined action of cyclins, cyclin- dependent kinases (CDKs), and CDK-inhibitors (CDKi), which are negative cell cycle regulators. p27KIPl is a CDKi key in cell cycle regulation, whose degradation is required for Gl/S transition. In spite of the absence of p27KIPl expression in proliferating lymphocytes, some aggressive B-cell lymphomas have been reported to show an anomalous p27KIPl staining. An abnormally high expression of p27KIPl was found in lymphomas of this type. Analysis of the clinical relevance of these findings showed that a high level of p27KIPl expression in this type of tumour is an adverse prognostic marker, in both univariate and multivariate analysis. These results show that there is abnormal p27KIPl expression in Diffuse Large B-cell Lymphomas (DLBCL), with adverse clinical significance, suggesting that this anomalous p27KIPl protein may be rendered nonfunctional through interaction with other cell cycle regulator proteins. (Br. J. Cancer. 1999 Jul;80(9): 1427-34. ρ27KIPl is abnormally expressed in Diffuse Large B-cell Lymphomas and is associated with an adverse clinical outcome. Saez A, Sanchez E, Sanchez-Beato M, Cruz MA, Chacon I, Munoz E, Camacho FI, Martinez-Montero JC, Mollejo M, Garcia JF, Piris MA. Department of Pathology, Virgen de Ia Salud Hospital, Toledo, Spain.)

Chronic Lymphocytic Leukemia

B-CeIl chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the Western hemisphere, with approximately 10,000 new cases diagnosed each year (Parker SL, Tong T, Bolden S, Wingo PA: Cancer statistics, 1997. Ca. Cancer. J. Clin. 47:5, (1997)). Relative to other forms of leukaemia, the overall prognosis of CLL is good, with even the most advanced stage patients having a median survival of 3 years.

The addition of fludarabine as initial therapy for symptomatic CLL patients has led to a higher rate of complete responses (27% v 3%) and duration of progression-free survival (33 v 17 months) as compared with previously used alkylator-based

therapies. Although attaining a complete clinical response after therapy is the initial step toward improving survival in CLL, the majority of patients either do not attain complete remission or fail to respond to fludarabine. Furthermore, all patients with CLL treated with fludarabine eventually relapse, making its role as a single agent purely palliative (Rai KR, Peterson B, Elias L, Shepherd L, Hines J, Nelson D, Cheson B, Kolitz J, Schiffer CA: A randomized comparison of fludarabine and chlorambucil for patients with previously untreated chronic lymphocytic leukemia. A CALGB SWOG, CTG/NCI-C and ECOG Inter-Group Study. Blood 88:141a, 1996 (abstr 552, suppl 1). Therefore, identifying new agents with novel mechanisms of action that complement fludarabine's cytotoxicity and abrogate the resistance induced by intrinsic CLL drug-resistance factors will be necessary if further advances in the therapy of this disease are to be realized.

The most extensively studied, uniformly predictive factor for poor response to therapy and inferior survival in CLL patients is aberrant p53 function, as characterized by point mutations or chromosome 17pl3 deletions. Indeed, virtually no responses to either alkylator or purine analog therapy have been documented in multiple single institution case series for those CLL patients with abnormal p53 function. Introduction of a therapeutic agent that has the ability to overcome the drug resistance associated with p53 mutation in CLL would potentially be a major advance for the treatment of the disease.

Flavopiridol and CYC 202, inhibitors of cyclin-dependent kinases induce in vitro apoptosis of malignant cells from B-cell chronic lymphocytic leukemia (B-CLL).

Flavopiridol exposure results in the stimulation of caspase 3 activity and in caspase- dependent cleavage of p27(kipl), a negative regulator of the cell cycle, which is overexpressed in B-CLL (Blood. 1998 Nov 15;92(10):3804-16 Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53. Byrd JC, Shinn C, Waselenko JK, Fuchs EJ, Lehman TA, Nguyen PL, Flinn IW, Diehl LF, Sausville E, Grever MR).

Aurora Kinases

Relatively recently, a new family of serine/threonine kinases known as the Aurora kinases has been discovered that are involved in the G2 and M phases of the cell cycle, and which are important regulators of mitosis.

The precise role of Aurora kinases has yet to be elucidated but that they play a part in mitotic checkpoint control, chromosome dynamics and cytokinesis (Adams et al, Trends Cell Biol, 11 : 49-54 (2001). Aurora kinases are located at the centrosomes of interphase cells, at the poles of the bipolar spindle and in the mid-body of the mitotic apparatus.

Three members of the Aurora kinase family have been found in mammals so far (E. A. Nigg, Nat. Rev. MoI. Cell Biol. 2: 21-32, (2001)). These are:

Aurora A (also referred to in the literature as Aurora 2); Aurora B (also referred to in the literature as Aurora 1); and Aurora C (also referred to in the literature as Aurora 3).

The Aurora kinases have highly homologous catalytic domains but differ considerably in their N-terminal portions (Katayama H, Brinkley WR, Sen S.; The Aurora kinases: role in cell transformation and tumorigenesis; Cancer Metastasis Rev. 2003 Dec;22(4):451-64).

The substrates of the Aurora kinases A and B have been identified as including a kinesin-like motor protein, spindle apparatus proteins, histone H3 protein, kinetochore protein and the tumour suppressor protein p53.

Aurora A kinases are believed to be involved in spindle formation and become localised on the centrosome during the early G2 phase where they phosphorylate spindle-associated proteins (Fxigent et al, Cell, 114: 531-535 (2003). Hirota etal, Cell, 114:585-598, (2003) found that cells depleted of Aurora A protein kinase were unable to enter mitosis. Furthermore, it has been found (Adams, 2001) that mutation or disruption of the Aurora A gene in various species leads to mitotic

abnormalities, including centrosome separation and maturation defects, spindle aberrations and chromosome segregation defects.

The Aurora kinases are generally expressed at a low level in the majority of normal tissues, the exceptions being tissues with a high proportion of dividing cells such as the thymus and testis. However, elevated levels of Aurora kinases have been found in many human cancers (Giet et ah, J. Cell. ScI 112: 3591-361, (1999) and Katayama (2003). Furthermore, Aurora A kinase maps to the chromosome 20ql 3 region that has frequently been found to be amplified in many human cancers.

Thus, for example, significant Aurora A over-expression has been detected in human breast, ovarian and pancreatic cancers (see Zhou et al, Nat. Genet. 20: 189- 193, (1998), Tanaka et al, Cancer Res., 59: 2041-2044, (1999) and Han et at, cancer Res., 62: 2890-2896, (2002).

Moreover, Isola, American Journal of Pathology 147,905-911 (1995) has reported that amplification of the Aurora A locus (20ql3) correlates with poor prognosis for patients with node-negative breast cancer.

Amplification and/or over-expression of Aurora- A is observed in human bladder cancers and amplification of Aurora- A is associated with aneuploidy and aggressive clinical behaviour, see Sen et al., J. Natl.Cancer Inst, 94: 1320-1329 (2002).

Elevated expression of Aurora- A has been detected in over 50% of colorectal cancers, (see Bischoff et al, EMBO J, 17: 3052-3065, (1998) and Takahashi et al., Jpn. J. Cancer Res. , 91: 1007-1014 (2000)) ovarian cancers (see Gritsko et al. Clin. Cancer Res., 9: 1420-1426 (2003), and gastric tumours Sakakura et al, British Journal of Cancer, 84: 824-831 (2001).

Tanaka et al Cancer Research, 59: 2041-2044 (1999) found evidence of over- expression of Aurora A in 94% of invasive duct adenocarcinomas of the breast.

High levels of Aurora A kinase have also been found in renal, cervical, neuroblastoma, melanoma, lymphoma, pancreatic and prostate tumour cell lines

Bischoff et al. (1998), EMBO J., 17: 3052-3065 (1998) ; Kimura et al. J. Biol. Chem., 274: 7334-7340 (1999) ; Zhou et al., Nature Genetics, 20: 189-193 (1998); Li et al., Clin Cancer Res. 9 (3): 991-7 (2003) ].

Aurora-B is highly expressed in multiple human tumour cell lines, including leukemic cells [Katayama et al., Gene 244: 1-7) ]. Levels of this enzyme increase as a function of Duke's stage in primary colorectal cancers [Katayama et al., J. Natl Cancer Inst, 91: 1160-1162 (1999)].

High levels of Aurora-3 (Aurora-C) have been detected in several tumour cell lines, even though this kinase tends to be restricted to germ cells in normal tissues (see Kimura et al. Journal of Biological Chemistry, 21 A: 7334-7340 (1999)). Over- expression of Aurora-3 in approximately 50% of colorectal cancers has also been reported in the article by Takahashi et al., Jpn J. Cancer Res. 91 : 1007-1014 (2001)].

Other reports of the role of Aurora kinases in proliferative disorders may be found in Bischoff et al., Trends in Cell Biology 9: 454-459 (1999); Giet et al. Journal of Cell Science, 112: 3591-3601 (1999) and Dutertre, et al. Oncogene, 21: 6175-6183 (2002).

Royce et al report that the expression of the Aurora 2 gene (known as STKl 5 or BTAK) has been noted in approximately one-fourth of primary breast tumours. (Royce ME, Xia W, Sahin AA, Katayama H, Johnston DA, Hortobagyi G, Sen S, Hung MC; STK15/Aurora-A expression in primary breast tumours is correlated with nuclear grade but not with prognosis; Cancer. 2004 Jan l;100(l):12-9).

Endometrial carcinoma (EC) comprises at least two types of cancer: endometrioid carcinomas (EECs) are estrogen-related tumours, which are frequently euploid and have a good prognosis. Nonendometrioid carcinomas (NEECs; serous and clear cell forms) are not estrogen related, are frequently aneuploid, and are clinically aggressive. It has also been found that Aurora was amplified in 55.5% of NEECs but not in any EECs (P <or= 0.001) (Moreno-Bueno G, Sanchez-Estevez C, Cassia R, Rodriguez-Perales S, Diaz-Uriarte R, Dominguez O, Hardisson D, Andujar M,

Prat J, Matias-Guiu X, Cigudosa JC, Palacios J. Cancer Res. 2003 Sep 15;63(18):5697-702).

Reichardt et al {Oncol Rep. 2003 Sep-Oct;10(5):1275-9) _. have reported that quantitative DNA analysis by PCR to search for Aurora amplification in gliomas revealed that five out of 16 tumours (31%) of different WHO grade (Ix grade II, Ix grade III, 3x grade IV) showed DNA amplification of the Aurora 2 gene. It was hypothesized that amplification of the Aurora 2 gene may be a non-random genetic alteration in human gliomas playing a role in the genetic pathways of tumourigenesis.

Results by Hamada et al {Br. J. Haematol. 2003 May;121(3):439-47) also suggest that Aurora 2 is an effective candidate to indicate not only disease activity but also tumourigenesis of non-Hodgkin's lymphoma. Retardation of tumour cell growth resulting from the restriction of this gene's functions could be a therapeutic approach for non-Hodgkin's lymphoma.

In a study by Gritsko et al {Clin Cancer Res. 2003 Apr; 9(4) : 1420-6)), the kinase activity and protein levels of Aurora A were examined in 92 patients with primary ovarian tumours. In vitro kinase analyses revealed elevated Aurora A kinase activity in 44 cases (48%). Increased Aurora A protein levels were detected in 52 (57%) specimens. High protein levels of Aurora A correlated well with elevated kinase activity.

Results obtained by Li et al {Clin. Cancer Res. 2003 Mar; 9(3):991-7) showed that the Aurora A gene is overexpressed in pancreatic tumours and carcinoma cell lines and suggest that overexpression of Aurora A may play a role in pancreatic carcinogenesis.

Similarly, it has been shown that Aurora A gene amplification and associated increased expression of the mitotic kinase it encodes are associated with aneuploidy and aggressive clinical behaviour in human bladder cancer. (J. Natl. Cancer Inst. 2002 Sep 4; 94(17):1320-9).

Investigation by several groups (Dutertre S, Prigent C.,Aurora-A overexpression leads to override of the microtubule-kinetochore attachment checkpoint; MoI. Interv. 2003 May; 3(3): 127-30 and Anand S, Penrhyn-Lowe S, Venkitaraman AR., Aurora-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol, Cancer Cell. 2003 Jan;3(l):51-62) suggests that overexpression of Aurora kinase activity is associated with resistance to some current cancer therapies. For example overexpression of Aurora A in mouse embryo fibroblasts can reduce the sensitivity of these cells to the cytotoxic effects of taxane derivatives. Therefore Aurora kinase inhibitors may find particular use in patients who have developed reistance to existing therapies.

On the basis of work carried out to date, it is envisaged that inhibition of Aurora kinases, particularly Aurora kinase A and Aurora kinase B, will prove an effective means of arresting tumour development.

Harrington et al (Nat Med. 2004 Mar; 10(3):262-7) have demonstrated that an inhibitor of the Aurora kinases suppresses tumour growth and induces tumour regression in vivo. In the study, the Aurora kinase inhibitor blocked cancer cell proliferation, and also triggered cell death in a range of cancer cell lines including leukaemic, colorectal and breast cell lines. In addition, it has shown potential for the treatment of leukemia by inducing apoptosis in leukemia cells. VX-680 potently killed treatment-refractory primary Acute Myelogenous Leukemia (AML) cells from patients (Andrews, Oncogene, 2005, 24, 5005-5015).

Cancers which may be particularly amenable to Aurora inhibitors include breast, bladder, colorectal, pancreatic, ovarian, non-Hodgkin's lymphoma, gliomas and nonendometrioid endometrial carcinomas. Leukemias particularly amenable to Aurora inhibitors include Acute Myelogenous Leukemia (AML), chronic myelogenous leukaemia (CML), B-cell lymphoma (Mantle cell), and Acute Lymphoblastic Leukemia (ALL).

Glycogen Synthase Kinase

Glycogen Synthase Kinase-3 (GSK3) is a serine-threonine kinase that occurs as two ubiquitously expressed isoforms in humans (GSK3α & beta GSK3β). GSK3 has been implicated as having roles in embryonic development, protein synthesis, cell proliferation, cell differentiation, microtubule dynamics, cell motility and cellular apoptosis. As such GSK3 has been implicated in the progression of disease states such as diabetes, cancer, Alzheimer's disease, stroke, epilepsy, motor neuron disease and/or head trauma. Phylogenetically GSK3 is most closely related to the cyclin dependent kinases (CDKs).

The consensus peptide substrate sequence recognised by GSK3 is (Ser/Thr)-X-X- X-(pSer/pThr), where X is any amino acid (at positions (n+1), (n+2), (n+3)) and pSer and pThr are phospho-serine and phospho-threonine respectively (n+4). GSK3 phosphorylates the first serine, or threonine, at position (n). Phospho-serine, or phospho-threonine, at the (n+4) position appear necessary for priming GSK3 to give maximal substrate turnover. Phosphorylation of GSK3α at Ser21, or GSK3β at Ser9, leads to inhibition of GSK3. Mutagenesis and peptide competition studies have led to the model that the phosphorylated N-terminus of GSK3 is able to compete with phospho-peptide substrate (S/TXXXpS/pT) via an autoinhibitory mechanism. There are also data suggesting that GSK3α and GSKβ may be subtly regulated by phosphorylation of tyrosines 279 and 216 respectively. Mutation of these residues to a Phe caused a reduction in in vivo kinase activity. The X-ray crystallographic structure of GSK3β has helped to shed light on all aspects of GSK3 activation and regulation.

GSK3 forms part of the mammalian insulin response pathway and is able to phosphorylate, and thereby inactivate, glycogen synthase. Upregulation of glycogen synthase activity, and thereby glycogen synthesis, through inhibition of GSK3, has thus been considered a potential means of combating type II, or non- insulin-dependent diabetes mellitus (NIDDM): a condition in which body tissues become resistant to insulin stimulation. The cellular insulin response in liver, adipose, or muscle tissues, is triggered by insulin binding to an extracellular insulin receptor. This causes the phosphorylation, and subsequent recruitment to the

plasma membrane, of the insulin receptor substrate (IRS) proteins. Further phosphorylation of the IRS proteins initiates recruitment of phosphoinositide-3 kinase (PBK) to the plasma membrane where it is able to liberate the second messenger phosphatidylinosityl 3,4,5-trisphosρhate (PIP3). This facilitates co- localisation of 3-phosphoinositide-dedependent protein kinase 1 (PDKl) and protein kinase B (PKB or Akt) to the membrane, where PDKl activates PKB. PKB is able to phosphorylate, and thereby inhibit, GSK3α and/or GSKβ through phosphorylation of Ser9, or ser21, respectively. The inhibition of GSK3 then triggers upregulation of glycogen synthase activity. Therapeutic agents able to inhibit GSK3 may thus be able to induce cellular responses akin to those seen on insulin stimulation. A further in vivo substrate of GSK3 is the eukaryotic protein synthesis initiation factor 2B (eIF2B). eIF2B is inactivated via phosphorylation and is thus able to suppress protein biosynthesis. Inhibition of GSK3, e.g. by inactivation of the "mammalian target of rapamycin" protein (mTOR), can thus upregulate protein biosynthesis. Finally there is some evidence for regulation of GSK3 activity via the mitogen activated protein kinase (MAPK) pathway through phosphorylation of GSK3 by kinases such as mitogen activated protein kinase activated protein kinase 1 (MAPKAP-Kl or RSK). These data suggest that GSK3 activity may be modulated by mitogenic, insulin and/or amino acid stimulii.

