Pyrrolnitrin derivatives

The present invention relates to the medical use of pyrrolnitrin derivatives and particularly their use in the treatment of cancer.

Background

Cancer (or neoplasm) covers a range of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). Cancer affects people at all ages with the risk for most types increasing with age and caused about 13% of all human deaths in 2007. Deaths from cancer worldwide are projected to continue rising, with an estimated 12 million deaths in 2030. (WHO, February 2009)

Traditionally cancer treatment is based around surgery, chemotherapy and radiotherapy. However, the effectiveness of surgery is often limited by the propensity of cancers to invade adjacent tissue or to spread to distant sites by microscopic metastasis. The effectiveness of chemotherapy is often limited by toxicity to other tissues in the body. Radiation can also cause damage to normal tissue.

More recently, targeted therapy has had a significant impact in the treatment of some types of cancer. This constitutes the use of agents specific for the deregulated proteins of cancer cells. Small molecule targeted therapy drugs are generally inhibitors of enzymatic domains on mutated, overexpressed, or otherwise critical proteins within the cancer cell.

The discovery and development of new agents for cancer treatment is both a very costly and very time consuming process. Traditionally agents are selected as having a promising activity against a particular biological target thought to be important in disease; however, little will be known about the safety, toxicity, pharmacokinetics and metabolism of this agent in humans. Therefore, it is traditionally necessary to assess all of these parameters prior to human clinical trials in order to be able to recommend a dose and schedule to be used the first time.

In addition, much drug development work is required to establish the physicochemical properties of a new agent, such as its chemical makeup, stability, solubility. The process l by which the chemical is made will be optimized and it will be further examined for its suitability to be made into capsules, tablets, aerosol, intramuscular injectable, subcuteneous injectable, or intravenous formulations. Many aspects of drug development are focused on satisfying the regulatory requirements of drug licensing authorities. These generally constitute a number of tests designed to determine the major toxicities of a novel compound prior to first use in man. It is a legal requirement that an assessment of major organ toxicity be performed (effects on the heart and lungs, brain, kidney, liver and digestive system), as well as effects on other parts of the body that might be affected by the drug (e.g. the skin if the new drug is to be delivered through the skin). While, increasingly, these tests can be made using in vitro methods (e.g. with isolated cells), many tests can only be made by using experimental animals, since it is only in an intact organism that the complex interplay of metabolism and drug exposure on toxicity can be examined.

The process of drug development does not stop once a novel agent begins human clinical trials. In addition to the tests required to move a novel drug into the clinic for the first time it is also important to ensure that long-term or chronic toxicities are determined, as well as effects on systems not previously monitored (fertility, reproduction, immune system, etc).

If a compound emerges from these tests with an acceptable toxicity and safety profile, and it can further be demonstrated to have the desired effect in clinical trials, then it can be submitted for marketing approval in the various countries where it will be sold. However, most new agents fail during drug development, often because they have some unacceptable toxicity.

Existing anti-cancer agents have considerable problems. A significant problem, which affects the majority of traditional anti-cancer agents, is the severe associated toxicities resulting in side effects such as depletion of the immune system, vomiting, malnutrition, hair loss, fatigue and anemia. Another significant problem is lack of efficacy. Current anti-cancer agents all have limited efficacy and cannot be guaranteed to cure a patient of disease. In some cancers a cure or halt of the disease can actually be very unlikely with the current anti-cancer drugs. Therefore, there remains a clear need to discover and develop new agents for use in the treatment of cancer which reduce the problems associated with current anti-cancer agents. To circumnavigate many of the developmental issues described above, the applicants have developed a method of screening compounds for potential pharmaceutical agents (preferably anti-cancer compounds) based on compounds for which bioactivity and toxicity data is already available. For example, such compounds might have previously been tested for use in unrelated fields, such as agrochemicals.

Here the applicants provide compounds that have been identified by this new screening method for use in medicine, and in particular, cancer treatment.

Pyrrolnitrin is an anti-fungal, secondary metabolite, produced by a range of bacteria. It was originally described as acting by inhibiting the electron transport chain (Lyr, H. 1977). Mutants defective in components of the HOG pathway are resistant to the synthetic derivatives of pyrrolnitrin, fludioxonil and fenpiclonil. These compounds normally induce cell death by activating the HOG pathway resulting in accumulation of intracellular glycerol, cell swelling and ultimately cell death.

There was an active research program on antifungal activity of pyrrolnitrin in- the early 1970's. Several papers arose describing the biosynthesis, metabolism, mechanism of action and effectiveness of pyrrolnitrin as an antibiotic (see Nishida 1965, Wente 1977, Yamada 1968, Macotela 1974, Vanbreuseghem 1977, Seidenari 1990, Uchida 1999, Tripathi 1969, Arima 1965).