It has also been shown that GSK3β is a key component in the vertebrate Wnt signalling pathway. This biochemical pathway has been shown to be critical for normal embryonic development and regulates cell proliferation in normal tissues. GSK3 becomes inhibited in response to Wnt stimulii. This can lead to the de- phosphorylation of GSK3 substrates such as Axin, the adenomatous polyposis coli (APC) gene product and β-catenin. Aberrant regulation of the Wnt pathway has been associated with many cancers. Mutations in APC, and/or β-catenin, are common in colorectal cancer and other tumours, β-catenin has also been shown to be of importance in cell adhesion. Thus GSK3 may also modulate cellular adhesion processes to some degree. Apart from the biochemical pathways already described there are also data implicating GSK3 in the regulation of cell division via phosphorylation of cyclin-Dl, in the phosphorylation of transcription factors such

as c-Jun, CCAAT/enhancer binding protein α (C/EBPα), c-Myc and/or other substrates such as Nuclear Factor of Activated T-cells (NFATc), Heat Shock Factor-1 (HSF-I) and the c-AMP response element binding protein (CREB). GSK3 also appears to play a role, albeit tissue specific, in regulating cellular apoptosis. The role of GSK3 in modulating cellular apoptosis, via a pro-apoptotic mechanism, may be of particular relevance to medical conditions in which neuronal apoptosis can occur. Examples of these are head trauma, stroke, epilepsy, Alzheimer's and motor neuron diseases, progressive supranuclear palsy, corticobasal degeneration, and Pick's disease. In vitro it has been shown that GSK3 is able to hyper- phosphorylate the microtubule associated protein Tau. Hyperphosphorylation of Tau disrupts its normal binding to microtubules and may also lead to the formation of intra-cellular Tau filaments. It is believed that the progressive accumulation of these filaments leads to eventual neuronal dysfunction and degeneration. Inhibition of Tau phosphorylation, through inhibition of GSK3, may thus provide a means of limiting and/or preventing neurodegenerative effects.

Prior Art

WO 02/34721 from Du Pont discloses a class of indeno [l,2-c]pyrazol-4-ones as inhibitors of cyclin dependent kinases.

WO 01/81348 from Bristol Myers Squibb describes the use of 5-thio-, sulfmyl- and sulfonylpyrazolo [3, 4-b] -pyridines as cyclin dependent kinase inhibitors.

WO 00/62778 also from Bristol Myers Squibb discloses a class of protein tyrosine kinase inhibitors.

WO 01 /72745 Al from Cyclacel describes 2-substituted 4-heteroaryl-pyrimidines and their preparation, pharmaceutical compositions containing them and their use as inhibitors of cyclin-dependant kinases (cdks) and hence their use in the treatment of proliferative disorders such as cancer, leukaemia, psoriasis and the like.

WO 99/21845 from Agouron describes 4-aminothiazole derivatives for inhibiting cyclin-dependent kinases (cdks), such as CDKl, CDK2, CDK4, and CDK6. The

invention is also directed to the therapeutic or prophylactic use of pharmaceutical compositions containing such compounds and to methods of treating malignancies and other disorders by administering effective amounts of such compounds.

WO 01/53274 from Agouron discloses as CDK kinase inhibitors a class of compounds which can comprise an amide-substituted benzene ring linked to an N- containing heterocyclic group. Although indazole compounds are not mentioned genetically, one of the exemplified compounds comprises an indazole 3-carboxylic acid anilide moiety linked via a methylsulfanyl group to a pyrazolopyrimidine.

WO 01/98290 (Pharmacia & Upjohn) discloses a class of 3-aminocarbonyl-2- carboxamido thiophene derivatives as protein kinase inhibitors. The compounds are stated to have multiple protein kinase activity.

WO 01/53268 and WO 01/02369 from Agouron disclose compounds that mediate or inhibit cell proliferation through the inhibition of protein kinases such as cyclin dependent kinase or tyrosine kinase. The Agouron compounds have an aryl or heteroaryl ring attached directly or though a CH=CH or CH=N group to the 3- position of an indazole ring.

WO 00/39108 and WO 02/00651 (both to Du Pont Pharmaceuticals) describe broad classes of heterocyclic compounds that are inhibitors of trypsin-like serine protease enzymes, especially factor Xa and thrombin. The compounds are stated to be useful as anticoagulants or for the prevention of thromboembolic disorders.

Heterocyclic compounds that have activity against factor Xa are also disclosed in WO 01/1978 Cor Therapeutics) and US 2002/0091116 (Zhu et al).

WO 03/035065 (Aventis) discloses a broad class of benzimidazole derivatives as protein kinase inhibitors but does not disclose activity against CDK kinases or GSK kinases.

WO 97/36585 and US 5,874,452 (both to Merck) disclose biheteroaryl compounds that are inhibitors of farnesyl transferase.

WO 03/037274 (Icagen) discloses pyrazole amides as inhibitors of sodium channels.

WO 00/43384 (Boehringer Ingelheim) discloses aryl and heteroaryl ureas as antiinflammatory agents.

WO 00/07996 (Chiron Corporation) discloses pyrazole compounds for use as oestrogen receptor modulators which may be useful in, for example, the treatment of breast and endometrial cancers.

WO 2004/000318 (Cellular Genomics) discloses amino-subsituted moncyclic compounds as kinase modulators that may be useful in the treatment of cancers.

WO 03/062392 (Ceretek LLC) discloses aryl imidazole amides as EDG receptor modulators that may be useful in the treatment of cancers.

WO 01/68585 (Fujisawa) discloses a class of amides for use as 5-HT anatagonists.

WO 97/40017 (Novo Nordisk) discloses a broad class of heterocyclic compounds for use as protein tyrosine phosphatase modulators.

Summary of the Invention

The invention provides compounds that have cyclin dependent kinase inhibiting or modulating activity and/or glycogen synthase kinase-3 (GSK3) inhibiting or modulating activity, and/or Aurora kinase inhibiting or modulating activity, and which it is envisaged will be useful in preventing or treating disease states or conditions mediated by the kinases.

Thus, for example, it is envisaged that the compounds of the invention will be useful in alleviating or reducing the incidence of cancer.

In a first aspect, the invention provides a compound of the formula (I):

or a salt, tautomer, N-oxide or solvate thereof; wherein

A is selected from a bond, CH ₂ and CH(CN); R ¹ is selected from:

(i) a cycloalkyl group of 3 to 6 ring members optionally substituted by one or more substituents selected from methyl, ethyl, hydroxy, methoxy, ethoxy, fluorine, amino and cyano;

(ii) a phenyl group optionally substituted by up to three substituents selected from methyl, ethyl, fluorine, chlorine, methoxy, ethoxy and methylsulphonyl, but excluding 2,6-difluorophenyl, 2-fluoro-6-methoxy and 5-chloro-2-methoxyphenyl; (iii) a monocyclic heterocyclic group selected from furyl and isoxazolyl, the heterocyclic group being optionally substituted by one or two groups selected from methyl, ethyl, and a group CH ₂ R ² where R ² is a five or six membered saturated heterocyclic ring containing one or two heteroatom ring members selected from O and N, the heterocyclic ring being optionally substituted by one or two methyl groups; and

(iv) a bicyclic heterocyclic group selected from 2,3-dihydrobenzofuranyl and benzo[c] isoxazolyl, the bicyclic group being optionally substituted by one or two substituents selected from methyl, ethyl, hydroxy, methoxy, ethoxy, fluorine, amino, cyano and chlorine, the bicyclic heterocyclic group being other than a 2,2-dimethyl-2,3-dihydrobenzofuran-7-yl group.

In one embodiment, R ¹ is an optionally substituted cycloalkyl group of 3 to 6 ring members. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups. In one sub-group of compounds, R ¹ is an optionally

substituted cycloalkyl group of 3 to 5 ring members, for example 3, or 4, or 5 ring members). In a particular sub-group of compounds, R ¹ can be an optionally substituted cyclopropyl group. More particularly, R ¹ can be an unsubstituted cyclopropyl group.

When R ¹ is a cycloalkyl group and in particular a cyclopropyl group, the moiety A can be a bond, or a group selected from CH ₂ and CH(CN). In one particular subgroup of compounds, A is selected from CH ₂ and CH(CN).

In another embodiment, R ¹ can be a phenyl group optionally substituted by up to three substituents selected from methyl, ethyl, fluorine, chlorine, methoxy, ethoxy and methylsulphonyl, but excluding 2,6-difluorophenyl, 2-fluoro-6-methoxy and 5- chloro-2-methoxyphenyl.

For example, the phenyl group may be substituted by one, two or three substituents selected from chlorine, fluorine, methoxy, ethoxy and methylsulphonyl.

More particularly, the phenyl group may be selected from 2-ethoxyphenyl, 2- methoxy-5-methylsulphonylphenyl, 2-fluoro-6-chorophenyl, 2,4,6-trifluorophenyl and 5-fiuoro-2-methoxyphenyl.

In one sub-group of compounds, the phenyl group is selected from 2-ethoxyphenyl, 2-methoxy-5-methylsulphonylphenyl and 5-fluoro-2-methoxyphenyl.

In another general embodiment, R ¹ is a monocyclic heterocyclic group selected from furyl and isoxazolyl, the heterocyclic group being optionally substituted by one or two groups selected from methyl, ethyl, and a group CH ₂ R ² where R ² is a five or six membered saturated heterocyclic ring containing one or two heteroatom ring members selected from O and N, the heterocyclic ring being optionally substituted by one or two methyl groups.

Where R ² is a five or six membered saturated heterocyclic ring, it can be, for example, selected from morpholine, piperidine (e.g. 1-piperidinyl, 2-piperidinyl 3- piperidinyl and 4-piperidinyl), N-methyl piperidine, pyrrolidine (e.g. 1-pyrrolidinyl,

2-pyrrolidinyl and 3-pyrrolidinyl), azetidine, pyran (2H-pyran or 4H-pyran), dioxane, tetrahydropyran (e.g. 4-tetrahydro pyranyl), piperazine, and N-methyl piperazine.

In one sub-group of compounds, R ¹ is an isoxazole group optionally substituted by one or more methyl groups.

In another sub-group of compounds, R ¹ is a furyl group optionally substituted by one or two groups selected from methyl, ethyl, and a group CH ₂ R ² where R ² is a five or six membered saturated heterocyclic ring containing one or two heteroatom ring members selected from O and N, the heterocyclic ring being optionally substituted by one or two methyl groups. Particular compounds are those in which the furyl group is an optionally substituted 2-furyl group. The furyl group is preferably other than a 3-methyl-2-furyl, 5 -(pyrrolidine- l-ylmethyl)-2-furyl or 5-(4- morpholinylmethyl)-2-furyl group.

In a further general embodiment, R ¹ is a bicyclic heterocyclic group selected from 2,3-dihydrobenzofuranyl and benzo[c]isoxazolyl optionally substituted by one or two substituents selected from methyl, ethyl, methoxy, ethoxy, fluorine and chlorine, the bicyclic heterocyclic group being other than a 2,2-dimethyl-2,3- dihydrobenzofuran-7-yl group.

In one sub-group of compounds, R ¹ is a 2,3-dihydrobenzofuranyl group optionally substituted by one or two substituents selected from methyl, ethyl, methoxy, ethoxy, fluorine and chlorine, but excluding a 2,2-dimethyl-2,3-dihydrobenzofuran- 7-yl group. The 2,3-dihydrobenzofuranyl group may be, for example, an unsubstituted 2,3-dihydrobenzofuranyl group, such as a 2,3-dihydrobenzofuran-6-yl group or 2,3-dihydrobenzofuran-7-yl group.

In another sub-group of compounds, R ¹ is a benzo[c]isoxazolyl group optionally substituted by one or two substituents selected from methyl, ethyl, methoxy, ethoxy, fluorine and chlorine. More particularly, the benzo[c]isoxazolyl group may be unsubstituted.

The moiety A is selected from a bond, CH ₂ and CH(CN).

In one general embodiment, the moiety A is a bond.

In another general embodiment, the moiety A is CH ₂ .

In a further general embodiment, the moiety A is CH(CN).

In one sub-group of compounds of the formula (I), A is selected from a bond and a CH ₂ group.

In another sub-group of compounds of the formula (I), A is selected from CH ₂ and CH(CN).

The various functional groups and substituents making up the compounds of the formula (I) are typically chosen such that the molecular weight of the compound of the formula (I) does not exceed 1000. More usually, the molecular weight of the compound will be less than 750, for example less than 700, or less than 650, or less than 600, or less than 550. More preferably, the molecular weight is less than 525 and, for example, is 500 or less.

Particular compounds of the formula (I) are:

5-methyl-4-morpholin-4-ylmethyl-furan-2-carboxylic acid [3-(5-methyl-4- trifluoromethy 1- 1 H-imidazol-2-yl)- 1 H-pyrazol-4-yl] amide ;

2-cyclopropyl-N-[3-(5-methyl-4-trifluoromethyl -lH-imidazol-2-yl)-lH-pyrazol-4- yl]-acetamide; N-[3-(5-methyl-4-trifluoromethyl -lH-imidazol-2-yl)-lH-pyrazol-4-yl]-5-fluoro-2- methoxy-benzamide;

2-chloro-6-fluoro-N-[3-(5-methyl-4-trifluoromethyl-lH-imi dazol-2-yl)-lH-pyrazol- 4-yl]-benzamide;

S-methyl-isoxazole-S-carboxylic acid [3-(5-methyl-4-trifluoromethyl-lH-imidazol- 2-yl)-l H-pyrazol-4-yl] -amide;

2,4,6-trifluoro-N-[3-(5-methyl-4-trifluoromethyl-lH-imidazol -2-yl)-lH-pyrazol-4- yl]-benzamide;

2 ₅ 3-dihydro-benzofuran-7-carboxylic acid [3-(5-methyl-4-trifluoiOmethyl-lH- imidazol-2-yl)-lH-pyrazol-4-yl]-amide; 2-ethoxy-N- [3 -(5 -methyl-4-trifluoromethyl- 1 H-imidazol-2-yl)- 1 H-pyrazol -4-yl]-benzamide; furan-2-carboxylic acid [3 -(5 -methyl-4-trifluoromethyl- 1 H-imidazol-2-yl)- 1 H- pyrazol-4-yl]-amide; benzo [c] isoxazole-3 -carboxylic acid [3 -(5 -methyl-4-trifluoromethyl- 1 H-imidazol- 2-yl)-lH-pyrazol-4-yl] -amide;

2,3-dihydro-benzofuran-6-carboxylic acid [3-(5-methyl-4-trifluoromethyl-lH- imidazol-2-yl)- 1 H-pyrazol-4-yl] -amide;

2-cyano-2-cyclopropyl-N- [3 -(5 -methyl-4-trifluoromethyl- 1 H-imidazol-2-yl)- 1 H- pyrazol-4-yl]-acetamide; and 5 -methanesulphonyl-2-methoxy-N- [3 -(5 -methyl-4-trifluoromethyl- 1 H-imidazol-2- yl)- 1 H-pyrazol-4-yl] -benzamide ; and salts, N-oxides, tautomers and solvates thereof.

In further aspects, the invention provides inter alia:

• The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of a disease state or condition mediated by a cyclin dependent kinase or glycogen synthase kinase-3.

• A method for the prophylaxis or treatment of a disease state or condition mediated by a cyclin dependent kinase or glycogen synthase kinase-3, which method comprises administering to a subject in need thereof a compound of the formula (I) as defined herein.

• A method for alleviating or reducing the incidence of a disease state or condition mediated by a cyclin dependent kinase or glycogen synthase kinase-3, which method comprises administering to a subject in need thereof a compound of the formula (I) as defined herein.

• A method for treating a disease or condition comprising or arising from abnormal cell growth in a mammal, which method comprises administering to the mammal a compound of the formula (I) as defined herein in an amount effective in inhibiting abnormal cell growth.

• A method for alleviating or reducing the incidence of a disease or condition comprising or arising from abnormal cell growth in a mammal, which method comprises administering to the mammal a compound of the formula (I) as defined herein in an amount effective in inhibiting abnormal cell growth.

• A method for treating a disease or condition comprising or arising from abnormal cell growth in a mammal, the method comprising administering to the mammal a compound of the formula (I) as defined herein in an amount effective to inhibit a cdk kinase (such as cdkl or cdk2) or glycogen synthase kinase-3 activity.

• A method for alleviating or reducing the incidence of a disease or condition comprising or arising from abnormal cell growth in a mammal, the method comprising administering to the mammal a compound of the formula (I) as defined herein in an amount effective to inhibit a cdk kinase (such as cdkl or cdk2) or glycogen synthase kinase-3 activity.

• A method of inhibiting a cyclin dependent kinase or glycogen synthase kinase-3, which method comprises contacting the kinase with a kinase- inhibiting compound of the formula (I) as defined herein.

• A method of modulating a cellular process (for example cell division) by inhibiting the activity of a cyclin dependent kinase or glycogen synthase kinase-3 using a compound of the formula (I) as defined herein.

• The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for prophylaxis or treatment of a disease or condition characterised by up-regulation of an Aurora kinase (e.g. Aurora A kinase or Aurora B kinase).

• The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of a cancer, the cancer being one which is characterised by up-regulation of an Aurora kinase (e.g. Aurora A kinase or Aurora B kinase).

• The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of cancer in a patient selected from a sub-population possessing the Ile31 variant of the Aurora A gene.

• The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of cancer in a patient who has been diagnosed as forming part of a sub-population possessing the Ile31 variant of the Aurora A gene.

• A method for the prophylaxis or treatment of a disease or condition characterised by up-regulation of an Aurora kinase (e.g. Aurora A kinase or Aurora B kinase), the method comprising administering a compound of the formula (I) as defined herein.

• A method for alleviating or reducing the incidence of a disease or condition characterised by up-regulation of an Aurora kinase (e.g. Aurora A kinase or

Aurora B kinase), the method comprising administering a compound of the formula (I) as defined herein.

• A method for the prophylaxis or treatment of (or alleviating or reducing the incidence of) cancer in a patient suffering from or suspected of suffering from cancer; which method comprises (i) subjecting a patient to a diagnostic test to determine whether the patient possesses the IleSl variant of the Aurora A gene; and (ii) where the patient does possess the said variant, thereafter administering to the patient a compound of the formula (I) as defined herein having Aurora kinase inhibiting activity.

• A method for the prophylaxis or treatment of (or alleviating or reducing the incidence of) a disease state or condition characterised by up-regulation of an Aurora kinase (e.g. Aurora A kinase or Aurora B kinase); which method comprises (i) subjecting a patient to a diagnostic test to detect a marker characteristic of up-regulation of the Aurora kinase and (ii) where the diagnostic test is indicative of up-regulation of Aurora kinase, thereafter administering to the patient a compound of the formula (I) as defined herein having Aurora kinase inhibiting activity.

• The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of a disease state as described herein.

• A compound of the formula (I) as defined herein for use in the prophylaxis or treatment of a disease state as described herein.

• A pharmaceutical composition comprising a compound of the formula (I) as defined herein and a pharmaceutically acceptable carrier.

• A pharmaceutical composition for administration in an aqueous solution form, the pharmaceutical composition comprising a compound of the formula (I), (II) or (III) or any sub-groups or examples thereof as defined herein in the form of a salt having a solubility in water of greater than 25 mg/ml, typically greater than 50 mg/ml and preferably greater than 100 mg/ml.

• A pharmaceutical composition for parenteral administration comprising a compound of the formula (I) as defined herein and a pharmaceutically acceptable carrier suitable for parenteral administration.

• A pharmaceutical composition for administration by intravenous, intramuscular, intraperitoneal or subcutaneous administration injection or for direct delivery into a target organ or tissue by injection or infusion, the composition comprising a compound of the formula (I) as defined herein and a pharmaceutically acceptable carrier suitable for parenteral administration.

• A pharmaceutical composition comprising a compound of the formula (I) as defined herein in the form of a sterile solution for injection.

• A compound of the formula (I) as defined for use in medicine.

• A compound as defined herein for any of the uses and methods set forth above, and as described elsewhere herein.

• A compound of formula (I) or a salt (e.g. an acid addition salt), solvate, tautomer or N-oxide thereof for use in the treatment of B-cell lymphoma.

• A compound of formula (I) or a salt (e.g. an acid addition salt), solvate, tautomer or N-oxide thereof for use in the treatment of chronic lymphocytic leukaemia.

• A compound of formula (I) or a salt (e.g. an acid addition salt), solvate, tautomer or N-oxide thereof for use in the treatment of diffuse large B cell lymphoma.

• A method of treatment of B-cell lymphoma, diffuse large B cell lymphoma or chronic lymphocytic leukaemia by administering to a patient in need of such treatment a compound of formula (I) or a salt (e.g. an acid addition salt), solvate, tautomer or N-oxide thereof.

• A compound of formula (I) or a salt (e.g. an acid addition salt), solvate, tautomer or N-oxide thereof for use in the treatment of leukaemia in particular relapsed or refractory acute myelogenous leukemia, myelodysplastic syndrome, acute lymphocytic leukemia and chronic myelogenous leukemia.

For the avoidance of doubt, it is noted that each reference to the Formula (I) in this application is to be understood as being a reference not only to Formula (I) per se but also a reference to all embodiments, preferences, sub-groups and examples of compounds within Formula (I) unless the context requires otherwise.

Salts, Solvates, Tautomers, Isomers, N-Oxides, Esters, Prodrugs and Isotopes

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms thereof, for example, as discussed below.

Many compounds of the formula (I) can exist in the form of salts, for example acid addition salts or, in certain cases salts of organic and inorganic bases such as carboxylate, sulphonate and phosphate salts. All such salts are within the scope of this invention, and references to compounds of the formula (I) include the salt forms of the compounds.

The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

Acid addition salts may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include salts formed with an acid selected

from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulphonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(lS)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane- 1,2- disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, (+)-L-lactic, (±)-DL-lactic, lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulphonic, naphthalene-2- sulphonic, naphthalene- 1,5-disulphonic, l-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L- tartaric, thiocyanic, p-toluenesulphonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

The acid addition salts salts may also be selected from aspartic (e.g. D-aspartic), carbonic, dodecanoate, isobutyric, laurylsulphonic, mucic, naphthalenesulphonic (e.g. naphthalene-2-sulphonic), toluenesulphonic (e.g. ^-toluenesulphonic), and xinafoic acids.

One particular group of salts consists of salts formed from hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulphonic, toluenesulphonic, methanesulphonic, ethanesulphonic, naphthalenesulphonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids.

One sub-group of salts consists of salts formed from hydrochloric, acetic, adipic, L- aspartic and DL-lactic acids.

Another sub-group of salts consists of the acetate, mesylate, ethanesulphonate, DL- lactate, adipate, D-glucuronate, D-gluconate and hydrochloride salts.

Salts such as acid addition salts have a number of advantages over the corresponding free base. For example, the salts will enjoy one or more of the following advantages over the free base in that they will:

• be more soluble and hence will be better for i.v. administration (e.g. by infusion) and will have improved pharmacokinetics;

• have better stability (e.g. improved shelf life);

• have better thermal stability;

• be less basic and therefore better for i.v. administration;

• have advantages for production; • have improved metabolic properties; and

• exhibit less clinical variation between patients.

Preferred salts for use in the preparation of liquid (e.g. aqueous) compositions of the compounds of formula (I) described herein are salts having a solubility in a given liquid carrier (e.g. water) of greater than 25 mg/ml of the liquid carrier (e.g. water), more typically greater than 50 mg/ml and preferably greater than 100 mg/ml.

In another embodiment preferred salts for use in the preparation of liquid (e.g. aqueous) compositions the compounds of formulae (I) as described herein are salts having a solubility in a given liquid carrier (e.g. water) greater than 1 mg/ml, typically greater than 5 mg/ml of the liquid carrier (e.g. water), more typically greater than 15 mg/ml, more typically greater than 20 mg/ml and preferably greater than 25 mg/ml.

In another embodiment the preferred acid addition salts are mesylate, ethanesulphonate, D- or L-lactate, and hydrochloride salts. In one particular embodiment the acid addition salt is the lactate salt, in particular L-lactate or D- lactate, preferably L-lactate.

In one embodiment of the invention, there is provided a pharmaceutical composition comprising an aqueous solution containing a compound of the formula

(I) as described herein in the form of a salt in a concentration of greater than 25 mg/ml, typically greater than 50 mg/ml and preferably greater than 100 mg/ml.

In another embodiment of the invention, there is provided a pharmaceutical composition comprising an aqueous solution containing a compound of the formula (I) as described herein in the form of a salt in a concentration of greater than 1 mg/ml, typically greater than 5 mg/ml of the liquid carrier (e.g. water), more typically greater than 15 mg/ml, more typically greater than 20 mg/ml and preferably greater than 25 mg/ml.

If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO ^" ), then a salt may be formed with a suitable cation.

Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH ₄ ⁺ ) and substituted ammonium ions (e.g., NH ₃ R ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

Where the compounds of the formula (I) contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of formula (I).

The salt forms of the compounds of the invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et ah, 1977, "Pharmaceutically Acceptable Salts," J. Pharm. ScI, Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically

acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.

Compounds of the formula (I) containing an amine function may also form N- oxides. A reference herein to a compound of the formula (I) that contains an amine function also includes the N-oxide.

Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N- oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen- containing heterocycle.

N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4 ^th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. 1977, 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.

Compounds of the formula (I) may exist in a number of different geometric isomeric, and tautomeric forms and references to compounds of the formula (I) include all such forms. For the avoidance of doubt, where a compound can exist in one of several geometric isomeric or tautomeric forms and only one is specifically described or shown, all others are nevertheless embraced by formula (I).

For example, in compounds of the formula (I), the imidazole group may take either of the following two tautomeric forms A and B. For simplicity, the general formula (I) illustrates form A but the formula is to be taken as embracing both tautomeric forms.

A B

The pyrazole ring may also exhibit tautomerism and can exist in the two tautomeric forms C and D below.

D

The general formula (I) illustrates form C but the formula is to be taken as embracing both form C and form D.

Other examples of tautomeric forms include, for example, keto-, enol-, and enolate- forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, and nitro/aci-nitro.

keto enol enolate

Where compounds of the formula (I) contain one or more chiral centres, and can exist in the form of two or more optical isomers, references to compounds of the formula (I) include all optical isomeric forms thereof (e.g. enantiomers, epimers and diastereoisomers), either as individual optical isomers, or mixtures (e.g. racemic mixtures) or two or more optical isomers, unless the context requires otherwise.

For example, the group A can include one or more chiral centres. Thus, when E and R ¹ are both attached to the same carbon atom on the linker group A, the said carbon atom is typically chiral and hence the compound of the formula (I) will exist as a pair of enantiomers (or more than one pair of enantiomers where more than one chiral centre is present in the compound).

The optical isomers may be characterised and identified by their optical activity (i.e. as + and - isomers, or d and / isomers) or they may be characterised in terms of their absolute stereochemistry using the "R and S" nomenclature developed by Cahn, Ingold and Prelog, see Advanced Organic Chemistry by Jerry March, 4 ^th Edition, John Wiley & Sons, New York, 1992, pages 109-114, and see also Cahn, Ingold & Prelog, Angew. Chem. Int. Ed. Engl, 1966, 5, 385-415.

Optical isomers can be separated by a number of techniques including chiral chromatography (chromatography on a chiral support) and such techniques are well known to the person skilled in the art.

As an alternative to chiral chromatography, optical isomers can be separated by forming diastereoisomeric salts with chiral acids such as (+)-tartaric acid, (-)- pyroglutamic acid, (-)-di-toluloyl-L-tartaric acid, (+)~mandelic acid, (-)-malic acid, and (-)-camphorsulphonic, separating the diastereoisomers by preferential crystallisation, and then dissociating the salts to give the individual enantiomer of the free base.

Where compounds of the formula (I) exist as two or more optical isomeric forms, one enantiomer in a pair of enantiomers may exhibit advantages over the other enantiomer, for example, in terms of biological activity. Thus, in certain circumstances, it may be desirable to use as a therapeutic agent only one of a pair of enantiomers, or only one of a plurality of diastereoisomers. Accordingly, the invention provides compositions containing a compound of the formula (I) having one or more chiral centres, wherein at least 55% (e.g. at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound of the formula (I) is present as a single optical isomer (e.g. enantiomer or diastereoisomer). In one general embodiment,

99% or more (e.g. substantially all) of the total amount of the compound of the formula (I) may be present as a single optical isomer (e.g. enantiomer or diastereoisomer).

The compounds of the invention include compounds with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes within its scope ¹ H, ² H (D), and ³ H (T). Similarly, references to carbon and oxygen include within their scope respectively ¹² C, ¹³ C and ¹⁴ C and ¹⁶ O and ¹⁸ O.

The isotopes may be radioactive or non-radioactive. In one embodiment of the invention, the compounds contain no radioactive isotopes. Such compounds are preferred for therapeutic use. In another embodiment, however, the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.

Esters such as carboxylic acid esters and acyloxy esters of the compounds of formula (I) bearing a carboxylic acid group or a hydroxyl group are also embraced by Formula (I). Examples of esters are compounds containing the group -C(=O)OR, wherein R is an ester substituent, for example, a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Particular examples of ester groups include, but are not limited to, -C(=O)OCH ₃ , -C(K))OCH ₂ CH ₃ , -C(=O)OC(CH ₃ ) ₃ , and -C(K ⁾ )OPh. Examples of acyloxy (reverse ester) groups are represented by -OC(=O)R, wherein R is an acyloxy substituent, for example, a C _1-7 alkyl group, a C _3-2O heterocyclyl group, or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Particular examples of acyloxy groups include, but are not limited to, -OC(=O)CH ₃ (acetoxy), -OC(=O)CH ₂ CH ₃ , -OC(=O)C(CH ₃ ) ₃ , -OC(K ⁾ )Ph, and -OC(K ⁾ )CH ₂ Ph.

Also encompassed by formula (I) are any polymorphic forms of the compounds, solvates (e.g. hydrates), complexes (e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals) of the compounds, and pro-drugs of the compounds. By "prodrugs" is meant for example any

compound that is converted in vivo into a biologically active compound of the formula (I).

For example, some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required.

Examples of such metabolically labile esters include those of the formula - C(=O)OR wherein R is:

C _1-7 alkyl

(e.g., -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu);

C _1-7 aminoalkyl

(e.g., aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4-morpholino)ethyl); and acyloxy-C _1-7 alkyl

(e.g., acyloxymethyl; acyloxyethyl; pivaloyloxymethyl; acetoxymethyl; 1-acetoxy ethyl;

1 -( 1 -methoxy- 1 -methyl)ethyl-carbonxyloxyethyl;

1 -(benzoyloxy)ethyl; isopropoxy-carbonyloxymethyl;

1 -isopropoxy-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl;

1 -cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl;

1-cyclohexyloxy-carbonyloxyethyl;

(4-tetrahydropyranyloxy) carbonyloxymethyl; l-(4-tetrahydropyranyloxy)carbonyloxyethyl;

(4-tetrahydropyranyl)carbonyloxymethyl; and 1 -(4-tetrahydropyranyl)carbonyloxyethyl).

Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in ADEPT, GDEPT, LIDEPT ₅ etc.). For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Biological Activity

The compounds of the formulae (I) and sub-groups thereof are inhibitors of cyclin dependent kinases. For example, compounds of the invention have activity against CDKl, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK7 kinases, and in particular cyclin dependent kinases selected from CDKl, CDK2, CDK3, CDK4, CDK5 and CDK6.

Preferred compounds are compounds that inhibit one or more CDK kinases selected from CDKl, CDK2, CDK4 and CDK5, for example CDKl and/or CDK2.

In addition, CDK4, CDK8 and/or CDK9 may be of interest.

Compounds of the invention also have activity against glycogen synthase kinase-3 (GSK-3).

Compounds of the invention also have activity against Aurora kinases. Preferred compounds of the invention are those having IC ₅₀ values of less than 0.1 μM.

Many of the compounds of the invention exhibit selectivity for the Aurora A kinase compared to CDKl and CDK2 and such compounds represent one preferred embodiment of the ivention. For example, many compounds of the invention have IC ₅₀ values against Aurora A that are between a tenth and a hundredth of the IC ₅ O against CDKl and CDK2.

As a consequence of their activity in modulating or inhibiting CDK and Aurora kinases and glycogen synthase kinase, they are expected to be useful in providing a means of arresting, or recovering control of, the cell cycle in abnormally dividing cells. It is therefore anticipated that the compounds will prove useful in treating or

preventing proliferative disorders such as cancers. It is also envisaged that the compounds of the invention will be useful in treating conditions such as viral infections, type II or non-insulin dependent diabetes mellitus, autoimmune diseases, head trauma, stroke, epilepsy, neurodegenerative diseases such as Alzheimer's, motor neurone disease, progressive supranuclear palsy, corticobasal degeneration and Pick's disease for example autoimmune diseases and neurodegenerative diseases.

One sub-group of disease states and conditions where it is envisaged that the compounds of the invention will be useful consists of viral infections, autoimmune diseases and neurodegenerative diseases.

CDKs play a role in the regulation of the cell cycle, apoptosis, transcription, differentiation and CNS function. Therefore, CDK inhibitors could be useful in the treatment of diseases in which there is a disorder of proliferation, apoptosis or differentiation such as cancer. In particular RB+ve tumours may be particularly sensitive to CDK inhibitors. RB-ve tumours may also be sensitive to CDK inhibitors.

Examples of cancers which may be inhibited 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, epidermis, 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 tumour 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 tumour of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma;

melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma.