However, ultimately the conclusion was drawn that the compound was less effective than rivals "amphotericin, hamcyin, 5-fluorocytosine and saramycetin". In addition, there were reports of cases of contact dermatitis induced by pyrrolnitrin reported from 1975 onwards (Meneghini 1975, Romaguera 1980, Valsecchi 1981 , Meneghini 1982, Balato 1983).

Pyrrolnitrin continues to be marketed in several countries for the treatment of fungal infections of the skin. For example, Pyrrolnitrin alone is marketed by Pharmacia in Italy as Micutrin and in combination with betametasone valerate as Beta Micutrin.

The applicant has now shown that pyrrolnitrin derivatives can be used in the treatment of cancer (such as cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases) and may also be useful in the treatment of vasospasm, pulmonary hypertension, post operative scar formation, multiple sclerosis, hypertension (such as pulmonary hypertension), atherosclerosis, restenosis, cerebral ischemia, neuronal degeneration, nerve (such as spinal cord) injury, thrombotic disorders, asthma, glaucoma osteoporosis, reperfusion injury, asthma, fibrosis, anti-inflammatory diseases, HIV infection, hearing loss, tinnitus, macular degeneration, and other indications mediated by Rho-kinase, e.g., coronary heart disease. In addition, the compounds of the invention are useful to treat erectile dysfunction, i.e. erectile dysfunction mediated by Rho-kinase. Erectile dysfunction can be defined as an inability to obtain or sustain an erection adequate for intercourse.

Fludioxonil and fenpiclonil are two such derivatives that originated from a lead optimisation programme at Ciba Geigy, who used pyrrolnitrin as the starting structure for a developmental chemistry program, with the aim of identifying new fungicides. This is described in Nyfeler and Ackermann (1992) Phenylpyrroles, a new class of agricultural fungicide related to the natural product pyrrolnitrin and in Baker, D. et al, Synthesis and Chemistry of Agrochemicals III (pp 395 - 404) Washington, D.C. American Chemical Society. Fludioxonil and fenpiclonil were further developed by Ciba Geigy and were initially registered as agricultural fungicides in 1991 and 1988 respectively. They are still used commercially for this purpose today.

Fenpiclonil was first registration as agricultural fungicide in 1988, in Switzerland. Commercial products include BERET and GALBAS sold by Syngenta.

Fludioxonil was first registration as agricultural fungicide in 1991, in France. Commercial products include SAPHIRE, CELEST and MAXIM sold by Syngenta. These compounds are not known or sold for the treatment of any non-agricultural use.

It has now been found that derivatives of pyrrolnitrin may be useful in medicine, particularly in the treatment of cancer (such as cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases) and may also be useful in the treatment of vasospasm, pulmonary hypertension, post operative scar formation, multiple sclerosis, hypertension (such as pulmonary hypertension), atherosclerosis, restenosis, cerebral ischemia, neuronal degeneration, nerve (such as spinal cord) injury, thrombotic disorders, asthma, glaucoma osteoporosis, reperfusion injury, asthma, fibrosis, antiinflammatory diseases, HIV infection, hearing loss, tinnitus, macular degeneration, and other indications mediated by Rho-kinase, e.g., coronary heart disease. In addition, the compounds of the invention are useful to treat erectile dysfunction, i.e. erectile dysfunction mediated by Rho-kinase. Erectile dysfunction can be defined as an inability to obtain or sustain an erection adequate for intercourse.

This invention is not intended to cover pyrrolnitrin itself, but only derivatives of pyrrolnitrin as described in the following aspects of the invention.

In a first aspect of the invention there is provided a pyrrolnitrin derivative of formula I or a salt or ester thereof for use in medicine. Formula I:

A = aromatic or heteroaromatic substituted with 1-3 N in the ring

B = aromatic or a partially or fully reduced five or six membered ring.

Either A or B rings can be optionally benzo fused. Q = bond, C1-4 alky, O, -(CH2)mO-(CH2)n- (m and n = 1, 2), S, -(CH2)mS-(CH2)n- (m and n = 1 , 2), NR (R = H or C1-4 alkyl), or -(pH2)mNR-(CH2)n- (m and n = 1 , 2; R = H or C1-4 alkyl) -

Y = (CX) ₂ , CX ₂ , NH, NR (R = H or C1-4 alkyl), S, or O. X = one or more substituents on ring A selected from: H, Halo (from F, CI, Br, I), C1-6 alkyl (linear and branched), C1-6 alkoxy, C2-4 alkenyl, C2-4 alkynyl, C1-4 haloalkyi (halo is 1-3 atoms from F, CI, Br, I), C1-4 haloalkoxy (halo is 1-3 atoms from F, CI, Br, I), OH, SR (R = H or C1-4 alkyl), NR2 (R = H or C1-4 alkyl), NRS02R (R = H or C1-4 alkyl), NRCOR (R = H or C1-4 alkyl), N02, CN, C02R (R = H or C1-4 alkyl), CONR2 (R = H or C1-4 alkyl), S02R (R = H or C1-4 alkyl), SOR (R = H or C1-4 alkyl), S02NR2 (R = H or C1-4 alkyl), OCOR (R = H or C1-4 alkyl), and COR (R = C1-4 alkyl).