The cancers may be cancers which are sensitive to inhibition of any one or more cyclin dependent kinases selected from CDKl, CDK2, CDK3, CDK4, CDK5 and CDK6, for example, one or more CDK kinases selected from CDKl , CDK2, CDK4 and CDK5, e.g. CDKl and/or CDK2.

Whether or not a particular cancer is one which is sensitive to inhibition by a cyclin dependent kinase or an aurora kinase may be determined by means of a cell growth assay as set out in Example 57 and 58 below or by a method as set out in the section headed "Methods of Diagnosis".

CDKs are also known to play a role in apoptosis, proliferation, differentiation and transcription and therefore CDK inhibitors could also be useful in the treatment of the following diseases other than cancer; viral infections, for example herpes virus, pox virus, Epstein-Barr virus, Sindbis virus, adenovirus, HIV, HPV, HCV and HCMV; prevention of AIDS development in HIV-infected individuals; chronic inflammatory diseases, for example systemic lupus erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus; cardiovascular diseases for example cardiac hypertrophy, restenosis, atherosclerosis; neurodegenerative disorders, for example Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotropic lateral sclerosis, retinitis pigmentosa, spinal muscular atropy and cerebellar degeneration; glomerulonephritis; myelodysplastic syndromes, ischemic injury associated myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, haematological diseases, for example, chronic anaemia and aplastic anaemia; degenerative diseases of the musculoskeletal system, for example, osteoporosis and arthritis, aspirin- sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain.

5 005109

39

It has also been discovered that some cyclin-dependent kinase inhibitors can be used in combination with other anticancer agents. For example, the cyclin- dependent kinase inhibitor flavopiridol has been used with other anticancer agents in combination therapy.

Thus, in the pharmaceutical compositions, uses or methods of this invention for treating a disease or condition comprising abnormal cell growth, the disease or condition comprising abnormal cell growth in one embodiment is a cancer.

One group of cancers includes human breast cancers (e.g. primary breast tumours, node-negative breast cancer, invasive duct adenocarcinomas of the breast, non- endometrioid breast cancers); and mantle cell lymphomas. In addition, other cancers are colorectal and endometrial cancers.

Another sub-set of cancers includes breast cancer, ovarian cancer, colon cancer, prostate cancer, oesophageal cancer, squamous cancer and non-small cell lung carcinomas.

In the case of compounds having activity against Aurora kinase, particular examples of cancers where it is envisaged that the Aurora kinase inhibiting compounds of the invnention will be useful include: human breast cancers (e.g. primary breast tumours, node-negative breast cancer, invasive duct adenocarcinomas of the breast, non-endometrioid breast cancers); ovarian cancers (e.g. primary ovarian tumours); pancreatic cancers; human bladder cancers; colorectal cancers (e.g. primary colorectal cancers); gastric tumours; renal cancers; cervical cancers: neuroblastomas;

melanomas; lymphomas; prostate cancers; leukemia; non-endometrioid endometrial carcinomas; gliomas; non-Hodgkin's lymphoma;

Cancers which may be particularly amenable to Aurora inhibitors include breast, bladder, colorectal, pancreatic, ovarian, non-Hodgkin's lymphoma, gliomas and nonendometrioid endometrial carcinomas.

A particular sub-set of cancers which may be particularly amenable to Aurora inhibitors consist of breast, ovarian, colon, liver, gastric and prostate cancers.

The activity of the compounds of the invention as inhibitors of cyclin dependent kinases, Aurora kinases and glycogen synthase kinase-3 can be measured using the assays set forth in the examples below and the level of activity exhibited by a given compound can be defined in terms of the IC ₅ 0 value. Preferred compounds of the present invention are compounds having an IC ₅₀ value of less than 1 micromolar, more preferably less than 0.1 micromolar.

Another subset of cancers that Aurora inhibitors may be particularly amenable to treat are hematological cancers, in particular leukemia. Therefore, in a further embodiment the compounds of formula (I) are used to treat hematological cancers, in particular leukemia. Particular leukemias are selected from Acute Myelogenous Leukemia (AML), chronic myelogenous leukaemia (CML), B-cell lymphoma (Mantle cell), and Acute Lymphoblastic Leukemia (ALL). In one embodiment the leukemias are selected from relapsed or refractory acute myelogenous leukemia, myelodysplastic syndrome, acute lymphocytic leukemia and chronic myelogenous leukemia.

One group of cancers includes human breast cancers (e.g. primary breast tumours, node-negative breast cancer, invasive duct adenocarcinomas of the breast, non- endometrioid breast cancers); and mantle cell lymphomas. In addition, other cancers are colorectal and endometrial cancers.

Another sub-set of cancers includes hematopoietic tumours of lymphoid lineage, for example leukemia, chronic lymphocytic leukaemia, mantle cell lymphoma and B- cell lymphoma (such as diffuse large B cell lymphoma).

One particular cancer is chronic lymphocytic leukaemia.

Another particular cancer is mantle cell lymphoma.

Another particular cancer is diffuse large B cell lymphoma.

It is further envisaged tha the compounds of the invention, and in particular those compounds having aurora kinase inhibitory activity, will be particularly useful in the treatment or prevention of cancers of a type associated with or characterised by the presence of elevated levels of aurora kinases, for example the cancers referred to in this context in the introductory section of this application.

Methods for the Preparation of Compounds of the Formula (D

Compounds of the formula (I) can be prepared in accordance with synthetic methods well known to the skilled person and as described herein.

In this section, unless stated, otherwise R ¹ and A are as herein defined.

One general route to compounds of the Formula (I) is illustrated in Scheme 1.

SCHEME 1

In Scheme 1, the starting material (X) is a protected form of 4-aminopyrazole-3- carboxylic acid methyl ester in which protecting group PG ¹ can be, for example a tetrahydropyranyl group and protecting group PG ² can be, for example, a tert- butoxycarbonyl (Boc) group.

The protected 4-aminopyrazole-3-carboxylic acid methyl ester (X) is converted to the aldehyde (XII) by a sequence of steps involving, firstly, the reduction of the ester to the alcohol (XI) and, secondly, the oxidation of the alcohol (XI) to the aldehyde (XII). Reduction of the ester (X) to the alcohol (XI) can be accomplished by a hydride reducing agent such as di-isobutylaluminium hydride (DIBAH). The reduction is typically carried out in a non-aqueous polar solvent such as tetrahydrofuran (THF) at a reduced temperature. Oxidation of the alcohol (XI) to the aldehyde (XII) can be achieved using an oxidising agent such as manganese dioxide in a polar solvent such as acetone.

The aldehyde (XII) can then be used to introduce an imidazole group by means of a ring-forming reaction with ammonia and the α,α-dibromoketone (XIII) to give the pyrazolylimidazole intermediate (XIV). The ring-forming reaction is typically carried out in a polar solvent such as an alcohol (e.g. methanol) at or near ambient temperature. Following formation of the imidazole ring, the protecting group PG ¹ at the pyrazole 1 -position and the amino-protecting group PG can be removed.

The conditions used to de-protect the two nitrogen atoms will depend on the precise nature of the protecting group but, in the case of a tetrahydropyranyl group and a Boc group, deprotection can be carried out using a mineral acid such as hydrochloric acid.

Following deprotection, the amine (XV) can be coupled with an acid of the formula R^-A-CO ₂ H or a reactive derivative or activated form thereof to form an amide of the formula (I).

Standard amide formation conditions may be used to form the amide (I). Thus, for example, the coupling reaction between the carboxylic acid and the amine (XV) can be carried out in the presence of a reagent of the type commonly used in the formation of peptide linkages. Examples of such reagents include 1,3- dicyclohexylcarbodiimide (DCC) (Sheehan et al, J. Amer. Chem Soc. 1955, 77, 1067), l-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (EDC) (Sheehan et al, J. Org. Chem., 1961, 26, 2525), uronium-based coupling agents such as O-(7- azabenzotriazol-l-y^-ΛζΛζiV^N'-tetramethyluronium hexafluorophosphate (HATU)

(L. A. Carpino, J. Amer. Chem. Soc, 1993, 115, 4397) and phosphonium-based coupling agents such as l-benzo-triazolyloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) (Castro et al, Tetrahedron Letters, 1990, 3_1, 205). Carbodiimide-based coupling agents are advantageously used in combination with 1-hydroxyazobenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) (Konig et al, Chem. Ber., 103, 708, 2024-2034). Preferred coupling reagents include EDC and DCC in combination with HOBt.

The coupling reaction is typically carried out in a non-aqueous, non-protic solvent such as acetonitrile, dioxan, dimethylsulphoxide (DMSO), dichloromethane, dimethylformamide (DMF) or N-methylpyrrolidine, or in an aqueous solvent optionally together with one or more miscible co-solvents. The reaction can be carried out at room temperature or, where the reactants are less reactive at an appropriately elevated temperature. The reaction may be carried out in the presence of a non-interfering base, for example a tertiary amine such as triethylamine or N,N- diisopropylethylamine.

As an alternative, a reactive derivative of the carboxylic acid, e.g. an anhydride or acid chloride, may be used. Reaction with a reactive derivative such an anhydride is typically accomplished by stirring the amine and anhydride at room temperature in the presence of a base such as pyridine.

The starting material for Scheme 1, the protected derivative (X) of 4- aminopyrazole-3 -carboxylic acid methyl ester, can be prepared by the sequence of steps shown in Scheme 2.

(XIX)

SCHEME 2

As shown in Scheme 2, 4-nitro-3-pyrazole carboxylic acid (XIX) is esterified by reaction with thionyl chloride to give the acid chloride intermediate followed by reaction with ethanol to form the ethyl ester (XX). Alternatively, the esterification can be carried out by reacting the alcohol and carboxylic acid in the presence of an acidic catalyst, one example of which is thionyl chloride. The reaction is typically carried out at room temperature using the esterifying alcohol (e.g. ethanol) as the solvent.

The pyrazole 1 -nitrogen atom is then protected by means of a suitable protecting group PG ¹ , for example a tetrahydropyranyl (THP) group or an optionally substituted benzyl group such as a pαrø-methoxybenzyl group. The THP group can be introduced by reaction with 3,4-dihydropyran in the presence of an acid such as p-toluene sulphonic acid in a non-aqueous solvent such as chloroform. The benzyl group (e.g. ;?αrα-methoxybenzyl group) can be introduced by reacting the nitro ester (XX) with the appropriate benzyl halide in the presence of a base such as sodium carbonate or potassium carbonate. The reaction is typically carried out in a polar solvent such as acetonitrile at ambient temperature.

The N-protected nitro ester (XXI) can be reduced to the corresponding amino compound (XXII) according to standard methods. The reduction may be effected, for example by catalytic hydrogenation in the presence of a catalyst such as palladium on carbon in a polar solvent such as ethanol or dimethylformamide at a temperature between about room temperature and about 55 ⁰ C. The 4-amino group in the compound (XXII) can then be protected by standard methods to give the protected compound of the formula (X). For example, a Boc protecting group can be introduced by reacting the amine (XXII) with tert-butyl carbonate in the presence of a non-interfering base such as triethylamine.

As an alternative to the route shown in Scheme 1 above, the aminopyrazole (XXII) (see Scheme 2 above) can be acylated by reaction with a compound of the formula R^-A-CO ₂ H under the amide-forming conditions described above to give a compound of the formula (XXIII).

The ester (XXIII) can then be subjected to reduction (e.g. with DIBAH) to the corresponding alcohol and subsequent oxidation with an oxidising agent (e.g. MnO ₂ ) to give an aldehyde of the formula (XXIV).

The aldehyde (XXIV) can then be cyclised by reaction with ammonia and the α,α- dibromoketone (XVI) under the conditions described above to give the N-protected compound (XV) which can be deprotected under standard conditions to yield a compound of the formula (I).

The carboxylic acids of the formula R -A-CO ₂ H used in the amide coupling step described in Scheme 1 above are commercially available or can be made by methods well known for the preparation of carboxylic acids.

In many of the reactions described above, it may be necessary to protect one or more groups to prevent reaction from taking place at an undesirable location on the molecule. Examples of protecting groups, and methods of protecting and deprotecting functional groups, can be found in Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).

A hydroxy group may be protected, for example, as an ether (-OR) or an ester (- OC(=O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (-OC(=O)CH ₃ , -OAc). An aldehyde or ketone group may be protected, for example, as an acetal (R-CH(OR) ₂ ) or ketal (R ₂ C(OR) ₂ ), respectively, in which the carbonyl group (>C=O) is converted to a diether (>C(0R) ₂ ), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid. An amine group may be protected, for example, as an amide (-NRC0-R) or a urethane (-NRC0-0R), for example, as: a methyl amide (-NHCO-CH ₃ ); a benzyloxy amide (-NHCO-OCH ₂ C ₆ H ₅ , -NH-Cbz); as a t-butoxy amide (-NHCO-OC(CH ₃ ) ₃ , -NH-Boc); a 2-biphenyl-2-ρropoxy amide (-NHCO-OC(CH ₃ ) ₂ C ₆ H ₄ C ₆ H ₅ , -NH- Bpoc), as a 9-fluorenylmethoxy amide (-NH-Fmoc), as a 6-nitroveratryloxy amide (-NH-Nvoc), as a 2-trimethylsilylethyloxy amide (-NH-Teoc), as a 2,2,2- trichloroethyloxy amide (-NH-Troc), as an allyloxy amide (-NH-Alloc), or as a 2(- phenylsulphonyl)ethyloxy amide (-NH-Psec). Other protecting groups for amines, such as cyclic amines and heterocyclic N-H groups, include toluenesulfonyl (tosyl) and methanesulfonyl (mesyl) groups and benzyl groups such as a. para- methoxybenzyl (PMB) group, or a tetrahydropyranyl (THP) group. A carboxylic acid group may be protected as an ester for example, as: an C _1-7 alkyl ester (e.g., a methyl ester; a t-butyl ester); a C _1-7 haloalkyl ester (e.g., a C _1-7 trihaloalkyl ester); a triC _1-7 alkylsilyl-C _1-7 alkyl ester; or a C _5-20 aryl-C _1-7 alkyl ester (e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide. A thiol group may be protected, for example, as a thioether (-SR), for example, as: a benzyl thioether; an acetamidomethyl ether (-S-CH ₂ NHC(=O)CH ₃ ).

Methods of Purification

The compounds may be isolated and purified by a number of methods well known to those skilled in the art and examples of such methods include chromatographic techniques such as column chromatography (e.g. flash chromatography) and HPLC. Preparative LC-MS is a standard and effective method used for the purification of

small organic molecules such as the compounds described herein. The methods for the liquid chromatography (LC) and mass spectrometry (MS) can be varied to provide better separation of the crude materials and improved detection of the samples by MS. Optimisation of the preparative gradient LC method will involve varying columns, volatile eluents and modifiers, and gradients. Methods are well known in the art for optimising preparative LC-MS methods and then using them to purify compounds. Such methods are described in Rosentreter U, Huber U.; Optimal fraction collecting in preparative LCMS; J Comb Chem.; 2004; 6(2), 159- 64 and Leister W, Strauss K ₃ Wisnoski D, Zhao Z, Lindsley C, Development of a custom high-throughput preparative liquid chromatography/mass spectrometer platform for the preparative purification and analytical analysis of compound libraries; J Comb Chem.; 2003; 5(3); 322-9.

One such system for purifying compounds via preparative LC-MS is described in the experimental section below although a person skilled in the art will appreciate that alternative systems and methods to those described could be used. In particular, normal phase preparative LC based methods might be used in place of the reverse phase methods described here. Most preparative LC-MS systems utilise reverse phase LC and volatile acidic modifiers, since the approach is very effective for the purification of small molecules and because the eluents are compatible with positive ion electrospray mass spectrometry. Employing other chromatographic solutions e.g. normal phase LC, alternatively buffered mobile phase, basic modifiers etc as outlined in the analytical methods described above could alternatively be used to purify the compounds.

Recrvstallisation

Methods of recrystallisation of compounds of formula (I) and salts thereof can be carried out by methods well known to the skilled person - see for example (P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Chapter 8, Publisher Wiley-VCH). Products obtained from an organic reaction are seldom pure when isolated directly from the reaction mixture. If the compound (or a salt

thereof) is solid, it may be purified and/or crystallized by recrystallisation from a suitable solvent. A good recrystallisation solvent should dissolve a moderate quantity of the substance to be purified at elevated temperatures but only a small quantity of the substance at lower temperature. It should dissolve impurities readily at low temperatures or not at all. Finally, the solvent should be readily removed from the purified product. This usually means that it has a relatively low boiling point and a person skilled in the art will know recrystallising solvents for a particular substance, or if that information is not available, test several solvents. To get a good yield of purified material, the minimum amount of hot solvent to dissolve all the impure material is used. In practice, 3-5% more solvent than necessary is used so the solution is not saturated. If the impure compound contains an impurity which is insoluble in the solvent it may then be removed by filtration and then allowing the solution to crystallise. In addition, if the impure compound contains traces of coloured material that are not native to the compound, it may be removed by adding a small amount of decolorizing charcoal to the hot solution, filtering it and then allowing it to crystallise. Usually crystallisation spontaneously occurs upon cooling the solution. If it is not, crystallisation may be induced by cooling the solution below room temperature or by adding a single crystal of pure material (a seed crystal). Recrystallisation can also be carried out and/or the yield optimized by the use of an anti-solvent. In this case, the compound is dissolved in a suitable solvent at elevated temperature, filtered and then an additional solvent in which the required compound has low solubility is added to aid crystallisation. The crystals are then typically isolated using vacuum filtration, washed and then dried, for example, in an oven or via desiccation.

Other examples of methods for crystallisation include crystallisation from a vapor, which includes an evaporation step for example in a sealed tube or an air stream, and crystallisation from melt (Crystallization Technology Handbook 2nd Edition, edited by A. Mersmann, 2001).

In particular the compound of formula (I) may subjected to recrystallisation (e.g. using 2-propanol or ethanol as the solvent) to increase the purity and to give a crystalline form.

Generally, the crystals obtained are analysed by an X-ray diffraction method such as X-ray powder diffraction (XRPD) or X-ray crystal diffraction.

Pharmaceutical Formulations

While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) comprising at least one active compound of the invention together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents; for example agents that reduce or alleviate some of the side effects associated with chemotherapy. Particular examples of such agents include anti-emetic agents and agents that prevent or decrease the duration of chemotherapy-associated neutropenia and prevent complications that arise from reduced levels of red blood cells or white blood cells, for example erythropoietin (EPO), granulocyte macrophage-colony stimulating factor (GM-CSF), and granulocyte-colony stimulating factor (G-CSF).

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials, as described herein.

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or

complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Accordingly, in a further aspect, the invention provides compounds of the formula (I) and sub-groups thereof such as formulae (II) and (III) and sub-groups thereof as defined herein in the form of pharmaceutical compositions.

The pharmaceutical compositions can be in any form suitable for oral, parenteral, topical, intranasal, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration. Where the compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery. The delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilisation of a suitable infusion pump.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Examples of these are described in R. G. Strickly, Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, VoI 21(2) 2004, p 201-230. In addition, they may contain co-solvents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilisation protectants and combinations of agents for, inter alia, stabilising, the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

A drug molecule that is ionizable can be solubilized to the desired concentration by pH adjustment if the drug's pKa is sufficiently away from the formulation pH value. The acceptable range is pH 2-12 for intravenous and intramuscular administration, but subcutaneously the range is pH 2.7-9.0. The solution pH is controlled by either the salt form of the drug, strong acids/bases such as hydrochloric acid or sodium hydroxide, or by solutions of buffers which include but are not limited to buffering solutions formed from glycine, citrate, acetate, maleate, succinate, histidine, phosphate, tris(hydroxymethyl)aminomethane (TRIS), or carbonate.

The combination of an aqueous solution and a water-soluble organic solvent/surfactant (i.e., a cosolvent) is often used in injectable formulations. The water-soluble organic solvents and surfactants used in injectable formulations include but are not limited to propylene glycol, ethanol, polyethylene glycol 300, polyethylene glycol 400, glycerin, dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP; Pharmasolve), dimethylsulphoxide (DMSO), Solutol HS 15, Cremophor EL, Cremophor RH 60, and polysorbate 80. Such formulations can usually be, but are not always, diluted prior to injection.

Propylene glycol, PEG 300, ethanol, Cremophor EL, Cremophor RH 60, and polysorbate 80 are the entirely organic water-miscible solvents and surfactants used in commercially available injectable formulations and can be used in combinations with each other. The resulting organic formulations are usually diluted at least 2-fold prior to rV bolus or FV infusion.

Alternatively increased water solubility can be achieved through molecular complexation with cyclodextrins

Liposomes are closed spherical vesicles composed of outer lipid bilayer membranes and an inner aqueous core and with an overall diameter of < 100 μm. Depending on the level of hydrophobicity, moderately hydrophobic drugs can be solubilized by liposomes if the drug becomes encapsulated or intercalated within the liposome. Hydrophobic drugs can also be solubilized by liposomes if the drug molecule becomes an integral part of the lipid bilayer membrane, and in this case, the

hydrophobic drag is dissolved in the lipid portion of the lipid bilayer. A typical liposome formulation contains water with phospholipid at -5-20 mg/ml, an isotonicifier, a pH 5-8 buffer, and optionally cholesterol.

The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried

(lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

The pharmaceutical formulation can be prepared by lyophilising a compound of Formula (I) or acid addition salt thereof. Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms. A typical process is to solubilise the compound and the resulting formulation is clarified, sterile filtered and aseptically transferred to containers appropriate for lyophilisation (e.g. vials). In the case of vials, they are partially stoppered with lyo-stoppers. The formulation can be cooled to freezing and subjected to lyophilisation under standard conditions and then hermetically capped forming a stable, dry lyophile formulation. The composition will typically have a low residual water content, e.g. less than 5% e.g. less than 1% by weight based on weight of the lyophile.

The lyophilisation formulation may contain other excipients for example, thickening agents, dispersing agents, buffers, antioxidants, preservatives, and tonicity adjusters. Typical buffers include phosphate, acetate, citrate and glycine. Examples of antioxidants include ascorbic acid, sodium bisulphite, sodium metabisulphite, monothioglycerol, thiourea, butylated hydroxytoluene, butylated hydroxyl anisole, and ethylenediamietetraacetic acid salts. Preservatives may include benzoic acid and its salts, sorbic acid and its salts, alkyl esters of para- hydroxybenzoic acid, phenol, chlorobutanol, benzyl alcohol, thimerosal, benzalkonium chloride and cetylpyridinium chloride. The buffers mentioned previously, as well as dextrose and sodium chloride, can be used for tonicity adjustment if necessary.

Bulking agents are generally used in lyophilisation technology for facilitating the process and/or providing bulk and/or mechanical integrity to the lyophilized cake. Bulking agent means a freely water soluble, solid particulate diluent that when co- lyophilised with the compound or salt thereof, provides a physically stable lyophilized cake, a more optimal freeze-drying process and rapid and complete reconstitution. The bulking agent may also be utilised to make the solution isotonic.

The water-soluble bulking agent can be any of the pharmaceutically acceptable inert solid materials typically used for lyophilisation. Such bulking agents include, for example, sugars such as glucose, maltose, sucrose, and lactose; polyalcohols such as sorbitol or mannitol; amino acids such as glycine; polymers such as polyvinylpyrrolidine; and polysaccharides such as dextran.

The ratio of the weight of the bulking agent to the weight of active compound is typically within the range from about 1 to about 5, for example of about 1 to about 3, e.g. in the range of about 1 to 2.

Alternatively they can be provided in a solution form which may be concentrated and sealed in a suitable vial. Sterilisation of dosage forms may be via filtration or by autoclaving of the vials and their contents at appropriate stages of the formulation process. The supplied formulation may require further dilution or preparation before delivery for example dilution into suitable sterile infusion packs.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

In one preferred embodiment of the invention, the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion.

Pharmaceutical compositions of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable

aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

If a compound is not stable in aqueous media or has low solubility in aqueous media, it can be formulated as a concentrate in organic solvents. The concentrate can then be diluted to a lower concentration in an aqueous system, and can be sufficiently stable for the short period of time during dosing. Therefore in another aspect, there is provided a pharmaceutical composition comprising a non aqueous solution composed entirely of one or more organic solvents, which can be dosed as is or more commonly diluted with a suitable IV excipient (saline, dextrose; buffered or not buffered) before administration (Solubilizing excipients in oral and injectable formulations, Pharmaceutical Research, 21(2), 2004, p201-230). Examples of solvents and surfactants are propylene glycol, PEG300, PEG400, ethanol, dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP, Pharmasolve), Glycerin, Cremophor EL, Cremophor RH 60 and polysorbate. Particular non aqueous solutions are composed of 70-80% propylene glycol, and 20-30% ethanol. One particular non aqueous solution is composed of 70% propylene glycol, and 30% ethanol. Another is 80% propylene glycol and 20% ethanol .Normally these

solvents are used in combination and usually diluted at least 2-fold before IV bolus or IV infusion. The typical amounts for bolus IV formulations are -50% for Glycerin, propylene glycol, PEG300, PEG400, and -20% for ethanol. The typical amounts for IV infusion formulations are -15% for Glycerin, 3% for DMA ₅ and ~10% for propylene glycol, PEG300, PEG400 and ethanol.

In one preferred embodiment of the invention, the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion. For intravenous administration, the solution can be dosed as is, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose), before administration.

In another preferred embodiment, the pharmaceutical composition is in a form suitable for sub-cutaneous (s.c.) administration.

Pharmaceutical dosage forms suitable for oral administration include tablets, capsules, caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches and buccal patches.

Pharmaceutical compositions containing compounds of the formula (I) can be formulated in accordance with known techniques, see for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

Thus, tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, eg; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g. stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents

such as citrate/bicarbonate mixtures. Such excipients are well known and do not need to be discussed in detail here.

Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form. Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.

The solid dosage forms (eg; tablets, capsules etc.) can be coated or un-coated, but typically have a coating, for example a protective film coating (e.g. a wax or varnish) or a release controlling coating. The coating (e.g. a Eudragit ™ type polymer) can be designed to release the active component at a desired location within the gastro-intestinal tract. Thus, the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the compound in the stomach or in the ileum or duodenum.

Instead of, or in addition to, a coating, the drug can be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract. Alternatively, the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract. As a further alternative, the active compound can be formulated in a delivery system that provides osmotic control of the release of the compound. Osmotic release and other delayed release or sustained release formulations may be prepared in accordance with methods well known to those skilled in the art.

The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, dragees, tablets or capsules.

Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.

Compositions for topical use include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.

Compositions for parenteral administration are typically presented as sterile aqueous or oily solutions or fine suspensions, or may be provided in finely divided sterile powder form for making up extemporaneously with sterile water for injection.

Examples of formulations for rectal or intra- vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active compound.

Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known. For administration by inhalation, the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose.

The pharmaceutical formulations may be presented to a patient in "patient packs" containing an entire course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.

The compounds of the inventions will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity. For example, a formulation intended for oral administration may contain from 0.1 milligrams to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient. Within this range, particular subranges of compound are 0.1 milligrams to 2 grams of active ingredient more usually from 10 milligrams to 1 gram, for example, 50 milligrams to 500 milligrams or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).

For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more typically 10 milligrams to 1 gram, for example 50 milligrams to 1 gram, e.g. 100 milligrams to 1 gram, of active compound.

The active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.

Methods of Treatment

It is envisaged that the compounds of the formula (I) as defined herein will be useful in the prophylaxis or treatment of a range of disease states or conditions mediated by cyclin dependent kinases, glycogen synthase kinase-3 and Aurora kinases. Examples of such disease states and conditions are set out above.

The compounds are generally administered to a subject in need of such administration, for example a human or animal patient, preferably a human.

The compounds will typically be administered in amounts that are therapeutically or prophylactically useful and which generally are non-toxic. However, in certain situations (for example in the case of life threatening diseases), the benefits of administering a compound of the formula (I) may outweigh the disadvantages of any toxic effects or side effects, in which case it may be considered desirable to administer compounds in amounts that are associated with a degree of toxicity.

The compounds may be administered over a prolonged term to maintain beneficial therapeutic effects or may be administered for a short period only. Alternatively they may be administered in a pulsatile or continuous manner.

A typical daily dose of the compound can be in the range from 100 picograms to 100 milligrams per kilogram of body weight, more typically 5 nanograms to 25 milligrams per kilogram of bodyweight, and more usually 10 nanograms to 15 milligrams per kilogram (e.g. 10 nanograms to 10 milligrams such as 1 micrograms to 10 milligrams) per kilogram of bodyweight although higher or lower doses may be administered where required. Ultimately, the quantity of compound administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.

The compounds of formula (I) as defined herein can be administered as the sole therapeutic agent or they can be administered in combination therapy with one of more other compounds for treatment of a particular disease state, for example a neoplastic disease such as a cancer as hereinbefore defined. Examples of other therapeutic agents or therapies that may be administered or used together (whether concurrently or at different time intervals) with the compounds of the invention include but are not limited to topoisomerase inhibitors, alkylating agents, antimetabolites, DNA binders, microtubule inhibitors (tubulin targeting agents), particular examples being cisplatin, cyclophosphamide, doxorubicin, irinotecan, fiudarabine, 5FU, taxanes, mitomycin C and radiotherapy.

Other examples of therapeutic agents that may be administered together (whether concurrently or at different time intervals) with the compounds of the formula (I) as defined herein include monoclonal antibodies and signal transduction inhibitors.

For the case of CDK or Aurora inhibitors combined with other therapies, the two or more treatments may be given in individually varying dose schedules and via different routes.

Where the compound of the formula (I) is administered in combination therapy with one, two, three, four or more other therapeutic agents (preferably one or two, more preferably one), 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 periods 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.

For use in combination therapy with another chemotherapeutic agent, the compound of the formula (I) and one, two, three, four or more other therapeutic agents can be, for example, formulated together in a dosage form containing two, three, four or more therapeutic agents. In an alternative, the individual therapeutic agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.

A person skilled in the art would know through his or her common general knowledge the dosing regimes and combination therapies to use.

Methods of Diagnosis

Prior to administration of a compound of the formula (I), a patient may be screened to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against Aurora and/or cyclin dependent kinases.

For example, a biological sample taken from a patient may be analysed to determine whether a condition or disease, such as cancer, that the patient is or may be suffering from is one which is characterised by a genetic abnormality or abnormal protein expression which leads to over-activation of CDKs or to

sensitisation of a pathway to normal CDK activity. Examples of such abnormalities that result in activation or sensitisation of the CDK2 signal include up-regulation of cyclin E, (Harwell RM, Mull BB, Porter DC, Keyomarsi K.; J Biol Chem. 2004 Mar 26;279(13):12695-705) or loss of p21 or p27, or presence of CDC4 variants (Rajagopalan H, Jallepalli PV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B, Lengauer C; Nature. 2004 Mar 4;428(6978):77-81). Tumours with mutants of CDC4 or up-regulation, in particular over-expression, of cyclin E or loss of p21 or p27 may be particularly sensitive to CDK inhibitors. Alternatively or in addition, a biological sample taken from a patient may be analysed to determine whether a condition or disease, such as cancer, that the patient is or may be suffering from is one which is characterised by upregulation of Aurora kinase and thus may be particularly to Aurora inhibitors. The term up-regulation includes elevated expression or over-expression, including gene amplification (i.e. multiple gene copies) and increased expression by a transcriptional effect, and hyperactivity and activation, including activation by mutations.

Thus, the patient may be subjected to a diagnostic test to detect a marker characteristic of over-expression, up-regulation or activation of Aurora kinase or the patient may be subjected to a diagnostic test to detect a marker characteristic of up-regulation of cyclin E, or loss of p21 or p27, or presence of CDC4 variants. The term diagnosis includes screening. By marker we include genetic markers including, for example, the measurement of DNA composition to identify mutations of Aurora or CDC4. The term marker also includes markers which are characteristic of up regulation of Aurora or cyclin E, including enzyme activity, enzyme levels, enzyme state (e.g. phosphorylated or not) and mRNA levels of the aforementioned proteins. Tumours with upregulation of cyclin E, or loss of p21 or p27 may be particularly sensitive to CDK inhibitors. Tumours may preferentially be screened for upregulation of cyclin E, or loss of p21 or p27 prior to treatment. Thus, the patient may be subjected to a diagnostic- test to detect a marker characteristic of upregulation of cyclin E, or loss of p21 or p27.

The diagnostic tests are typically conducted on a biological sample selected from tumour biopsy samples, blood samples (isolation and enrichment of shed tumour cells), stool biopsies, sputum, chromosome analysis, pleural fluid, peritoneal fluid, or urine.

It has been found, see Ewart-Toland et al, (Nat Genet. 2003 Aug;34(4):403-12), that individuals forming part of the sub-population possessing the Ile31 variant of the STK gene (the gene for Aurora kinase A) may have an increased susceptibility to certain forms of cancer. It is envisaged therefore that such individuals suffering from cancer will benefit from the administration of compounds having Aurora kinase inhibiting activity. A patient suffering from, or suspected of suffering from, a cancer may therefore be screened to determine whether he or she forms part of the Ile31 variant sub-population. In addition, it has been found, Rajagopalan et al (Nature. 2004 Mar 4;428(6978):77-81), that there were mutations present in CDC4 (also known as Fbw7 or Archipelago) in human colorectal cancers and endometrial cancers (Spruck et al, Cancer Res. 2002 Aug 15;62(16):4535-9). Identification of individual carrying a mutation in CDC4 may mean that the patient would be particularly suitable for treatment with a CDK inhibitor. Tumours may preferentially be screened for presence of a CDC4 variant prior to treatment. The screening process will typically involve direct sequencing, oligonucleotide microarray analysis, or a mutant specific antibody.

Tumours with activating mutants of Aurora or up-regulation of Aurora including any of the isoforms thereof, may be particularly sensitive to Aurora inhibitors. Tumours may preferentially be screened for up-regulation of Aurora or for Aurora possessing the Ile31 variant prior to treatment (Ewart-Toland et al., Nat Genet. 2003 Aug;34(4):403-12). Ewart-Toland et al identified a common genetic variant in STKl 5 (resulting in the amino acid substitution F31I) that is preferentially amplified and associated with the degree of aneuploidy in human colon tumors. These results are consistent with an important role for the Ile31 variant of STKl 5 in human cancer susceptibility. In particular, this polymorphism in Aurora A has been

suggested to be a genetic modifier fir developing breast carcinoma (Sun et al, Carcinogenesis, 2004, 25(11), 2225-2230).

The aurora A gene maps to the chromosome 20ql 3 region that is frequently amplified in many cancers e.g. breast, bladder, colon, ovarian, pancreatic. Patients ^■ with a tumour that has this gene amplification might be particularly sensitive to treatments targeting aurora kinase inhibition

Methods of identification and analysis of mutations and up-regulation of protein e.g. Aurora isoforms and chromosome 20ql 3 amplification are known to a person skilled in the art. Screening methods could include, but are not limited to, standard methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) or in- situ hybridisation.

In screening by RT-PCR, the level of mRNA in the tumour is assessed by creating a cDNA copy of the mRNA followed by amplification of the cDNA by PCR. Methods of PCR amplification, the selection of primers, and conditions for amplification, are known to a person skilled in the art. Nucleic acid manipulations and PCR are carried out by standard methods, as described for example in Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc., or Innis, M.A. et-al., eds. PCR Protocols: a guide to methods and applications, 1990, Academic Press, San Diego. Reactions and manipulations involving nucleic acid techniques are also described in Sambrook et al., 2001, 3 ^rd Ed, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Alternatively a commercially available kit for RT-PCR (for example Roche Molecular Biochemicals) may be used, or methodology as set forth in United States patents 4,666,828; 4,683,202; 4,801,531; 5,192,659, 5,272,057, 5,882,864, and 6,218,529 and incorporated herein by reference.

An example of an in-situ hybridisation technique for assessing mRNA expression would be fluorescence in-situ hybridisation (FISH) (see Angerer, 1987 Meth. Enzymol., 152: 649).

Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue to be analyzed; (2) prehybridization treatment of the sample to increase accessibility of target nucleic acid, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization, and (5) detection of the hybridized nucleic acid fragments. The probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions. Standard methods for carrying out FISH are described in Ausubel, F.M. et al, eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc and Fluorescence In Situ Hybridization: Technical Overview by John M. S. Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd ed.; ISBN: 1-59259-760-2; March 2004, pps. 077-088; Series: Methods in Molecular Medicine.

Alternatively, the protein products expressed from the mRNAs may be assayed by immunohistochemistry of tumour samples, solid phase immunoassay with microtiter plates, Western blotting, 2-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and other methods known in the art for detection of specific proteins. Detection methods would include the use of site specific antibodies. The skilled person will recognize that all such well-known techniques for detection of upregulation of cyclin E, or loss of p21 or p27, or detection of CDC4 variants, Aurora up-regulation and mutants of Aurora could be applicable in the present case.

Therefore, all of these techniques could also be used to identify tumours particularly suitable for treatment with the compounds of the invention.

Tumours with mutants of CDC4 or up-regulation, in particular over-expression, of cyclin E or loss of p21 or p27 may be particularly sensitive to CDK inhibitors. Tumours may preferentially be screened for up-regulation, in particular over-

expression, of cyclin E (Harwell RM, Mull BB, Porter DC, Keyomarsi K.; J Biol Chem. 2004 Mar 26;279(13): 12695-705) or loss of p21 or p27 or for CDC4 variants prior to treatment (Rajagopalan H, Jallepalli PV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B, Lengauer C; Nature. 2004 Mar 4;428(6978):77-81).

Patients with mantle cell lymphoma (MCL) could be selected for treatment with a compound of the invention using diagnostic tests outlined herein. MCL is a distinct clinicopathologic entity of non-Hodgkin's lymphoma, characterized by proliferation of small to medium-sized lymphocytes with co-expression of CD5 and CD20, an aggressive and incurable clinical course, and frequent t(l I;14)(ql3;q32) translocation. Over-expression of cyclin Dl mRNA, found in mantle cell lymphoma (MCL), is a critical diagnostic marker. Yatabe et al (Blood. 2000 Apr 1;95(7):2253-61) proposed that cyclin Dl-positivity should be included as one of the standard criteria for MCL, and that innovative therapies for this incurable disease should be explored on the basis of the new criteria. Jones et al (J MoI Diagn. 2004 May;6(2):84-9) developed a real-time, quantitative, reverse transcription PCR assay for cyclin Dl (CCNDl) expression to aid in the diagnosis of mantle cell lymphoma (MCL). Howe et al (Clin Chem. 2004 Jan;50(l):80-7) used real-time quantitative RT-PCR to evaluate cyclin Dl mRNA expression and found that quantitative RT-PCR for cyclin Dl mRNA normalized to CD 19 mRNA can be used in the diagnosis of MCL in blood, marrow, and tissue. Alternatively, patients with breast cancer could be selected for treatment with a CDK inhibitor using diagnostic tests outline above. Tumour cells commonly overexpress cyclin E and it has been shown that cyclin E is over-expressed in breast cancer (Harwell et al, Cancer Res, 2000, 60, 481-489). Therefore breast cancer may in particular be treated with a CDK inhibitor as provided herein.

Antifungal Use

In a further aspect, the invention provides the use of the compounds of the formula (I) as hereinbefore defined as antifungal agents.

The compounds of the formula (I) may be used in animal medicine (for example in the treatment of mammals such as humans), or in the treatment of plants (e.g. in agriculture and horticulture),. or as general antifungal agents, for example as preservatives and disinfectants.

In one embodiment, the invention provides a compound of the formula (I) as hereinbefore defined for use in the prophylaxis or treatment of a fungal infection in a mammal such as a human.

Also provided is the use of a compound of the formula (I) for the manufacture of a medicament for use in the prophylaxis or treatment of a fungal infection in a mammal such as a human.

For example, compounds of the invention may be administered to human patients suffering from, or at risk of infection by, topical fungal infections caused by among other organisms, species of Candida, Trichophyton, Microsporum or Epidermophyton, or in mucosal infections caused by Candida albicans (e.g. thrush and vaginal candidiasis). The compounds of the invention can also be administered for the treatment or prophylaxis of systemic fungal infections caused by, for example, Candida albicans, Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus, Coccidiodies, Paracoccidioides, Histoplasma or Blastomyces.

In another aspect, the invention provides an antifungal composition for agricultural (including horticultural) use, comprising a compound of the formula (I) together with an agriculturally acceptable diluent or carrier.

The invention further provides a method of treating an animal (including a mammal such as a human), plant or seed having a fungal infection, which comprises treating said animal, plant or seed, or the locus of said plant or seed, with an effective amount of a compound of the formula (I).

The invention also provides a method of treating a fungal infection in a plant or seed which comprises treating the plant or seed with an antifungally effective

amount of a fungicidal composition containing a compound of the formula (I) as hereinbefore defined.

Differential screening assays may be used to select for those compounds of the present invention with specificity for non-human CDK enzymes. Compounds which act specifically on the CDK enzymes of eukaryotic pathogens can be used as antifungal or anti-parasitic agents. Inhibitors of the Candida CDK kinase, CKSI, can be used in the treatment of candidiasis. Antifungal agents can be used against infections of the type hereinbefore defined, or opportunistic infections that commonly occur in debilitated and immunosuppressed patients such as patients with leukemias and lymphomas, people who are receiving immunosuppressive therapy, and patients with predisposing conditions such as diabetes mellitus or AIDS, as well as for non-immunosuppressed patients.

Assays described in the art can be used to screen for agents which may be useful for inhibiting at least one fungus implicated in mycosis such as candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidiodomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, or sporotrichosis. The differential screening assays can be used to identify anti-fungal agents which may have therapeutic value in the treatment of aspergillosis by making use of the CDK genes cloned from yeast such as

Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus, or where the mycotic infection is mucon-nycosis, the CDK assay can be derived from yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, or Mucorpusillus. Sources of other CDK enzymes include the pathogen Pneumocystis carinii.

By way of example, in vitro evaluation of the antifungal activity of the compounds can be performed by determining the minimum inhibitory concentration (M.I.C.) which is the lowest concentration of the test compounds, in a suitable medium, at which growth of the particular microorganism fails to occur. In practice, a series of agar plates, each having the test compound incorporated at a particular

concentration is inoculated with a standard culture of, for example, Candida albicans and each plate is then incubated for an appropriate period at 37 °C. The plates are then examined for the presence or absence of growth of the fungus and the appropriate M.I.C. value is noted. Alternatively, a turbidity assay in liquid cultures can be performed and a protocol outlining an example of this assay can be found in Example 51.

The in vivo evaluation of the compounds can be carried out at a series of dose levels by intraperitoneal or intravenous injection or by oral administration, to mice that have been inoculated with a fungus, e.g. a strain of Candida albicans or Aspergillus flavus. The activity of the compounds can be assessed by monitoring the growth of the fungal infection in groups of treated and untreated mice (by histology or by retrieving fungi from the infection). The activity may be measured in terms of the dose level at which the compound provides 50% protection against the lethal effect of the infection (PD ₅ o).

For human antifungal use, the compounds of the formula (I) can be administered alone or in admixture with a pharmaceutical carrier selected in accordance with the intended route of administration and standard pharmaceutical practice. Thus, for example, they may be administered orally, parenterally, intravenously, intramuscularly or subcutaneously by means of the formulations described above in the section headed "Pharmaceutical Formulations".

For oral and parenteral administration to human patients, the daily dosage level of the antifungal compounds of the formula (I) can be from 0.01 to 10 mg/kg (in divided doses), depending on inter alia the potency of the compounds when administered by either the oral or parenteral route. Tablets or capsules of the compounds may contain, for example, from 5 mg to 0.5 g of active compound for administration singly or two or more at a time as appropriate. The physician in any event will determine the actual dosage (effective amount) which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient.

Alternatively, the antifungal compounds of formula (I) can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. For example, they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin; or they can be incorporated, at a concentration between 1 and 10%, into an ointment consisting of a white wax or white soft paraffin base together with such stabilizers and preservatives as may be required.

In addition to the therapeutic uses described above, anti-fungal agents developed with such differential screening assays can be used, for example, as preservatives in foodstuff, feed supplement for promoting weight gain in livestock, or in disinfectant formulations for treatment of non-living matter, e.g., for decontaminating hospital equipment and rooms. In similar fashion, side by side comparison of inhibition of a mammalian CDK and an insect CDK, such as the Drosophilia CDK5 gene (Heimlich et al. (1994) FEBS Lett 356:317-21), will permit selection amongst the compounds herein of inhibitors which discriminate between the human/mammalian and insect enzymes. Accordingly, the present invention expressly contemplates the use and formulation of the compounds of the invention in insecticides, such as for use in management of insects like the fruit fly.

In yet another embodiment, certain of the subject CDK inhibitors can be selected on the basis of inhibitory specificity for plant CDK's relative to the mammalian enzyme. For example, a plant CDK can be disposed in a differential screen with one or more of the human enzymes to select those compounds of greatest selectivity for inhibiting the plant enzyme. Thus, the present invention specifically contemplates formulations of the subject CDK inhibitors for agricultural applications, such as in the form of a defoliant or the like.

For agricultural and horticultural purposes the compounds of the invention may be used in the form of a composition formulated as appropriate to the particular use and intended purpose. Thus the compounds may be applied in the form of dusting powders, or granules, seed dressings, aqueous solutions, dispersions or emulsions, dips, sprays, aerosols or smokes. Compositions may also be supplied in the form of

dispersible powders, granules or grains, or concentrates for dilution prior to use. Such compositions may contain such conventional carriers, diluents or adjuvants as are known and acceptable in agriculture and horticulture and they can be manufactured in accordance with conventional procedures. The compositions may also incorporate other active ingredients, for example, compounds having herbicidal or insecticidal activity or a further fungicide. The compounds and compositions can be applied in a number of ways, for example they can be applied directly to the plant foliage, stems, branches, seeds or roots or to the soil or other growing medium, and they may be used not only to eradicate disease, but also prophylactically to protect the plants or seeds from attack. By way of example, the compositions may contain from 0.01 to 1 wt.% of the active ingredient. For field use, likely application rates of the active ingredient may be from 50 to 5000 g/hectare.

The invention also contemplates the use of the compounds of the formula (I) in the control of wood decaying fungi and in the treatment of soil where plants grow, paddy fields for seedlings, or water for perfusion. Also contemplated by the invention is the use of the compounds of the formula (I) to protect stored grain and other non-plant loci from fungal infestation.

EXAMPLES

The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.

In the examples, the following abbreviations are used.

AcOH acetic acid

BOC ført-butyloxycarbonyl CDI 1,1-carbonyldiimidazole

DMAW90 Solvent mixture: DCM: MeOH, AcOH, H ₂ O (90: 18:3 :2)

DMAW120 Solvent mixture: DCM: MeOH, AcOH, H ₂ O (120:18:3:2)

DMAW240 Solvent mixture: DCM: MeOH, AcOH, H ₂ O (240:20:3 :2)

DCM dichloromethane

DMF dimethylformamide

DMSO dimethyl sulphoxide

EDC 1 -ethyl-3-(3 ' -dimethylaminopropy^-carbodiimide

Et ₃ N triethylamine EtOAc ethyl acetate

Et ₂ O diethyl ether

HOAt 1-hydroxyazabenzotriazole

HOBt 1-hydroxybenzotriazole

MeCN acetonitrile MeOH methanol

SiO ₂ silica

TBTU N,N,N^N'-tetramethyl~O-(benzotriazol- 1 -yl)uronium tetrafluoroborate

THF tetrahydrofuran

Analytical LC-MS System and Method Description

In the examples, the compounds prepared were characterised by liquid chromatography and mass spectroscopy using the systems and operating conditions set out below. Where atoms with different isotopes are present, and a single mass quoted, the mass quoted for the compound is the monoisotopic mass (i.e. ³⁵ Cl; ⁷⁹ Br etc.). Several systems were used, as described below, and these were equipped with, and were set up to run under, closely similar operating conditions. The operating conditions used are also described below.

Waters Platform LC-MS system;

HPLC System: Waters 2795 Mass Spec Detector: Micromass Platform LC

PDA Detector: Waters 2996 PDA

Analytical Acidic conditions;

Eluent A: H ₂ O (0.1 % Formic Acid) Eluent B : CH ₃ CN (0.1 % Formic Acid)

Gradient: 5-95% eluent B over 3.5 minutes

Flow: 0.8 ml/min

Column: Phenomenex Synergi 4μ MAX-RP 8OA, 2.0 x 50 mm

Analytical Basic conditions: Eluent A: H ₂ O (1OmM NH ₄ HCO ₃ buffer adjusted to pH=9.2 with NH ₄ OH)

Eluent B: CH ₃ CN

Gradient: 05-95% eluent B over 3.5 minutes

Flow: 0.8 ml/min

Column: Phenomenex Luna Cl 8(2) 5μm 2.0 x 50 mm

Analytical Polar conditions;

Eluent A: H ₂ O (0.1 % Formic Acid)

Eluent B: CH ₃ CN (0.1% Formic Acid)

Gradient: 00-50% eluent B over 3 minutes

Flow: 0.8 ml/min Column: Phenomenex Synergi 4μ MAX-RP 8OA, 2.0 x 50 mm

Analytical Lipophilic conditions:

Eluent A: H ₂ O (0.1 % Formic Acid)

Eluent B : CH ₃ CN (0.1 % Formic Acid)

Gradient: 55-95% eluent B over 3.5 minutes Flow: 0.8 ml/min

Column: Phenomenex Synergi 4μ MAX-RP 8OA, 2.0 x 50 mm

Analytical Long Acidic conditions:

Eluent A: H ₂ O (0.1 % Formic Acid)

Eluent B: CH ₃ CN (0.1% Formic Acid) Gradient: 05-95% eluent B over 15 minutes

Flow: 0.4 ml/min

Column: Phenomenex Synergi 4μ MAX-RP 8OA, 2.0 x 150 mm

Analytical Long Basic Conditions:

Eluent A: H ₂ O (1 OmM NH ₄ HCO ₃ buffer adjusted to pH=9.2 with NH ₄ OH)

Eluent B: CH ₃ CN

Gradient: 05-95% eluent B over 15 minutes

Flow: 0.8 ml/min

Column: Phenomenex Luna C18(2) 5μm 2.0 x 50 mm

Platform MS conditions:

Capillary voltage: 3.6 kV (3.40 kV on ES negative)

Cone voltage: 25 V Source Temperature: 120 °C

Scan Range: 100-800 amu

Ionisation Mode: ElectroSpray Positive or

Electro Spray Negative or ElectroSpray Positive & Negative

Waters Fractionlynx LC-MS system:

HPLC System: 2767 autosampler - 2525 binary gradient pump

Mass Spec Detector: Waters ZQ

PDA Detector: Waters 2996 PDA

Analytical Acidic conditions: Eluent A: H ₂ O (0.1 % Formic Acid)

Eluent B : CH ₃ CN (0.1 % Formic Acid)

Gradient: 5-95% eluent B over 4 minutes

Flow: 2.0 ml/min

Column: Phenomenex Synergi 4μ MAX-RP 8OA, 4.6 x 50 mm

Analytical Polar conditions:

Eluent A: H ₂ O (0.1 % Formic Acid) Eluent B : CH ₃ CN (0.1 % Formic Acid) Gradient: 00-50% eluent B over 4 minutes

Flow: 2.0 ml/min

Column: Phenomenex Synergi 4μ MAX-RP 8OA, 4.6 x 50 mm

Analytical Lipophilic conditions;

Eluent A: H ₂ O (0.1 % Formic Acid) Eluent B : CH ₃ CN (0.1 % Formic Acid)

Gradient: 55-95% eluent B over 4 minutes

Flow: 2.0 ml/min

Column: Phenomenex Synergi 4μ MAX-RP 8OA, 4.6 x 50 mm

Fractionlynx MS conditions: Capillary voltage: 3.5 kV (3.2 kV on ES negative)

Cone voltage: 25 V (30 V on ES negative)

Source Temperature: 120 ⁰ C

Scan Range: 100-800 amu

Ionisation Mode: ElectroSpray Positive or ElectroSpray Negative or

ElectroSpray Positive & Negative

Mass Directed Purification LC-MS System

Preparative LC-MS is a standard and effective method used for the purification of small organic molecules such as the compounds described herein. The methods for the liquid chromatography (LC) and mass spectrometry (MS) can be varied to provide better separation of the crude materials and improved detection of the samples by MS. Optimisation of the preparative gradient LC method will involve varying columns, volatile eluents and modifiers, and gradients. Methods are well known in the art for optimising preparative LC-MS methods and then using them to purify compounds. Such methods are described in Rosentreter U, Huber U.;

Optimal fraction collecting in preparative LC/MS; J Comb Chem.; 2004; 6(2), 159- 64 and Leister W, Strauss K, Wisnoski D, Zhao Z, Lindsley C, Development of a custom high-throughput preparative liquid chromatography/mass spectrometer

platform for the preparative purification and analytical analysis of compound libraries; J Comb Chem.; 2003; 5(3); 322-9.

One such system for purifying compounds via preparative LC-MS is described below although a person skilled in the art will appreciate that alternative systems and methods to those described could be used. In particular, normal phase preparative LC based methods might be used in place of the reverse phase methods described here. Most preparative LC-MS systems utilise reverse phase LC and volatile acidic modifiers, since the approach is very effective for the purification of small molecules and because the eluents are compatible with positive ion electrospray mass spectrometry. Employing other chromatographic solutions e.g. normal phase LC, alternatively buffered mobile phase, basic modifiers etc as outlined in the analytical methods described above could alternatively be used to purify the compounds.

Preparative LC-MS Systems:

Waters Fractionlynx System:

• Hardware:

2767 Dual Loop Autosampler/Fraction Collector 2525 preparative pump

CFO (column fluidic organiser) for column selection RMA (Waters reagent manager) as make up pump Waters ZQ Mass Spectrometer Waters 2996 Photo Diode Array detector Waters ZQ Mass Spectrometer

• Software: Masslynx 4.0

• Waters MS running conditions:

Capillary voltage: 3.5 kV (3.2 kV on ES Negative)

Cone voltage: 25 V

Source Temperature: 120 °C

Multiplier: 500 V

Scan Range: 125-800 amu

Ionisation Mode: ElectroSpray Positive or ElectroSpray Negative

Agilent 1100 LC-MS preparative system:

• Hardware:

Autosampler: 1100 series "prepALS"

Pump: 1100 series "PrepPump" for preparative flow gradient and 1100 series "QuatPump" for pumping modifier in prep flow

UV detector: 1100 series "MWD" Multi Wavelength Detector

MS detector: 1100 series "LC-MSD VL"

Fraction Collector: 2 x "Prep-FC"

Make Up pump: "Waters RMA" Agilent Active Splitter

• Software:

Chemstation: Chem32

• Agilent MS running conditions:

Capillary voltage: 4000 V (3500 V on ES Negative) Fragmentor/Gain: 150/1

Drying gas flow: 13.0 L/min

Gas Temperature: 350 ⁰ C

Nebuliser Pressure: 50 psig

Scan Range: 125-800 amu Ionisation Mode: ElectroSpray Positive or

ElectroSpray Negative

Chromatographic Conditions :

• Columns:

1. Low pH chromatography:

Phenomenex Synergy MAX-RP, lOμ, 100 x 21.2mm

(alternatively used Thermo Hypersil-Keystone HyPurity Aquastar, 5μ, 100 x

21.2mm for more polar compounds) 2. High pH chromatography:

Phenomenex Luna C18 (2), lOμ, 100 x 21.2mm (alternatively used Phenomenex Gemini, 5μ, 100 x 21.2mm)

• Eluents:

1. Low pH chromatography: Solvent A: EbO + 0.1% Formic Acid, ρH~1.5 Solvent B: CH ₃ CN + 0.1% Formic Acid

2. High pH chromatography:

Solvent A: H ₂ O + 10 mM NH ₄ HCO ₃ + NH4OH, pH-9.2 Solvent B: CH ₃ CN 3. Make up solvent:

MeOH + 0.2% Formic Acid (for both chromatography type)

• Methods:

According to the analytical trace the most appropriate preparative chromatography type was chosen. A typical routine was to run an analytical LC-MS using the type of chromatography (low or high pH) most suited for compound structure. Once the analytical trace showed good chromatography a suitable preparative method of the same type was chosen. Typical running condition for both low and high pH chromatography methods were:

Flow rate: 24 ml/min Gradient: Generally all gradients had an initial 0.4 min step with 95% A + 5% B. Then according to analytical trace a 3.6 min gradient was chosen in order to achieve good separation (e.g. from 5% to 50% B for early retaining compounds; from 35% to 80% B for middle retaining compounds and so on) Wash: 1.2 minute wash step was performed at the end of the gradient

Re-equilibration: 2.1 minutes re-equilibration step was ran to prepare the system for the next run

Make Up flow rate: 1 ml/min

• Solvent: All compounds were usually dissolved in 100% MeOH or 100% DMSO

From the information provided someone skilled in the art could purify the compounds described herein by preparative LC-MS.

The starting materials for each of the Examples are commercially available unless otherwise specified.

EXAMPLE 1

5 -methyl-4-morpholin-4- ylmethyl-furan-2-carboxylic acid [3 -(5 -methyl-4- trifluoromethyl- 1 H-imidazol-2-ylV 1 H-pyrazol-4-yl] amide

IA. Synthesis of 4-Nitro-lH-pyrazole-3-carboxylic acid methyl ester

Thionyl chloride (3.8 ml, 52.5 mmol) was added cautiously to a stirred, ice-cold mixture of 4-nitropyrazole-3-carboxylic acid (7.5 g, 47.7 mmol) in MeOH (150 ml), the mixture stirred at ambient temperature for 1 hour then heated at reflux for 3 hours. The reaction mixture was cooled, evaporated in vacuo then azeotroped with toluene to give 4-nitro-lH-pyrazole-3-carboxylic acid ethyl ester (8.8 g).

IB. Synthesis of 4-Nitro-l-(tetrahvdro-pyran-2-vD-lH-pyrazole-3-carboxylic acid methyl ester

A suspension of 4-nitro-lH-pyrazole-3-carboxylic acid methyl ester (5 g, 29.24 mmol) and p-toluene sulphonic acid (555 mg, 2.92 mmol) in chloroform (100 ml) at 0 ⁰ C was treated with 3,4-dihydropyran (4 ml, 43.8 mmol) dropwise. The reaction mixture was allowed to warm to ambient temperature, and then stirred for a further 2 hours. The reaction mixture was diluted with Et ₂ O, washed sequentially with saturated NaHCO ₃ solution and brine. The organic portion was dried (MgSO ₄ ), filtered and evaporated in vacuo. The residue was purified by flash chromatography [silica, ethyl acetate/petrol (1:2)] to give 4-nitro-l-(tetrahydro-pyran-2-yl)-lH- pyrazole-3-carboxylic acid methyl ester (7.1 g, 95%) as a colourless oil. (LC/MS: R _t 2.86, [M+H] ⁺ 256.00).

1C. Synthesis of 4-amino-l-(tetrahvdro-pyran-2-yl)-lH-pyrazole-3-carboxylic acid methyl ester.

To a stirred solution of 4-nitro- 1 -(tetrahydro-pyran-2-yl)- 1 H-pyrazole-3 -carboxylic acid methyl ester (16.Og, 62.75 mmoles, Example IB) and ammonium formate (39.6 g, 627.45 mmoles) and in ethanol (200 ml) and water (20 ml) under nitrogen was added palladium on carbon (10%, 0.8g). The reaction mixture was heated at 50 ⁰ C for 2 hours. The suspension was filtered through Celite®, and the filtrate was partitioned between ethyl acetate and water. The organic portion was dried

(MgSO ₄ ) to give 4-amino-l -(tetrahydro-pyran-2-yl)- 1 H-pyrazole-3 -carboxylic acid methyl ester as a yellow oil (12.5g, 89%). (LC/MS: R _t 1.84, [M+H] ⁺ 226.06).

ID. Synthesis of 4-fert-butoxycarbonylamino- 1 -(tetrahydro-pyran-2-ylV 1 H- PVrazole-3-carboxylic acid methyl ester

To a solution of 4-amino-l-(tetrahydro-pyran-2-yl)-lH-pyrazole carboxylic acid (23.5g, 104 mmoles) in dichloromethane (250 ml) was added triethylamine (17.4 ml, 125 mmoles) and di-tert-butyl carbonate (25.0 g, 115 mmoles) and the resulting solution was stirred at ambient temperature for 24 hours. The reaction mixture was partitioned between ethyl acetate and water. The organic portion was dried (MgSO ₄ ), filtered and evaporated in vacuo. The residue was purified by flash chromatography (Biotage SP4, 3x40M, flow rate 40 ml/min, gradient 1 :4 ethyl acetate/petrol to 7:3 ethyl acetate/petrol) to give 4-fert-butoxycarbonylamino-l- (tetrahydro-pyran-2-yl)-lH-pyrazole-3 -carboxylic acid methyl ester as a colourless oil (12.0 g, 36%). (LC/MS: R _t 3.28, [M+H] ⁺ 326.37).

IE. Synthesis of [3-hydroxymethyl-l-rtetrahvdro-pyran-2-yl)-lH-pyrazol-4-yll- carbamic acid ferf-butyl ester

A solution of 4-ført-butoxycarbonylamino-l-(tetrahydro-pyran-2-yl)-lH-pyr azole- 3-carboxylic acid methyl ester (6.6 g, 20.31 mmoles) in THF (150 ml) under nitrogen at -78 ¹⁰ C was treated dropwise with a solution of diisobutylaluminium hydride in THF (142 ml, IM). The reaction mixture was stirred at -78 ⁰ C for 30 minutes and then warmed to ambient temperature. A saturated aqueous solution of sodium sulphate was added to the reaction mixture. The resulting suspension was filtered through Celite®. The filtrate was partitioned between ethyl acetate and brine. The organic portion was dried (MgSO ₄ ), filtered and evaporated in vacuo.

The residue was purified by flash chromatography (Biotage™ SP4, 4OM, flow rate 40 ml/min, gradient 1:2 ethyl acetate /petrol to 4:1 ethyl acetate /petrol) to give [3- hydroxymethyl-1 -(tetrahydro-pyran-2-yl)-lH-pyrazol-4-yl]-carbamic acid tert-butyl ester as a white solid (2.2g, 37%). (LC/MS: R _t 2.48, [M+H] ⁺ 298.32).

IF. Synthesis of [3-formyl-l-(tetrahvdro-pyran-2-ylVlH-pyrazol-4-yl]-carbamic acid tert-bntyl ester

To a stirred solution of [3-hydroxymethyl-l-(tetrahydro-pyran-2-yl)-lH-pyrazol-4- yl]-carbamic acid tert-butyl ester (2.2 g, 7.41 mmoles) in acetone (40 ml) was added manganese dioxide (12.9 g, 148.15 mmoles). The resulting black suspension was stirred at ambient temperature for 24 hours. The reaction mixture was filtered through Celite, and the filtrate evaporated in vacuo to give [3-formyl-l-(tetrahydro- ρyran-2-yl)-lH-pyrazol-4-yl]-carbamic acid tert-butyl ester as a white solid (2.2 g, 100%). (LC/MS: R _t 3.29, [M-THP+H] ⁺ 212.22).

IG. Synthesis of 33-dibromo-l J J-trifluoro-butan-2-one

A suspension of l,l,l-trifluoro-butan-2-one (10 g, 79.4 mmoles), N- bromosuccinimide (36.7 g, 206.3 mmoles) and benzoyl peroxide (25 mg, 0.1 mmoles) in carbon tetrachloride (100 ml) was irradiated at 70 ⁰ C with a tungsten lamp for 36 hours. The suspension was filtered and the filtrate was evaporated in vacuo. The residue was triturated with petrol, filtered and the filtrate was evaporated in vacuo to give 3,3-dibromo-l,l,l-trifluoro-butan-2-one as a pale yellow liquid (13.5 g, 32%) which was a 1:1 mixture of the desired product with residual NBS. The crude product was taken into the next step without purification.

IH. Synthesis of [3-(4-methyl-5-trifluoromethyl-lH-imidazol-2-yl)-l-(tetrahyd ro- pyran-2-yl)-lH-pyrazol-4-yll-carbamic acid fert-butyl ester.

A solution of [3-formyl-l-(tetrahydro-pyran-2-yl)-lH-pyrazol-4-yl]-carbami c acid tert-butyl ester (2.2 g, 7.46 mmoles) and the crude 3,3-dibromo-l,l,l-trifluoro- butan-2-one from Example IG (13.5 g) in methanolic ammonia (40 ml, 2N) was stirred at ambient temperature for 24 hours. The reaction mixture was evaporated in vacuo. The residue was purified by flash chromatography (Biotage SP4, 4OM, flow rate 40 ml/min, gradient 3:17 ethyl acetate/petrol to 3:2 ethyl acetate /petrol) to give [3 -(4-methyl-5 -trifluoromethyl- lH-imidazol-2-yl)- 1 -(tetrahydro-pyran-2-yl)- 1 H- pyrazol-4-yl]-carbamic acid tert-butyl ester as a pale yellow oil (2.2 g, 71%). (LC/MS: R _t 3.69, [M+H] ⁺ 416.39).

II. Synthesis of 3-(5-methyl-4-trifluoromethyl-lH-imidazol-2-yl)- lH-pyrazol-4- ylamine

A solution of [3-(4-methyl-5-trifluoromethyl-lH-imidazol-2-yl)-l-(tetrahyd ro- pyran-2-yl)-lH-pyrazol-4-yi]-carbamic acid tert-butyl ester (2.2 g, 5.30 mmoles) in HCl in dioxane (30 ml, 4M) was stirred at ambient temperature for 24 hours. The reaction mixture was evaporated in vacuo. The residue was co-evaporated in vacuo with a 1 :1 mixture of toluene/methanol. The residue was purified by trituration with petrol to give 3-(5-methyl-4-trifluoromethyl-lH-imidazol-2-yl)-lH-pyrazol-4 - ylamine as a yellow mono hydrochloride solid (1.4 g, 100%). (LC/MS: R _t 1.56, [M+H] ⁺ 232.21).

IJ. Synthesis of 5-methyl-4-morpholin-4-ylmethyl-furan-2-carboxylic acid[3-(5- methyl-4-trifluoromethyl- 1 H-imidzaol-2-yD- 1 H-pyrazol-4-vH -amide

A solution of 3-(5-methyl-4-trifluoromethyl-lH-imidazol-2-yl)-lH-pyrazol-4 - ylamine (100 mg, 0.29 mmoles), EDC (68 mg, 0.35 mmoles), HOBt (48 mg, 0.35 mmoles), 5-methyl-4-morpholin-4-ylmethyl-furan-2-carboxylic acid (65 mg, 0.29 mmoles) and triethylamine (133 μl, 0.96 mmoles) in DMF (2 ml) was heated at 70 ⁰ C for 1 hour. The reaction mixture was partitioned between ethyl acetate and a saturated aqueous solution of sodium hydrogen carbonate. The organic portion was washed with water (x3), brine, dried (MgSO ₄ ), filtered and evaporated in vacuo. The residue was purified by flash chromatography (Biotage SP4, 25S, flow rate 25 ml/min, gradient 3:2 ethyl acetate /petrol to ethyl acetate and then isocratically with 1:9 methanol/ ethyl acetate) to give 5-methyl-4-morpholin-4-ylmethyl-furan-2- carboxylic acid [3 -(5-methyl-4-trifluoromethyl- 1 H-imidzaol-2-yl)- 1 H-pyrazol-4- yl]-amide as a pale yellow solid (10 mg, 8%). (LC/MS: R _t 2.09, [M+H] ⁺ 439.34).

EXAMPLES 2 TO 13

By following the procedure set out in Example IF, using the appropriate starting materials, the compounds of Examples 2 to 13 set out in the table below were prepared.

BIOLOGICAL ACTIVITY

EXAMPLE 14

Measurement of CDK2 Kinase Inhibitory Activity (ICW)

Compounds of the invention were tested for kinase inhibitory activity against CDK- 2 using the following protocol.

Activated CDK2/CyclinA (Brown et al, Nat. Cell Biol, 1, ρp438-443 ₅ 1999; Lowe, E.D., et al Biochemistry, 41, ppl5625-15634, 2002) is diluted to 125pM in 2.5X strength assay buffer (5OmM MOPS pH 7.2, 62.5 mM β-glycerophosphate, 12.5mM EDTA, 37.5mM MgCl ₂ , 112.5 mM ATP, 2.5 mM DTT, 2.5 mM sodium orthovanadate, 0.25 mg/ml bovine serum albumin), and 10 μl mixed with 10 μl of histone substrate mix (60 μl bovine histone Hl (Upstate Biotechnology, 5 mg/ml), 940 μl H ₂ O, 35 μCi γ ³³ P-ATP) and added to 96 well plates along with 5 μl of various dilutions of the test compound in DMSO (up to 2.5%). The reaction is

allowed to proceed for 2 to 4 hours before being stopped with an excess of ortho- phosphoric acid (5 μl at 2%).

γ ³³ P-ATP which remains unincorporated into the histone Hl is separated from phosphorylated histone Hl on a Millipore MAPH filter plate. The wells of the MAPH plate are wetted with 0.5% orthophosphoric acid, and then the results of the reaction are filtered with a Millipore vacuum filtration unit through the wells. Following filtration, the residue is washed twice with 200 μl of 0.5% orthophosphoric acid. Once the filters have dried, 20 μl of Microscint 20 scintillant is added, and then counted on a Packard Topcount for 30 seconds.

The % inhibition of the CDK2 activity is calculated and plotted in order to determine the concentration of test compound required to inhibit 50% of the CDK2 activity (IC ₅₀ ). The compounds of Examples 1 to 13 all have IC ₅₀ values no greater than 1 μM.

EXAMPLE 15 CDKl/CvclinB Assay.

The CDKl/CyclinB assay. is identical to the CDK2/CyclinA above except that CDKl/CyclinB (Upstate Discovery) is used and the enzyme is diluted to 6.25 nM.

In the CDKl assay, the compounds of Examples 2 to 5, 7, 10 and 12 all have IC ⁵⁰ values of less than 1 μM, the compounds of Examples 1, 6, 8 and 13 have IC ⁵⁰ values of less than 10 μM and the compounds of Examples 9 and 11 exhibit greater than 35% inhibition at a concentration of 1 μM.

EXAMPLE 16

GSK3 -B/ Aurora Kinase Inhibitory Activity Assay

AuroraA (Upstate Discovery) or GSK3-β (Upstate Discovery) are diluted to 1OnM and 7.5nM respectively in 25mM MOPS, pH 7.00, 25mg/ml BSA, 0.0025% Brij- 35, 1.25% glycerol, 0.5mM EDTA, 25mM MgCl ₂ , 0.025% β-mercaptoethanol, 37.5mM ATP and and 10 μl mixed with 10 μl of substrate mix. The substrate mix

for Aurora is 500μM Kemptide peptide (LRRASLG ₅ Upstate Discovery) in ImI of water with 35 μCi γ ³³ P-ATP. The substrate mix for GSK3-β is 12.5 μM phospho- glycogen synthase peptide-2 (Upstate Discovery) in ImI of water with 35 μCi γ ³³ P- ATP. Enzyme and substrate are added to 96 well plates along with 5 μl of various dilutions of the test compound in DMSO (up to 2.5%). The reaction is allowed to proceed for 30 minutes (Aurora) or 3 hours (GSK3-β) before being stopped with an excess of ortho-phosphoric acid (5 μl at 2%). The filtration procedure is as for Activated CDK2/CyclinA assay above.

EXAMPLE 17 CDK Selectivity Assays

Compounds of the invention were tested for kinase inhibitory activity against a number of different kinases using the general protocol described in Example 16, but modified as set out below.

Kinases are diluted to a 1Ox working stock in 20 mM MOPS pH 7.0, ImM EDTA, 0.1% γ-mercaptoethanol, 0.01% Brij-35, 5% glycerol, lmg/ml BSA. One unit equals the incorporation of lnmol of phosphate per minute into 0.1 mg/ml histone Hl, or CDK7 substrate peptide at 30 °C with a final ATP concentration of 100 μM.

The substrate for all the CDK assays (except CDK7) is histone Hl, diluted to 1OX working stock in 2OmM MOPS pH 7.4 prior to use. The substrate for CDK7 is a specific peptide diluted to 1OX working stock in deionised water.

Assay Procedure for CDKl/cvclinB. CDK2/cvclinA. CDK2/cvclinE. CDK3/cvclinE. CDK5/p35, CDK6/cvclinD3:

In a final reaction volume of 25 μl, the enzyme (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA ₅ 0.1 mg/ml histone Hl, 10 mM MgAcetate and [γ- ³³ P-ATP] (specific activity approx 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of Mg ²⁺ [γ- ³³ P-ATP]. After incubation for 40 minutes at room temperature the reaction is stopped by the addition of 5 μl of a 3% phosphoric acid solution. 10 ml of the reaction is spotted onto a P30 filter mat and

washed 3 times for 5 minutes in 75mM phosphoric acid and once in methanol prior to drying and counting.

Assay procedure for CDK7/cyclinH/MATl

In a final reaction volume of 25μl, the enzyme (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA ₅ 500 μM peptide, 10 mM MgAcetate and [γ- ³³ P- ATP] (specific activity approx 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of Mg ² +[γ- ³³ P-ATP]. After incubation for 40 minutes at room temperature the reaction is stopped by the addition of 5 μl of a 3% phosphoric acid solution. 10 ml of the reaction is spotted onto a P30 filtermat and washed 3 times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and counting.

EXAMPLE 18 Anti-proliferative Activity

The anti-proliferative activities of compounds of the invention can be determined by measuring the ability of the compounds to inhibition of cell growth in a number of cell lines. Inhibition of cell growth is measured using the Alamar Blue assay (Nociari, M. M, Shalev, A., Benias, P., Russo, C. Journal of Immunological Methods 1998, 213, 157-167). The method is based on the ability of viable cells to reduce resazurin to its fluorescent product resorufm. For each proliferation assay cells are plated onto 96 well plates and allowed to recover for 16 hours prior to the addition of inhibitor compounds for a further 72 hours. At the end of the incubation period 10% (v/v) Alamar Blue is added and incubated for a further 6 hours prior to determination of fluorescent product at 535nM ex / 59OnM em. hi the case of the non-proliferating cell assay cells are maintained at confluence for 96 hour prior to the addition of inhibitor compounds for a further 72 hours. The number of viable cells is determined by Alamar Blue assay as before. In addition, any morphological changes are recorded. Cell lines are obtained from ECACC (European Collection of cell Cultures).

By following the protocol set out above, compounds of the invention were found to inhibit cell growth in a number of cell lines.

In tests against the human colorectal carcinoma cell line HCT-116, the compounds of Examples 2 to 13 had IC ₅ O values of less than 15 μM.

EXAMPLE 19

Measurement of inhibitory activity against Glycogen Synthase Kinase-3 (GSK-3)

GSK3β (human) is diluted to a 1Ox working stock in 5OmM Tris pH 7.5, 0.ImM EGTA, O.lmM sodium vanadate, 0.1% β-mercaptoethanol, lmg/ml BSA. One unit equals the incorporation of lnmol of phosphate per minute phospho-glycogen synthase peptide 2 per minute.

In a final reaction volume of 25μl, GSK3β (5-10 mU) is incubated with 8mM MOPS 7.0, 0.2mM EDTA, 20μM YRRAAVPPSPSLSRHSSPHQS(p)EDEEE (phospho GS2 peptide) , 1OmM MgAcetate and [γ- ³³ P-ATP] (specific activity approx 500cpm/pmol, concentration as required). The reaction is initiated by the addition of Mg ² +[γ- ³³ P-ATP] . After incubation for 40 minutes at room temperature the reaction is stopped by the addition of 5μl of a 3% phosphoric acid solution. lOμl of the reaction is spotted onto a P30 filter mat and washed 3 times for 5 minutes in 5OmM phosphoric acid and once in methanol prior to drying and counting.

EXAMPLE 20

Aurora A Kinase assays

Aurora A kinase activity can be determined using a Dissociative Enhanced Lanthanide Fluoro Immuno Assay (DELFIA) with a GSK3-derived biotinylated peptide. The amount of phosphorylated peptide produced is quantified by means of a phospho-specific primary antibody and europium-labelled anti-rabbit IgG antibody using time-resolved fluorescence at λ _ex =337nm, λ _em ⁼⁼ 620nm.

Kinase reaction:

Assay reactions are set up in 96 well plates in a total reaction volume of 25 μl with 0.5 nM Aurora A (Upstate Discovery), 3μM Biotin-

CGPKGPGRRGRRRTSSFAEG, 15μM ATP and various dilutions of compound in 1OmM MOPS, pH 7.0, O.lmg/ml BSA, 0.001% Brij-35, 0.5% glycerol, 0.2mM EDTA, 1OmM MgCl ₂ , 0.01% β-mercaptoethanol & 2.5% DMSO. The reaction is allowed to proceed for 60 minutes at room temperature before stopping with lOOμl STOP buffer containing 10OmM EDTA, 0.05% Surfact-Amps20 (Pierce) and Ix Blocker™ BSA in TBS (Pierce).

Detection step: The reaction mixture is then transferred to a 96-well Neutravidin-coated plate (Pierce) and incubated for 30 minutes to capture the biotinylated peptide. After washing 5 times with 200μl TBST buffer per well, a mixture of anti-phospho- (Ser/Thr)-AKT substrate antibody (Cell Signalling Technology) and Eu-N ₁ anti- rabbit IgG (Perkin Elmer) is added to all wells and left for lhour. After a further washing step, DELFIA enhancement solution (Perkin Elmer) is added to all wells. After an incubation of 5 minutes, the wells are counted on a Fusion plate reader.

EXAMPLE 21 Aurora B Kinase assays

Kinase reaction: Assay reactions are set up in 96 well plates in a total reaction volume of 25 μl with 5nM AuroraB (ProQinase), 3μM Biotin-CGPKGPGRRGRRRTSSFAEG, 15μM ATP and various dilutions of compound in 25mM TRIS pH 8.5, O.lmg/ml BSA, 0.025% Surfact-Amps 20, 5mM MgCl ₂ , ImM DTT, & 2.5% DMSO. The reaction is allowed to proceed for 90 minutes at room temperature before stopping with lOOμl STOP buffer containing 10OmM EDTA, 0.05% Surfact-amρs20 (Pierce) and Ix Blocker™ BSA in TBS (Pierce).

The detection step is carried out as described for AuroraA.

EXAMPLE 22

Determination of Antifungal Activity

The antifungal activity of the compounds of the formula (I) is determined using the following protocol.

The compounds are tested against a panel of fungi including Candida parpsilosis, Candida tropicalis, Candida albicans-ATCC 36082 and Cryptococcus neoformans. The test organisms are maintained on Sabourahd Dextrose Agar slants at 4 °C. Singlet suspensions of each organism are prepared by growing the yeast overnight at 27 °C on a rotating drum in yeast-nitrogen base broth (YNB) with amino acids (Difco, Detroit, Mich.), pH 7.0 with 0.05 M morpholine propanesulphonic acid (MOPS). The suspension is then centrifuged and washed twice with 0.85% NaCl before sonicating the washed cell suspension for 4 seconds (Branson Sonifier, model 350, Danbury, Conn.). The singlet blastospores are counted in a haemocytometer and adjusted to the desired concentration in 0.85% NaCl.

The activity of the test compounds is determined using a modification of a broth microdilution technique. Test compounds are diluted in DMSO to a 1.0 mg/ml ratio then diluted to 64 μg/ml in YNB broth, pH 7.0 with MOPS (Fluconazole is used as the control) to provide a working solution of each compound. Using a 96-well plate, wells 1 and 3 through 12 are prepared with YNB broth, ten fold dilutions of the compound solution are made in wells 2 to 11 (concentration ranges are 64 to 0.125 μg/ml). Well 1 serves as a sterility control and blank for the spectrophotometric assays. Well 12 serves as a growth control. The microtitre plates are inoculated with 10 μl in each of well 2 to 11 (final inoculum size is 10 ⁴ organisms/ml). Inoculated plates are incubated for 48 hours at 35 °C. The IC50 values are determined spectrophotometrically by measuring the absorbance at 420 run (Automatic Microplate Reader, DuP ont Instruments, Wilmington, Del.) after agitation of the plates for 2 minutes with a vortex-mixer (Vorte-Genie 2 Mixer, Scientific Industries, Inc., Bolemia, N. Y.). The IC50 endpoint is defined as the lowest drug concentration exhibiting approximately 50% (or more) reduction of the growth compared with the control well. With the turbidity assay this is defined as the lowest drug concentration at which turbidity in the well is <50% of the control

(IC50). Minimal Cytolytic Concentrations (MCC) are determined by sub-culturing all wells from the 96-well plate onto a Sabourahd Dextrose Agar (SDA) plate, incubating for 1 to 2 days at 35 °C and then checking viability.

EXAMPLE 23 Protocol for the Biological Evaluation of Control of in vivo Whole Plant Fungal Infection

Compounds of the formula (I) are dissolved in acetone, with subsequent serial dilutions in acetone to obtain a range of desired concentrations. Final treatment volumes are obtained by adding 9 volumes of 0.05% aqueous Tween-20 ™ or 0.01% Triton X-100™, depending upon the pathogen.

The compositions are then used to test the activity of the compounds of the invention against tomato blight (Phytophthora infestans) using the following protocol. Tomatoes (cultivar Rutgers) are grown from seed in a soil-less peat-based potting mixture until the seedlings are 10-20 cm tall. The plants are then sprayed to run-off with the test compound at a rate of 100 ppm. After 24 hours the test plants are inoculated by spraying with an aqueous sporangia suspension of Phytophthora infestans, and kept in a dew chamber overnight. The plants are then transferred to the greenhouse until disease develops on the untreated control plants.

Similar protocols are also used to test the activity of the compounds of the invention in combatting Brown Rust of Wheat (Puccinia), Powdery Mildew of Wheat (Ervsiphe vraminis), Wheat (cultivar Monon), Leaf Blotch of Wheat (Septoria tritici), and Glume Blotch of Wheat (Leptosphaeria nodorum).

PHARMACEUTICAL FORMULATIONS

EXAMPLE 24

(i) Tablet Formulation

A tablet composition containing a compound of the formula (I) is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg

magnesium stearate as a lubricant and compressing to form a tablet in known manner.

(ii) Capsule Formulation

A capsule formulation is prepared by mixing 100 mg of a compound of the formula (I) with 100 mg lactose and filling the resulting mixture into standard opaque hard gelatin capsules.

(lip Injectable Formulation I

A parenteral composition for administration by injection can be prepared by dissolving a compound of the formula (I) (e.g. in a salt form) in water containing 10% propylene glycol to give a concentration of active compound of 1.5 % by weight. The solution is then sterilised by filtration, filled into an ampoule and sealed.

(Tv) Injectable Formulation II

A parenteral composition for injection is prepared by dissolving in water a compound of the formula (I) (e.g. in salt form) (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.

(v) Subcutaneous Injection Formulation

A composition for sub-cutaneous administration is prepared by mixing a compound of the formula (I) with pharmaceutical grade corn oil to give a concentration of 5 mg/ml. The composition is sterilised and filled into a suitable container.

(Vi) Injectable formulation III

A formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (I) (e.g. in a salt form) in water at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.

(ViD Injectable formulation IV

A formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (I) (e.g. in a salt form) in water containing a buffer (e.g. 0.2 M acetate pH 4.6) at 20mg/ml. The vial is then sealed and sterilised by autoclaving.

fviii) Subcutaneous Injection Formulation

A composition for sub-cutaneous administration is prepared by mixing a compound of the formula (I) with pharmaceutical grade corn oil to give a concentration of 5 mg/ml. The composition is sterilised and filled into a suitable container.

fix) Lyophilised formulation Aliquots of formulated compound of formula (I) or a salt thereof as defined herein are put into 50 mL vials and lyophilized. During lyophilisation, the compositions are frozen using a one-step freezing protocol at (-45 ⁰ C). The temperature is raised to -10 ⁰ C for annealing, then lowered to freezing at -45 ⁰ C, followed by primary drying at +25 ⁰ C for approximately 3400 minutes, followed by a secondary drying with increased steps if temperature to 50 ⁰ C. The pressure during primary and secondary drying is set at 80 millitor.

Equivalents

The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.