Ring A may also be optionally substituted by 2 O atoms that when are adjacent on the ring may together represent the moiety -O-CR'2-O- where R' = H, C1-4 alkyl or F.

Z = one or more substituents on ring B selected from: H, Halo (from F, CI, Br, I), C1-6 alkyl, C1-6 alkoxy, C2-4 alkenyl, C2-4 alkynyl, C1-4 haloalkyi (halo is 1-3 atoms from F, CI, Br, I), C1-4 haloalkoxy (halo is 1-3 atoms from F, CI, Br, I), OH, SR (R = H or C1-4 alkyl), NR2 (R = H or C1-4 alkyl), NRS02R (R = H or C1-4 alkyl), NRCOR (R = H or C1- 4 alkyl), N02, CN, C02R (R = H or C1-4 alkyl), CONR2 (R = H or C1-4 alkyl), S02R (R = H or C1-4 alkyl), SOR (R = H or C1-4 alkyl), S02NR2 (R = H or C1-4 alkyl), OCOR (R = H or C1-4 alkyl), and COR (R = C1-4 alkyl). In one embodiment of the invention the pyrrolnitrin derivative is fludioxonil or a salt or an ester thereof.

Fludioxonil (CAS number: 131341-86-1) is represented by the following structure:

Synonyms for Fludioxonil

1 ) 4-(2,2-difluoro-1 ,3-benzdioxol-4-yl)-1 h-pyrrole-3-carbonitrile

2) 4-(2,2-difluoro-1 ,3-benzodioxol-4-yl)-1h-pyrrole-3-carbonitril

3) 4-(2,2-difluoro-1 ,3-benzodioxol-4-yl)-1h-pyrrole-3-carbonitrile

4) cgal 73506

5) CELEST 6) FLUDIOXONIL

7) MAXIM

8) 1 H-Pyrrole-3-carbonitrile, 4-(2,2-difluoro-1 ,3-benzodioxol-4-yl)-

9) fludioxonil (bsi, pa e-iso)

10) 4-(2,2-difluoro-1 ,3-benzodioxol-4-vl) pyrrole-3-carbonitrile

11) Celest Saphire

12) Fludioxinil

In a further embodiment of the invention, the pyrrolnitrin derivative is fenpiclonil or a salt or an ester thereof. Fenpiclonil (CAS number: 74738-17-3) is represented by the following structure:

Synonyms for Fenpiclonil

1) GAMBIT

2) BERET

3) 3-(2,3-dichlorophenyl)-4-cyanopyrrole

4) 4-(2,3-dichlorophenyl)-1 h-pyrrole-3-carbonitril

5) 4-(2,3-dichlorophenyl)-1 H-pyrrole-3- carbonitrile

6) 4-cyano-3-(2,3-dichlorophenyl)pyrrole

7) cga142705

It has now been found that Fludioxonil and Fenpiclonil are inhibitors of PIM kinase family members (PIM1 , PIM2 and PIM3) and also of ROCKIl kinase.

PIM Kinase activity

PIM kinases are cytoplasmic serine/threonine kinases that are known to be involved in regulation of apoptosis and cellular metabolism. Certain PIM kinases have been shown to be upregulated in cancers and as such their inhibition represents a mechanism of action by which Fludioxonil and Fenpiclonil can have an anti-tumour effect in conditions such as leukaemia, lymphoma, prostate cancer, colon cancer and pancreatic cancer. The below studies have shown this link:

Liver cancer: Gong (2009), Fujii (2005) and Wu (2010) have shown PIM-2 to promote humourigenesis and PIM-3 to accelerate hepatocellular carcinoma development when induced by hepatocarcinogen.

Gastric cancer: Zhen (2008) and Warnecke-Eberz (2009) have shown overexpression of PIM-1 in gastric glands to be associated with lymph node metastases.

Head and neck cancer: Beier (2007) has shown PIM-1 overexpression in head and neck squamous cell carcinomas.

Colon cancer: Popivanova (2007) has shown PIM-3 to be aberrantly expressed in human colon cancer cells but not normal colon mucosa.

Pancreatic cancer: Li (2006), Chen (2009) and Reiser-Erkan (2008) have shown

PIM-3 expression occurs in human pancreatic cancer but not normal cells and PIM-1 blockage using siRNA resensitises pancreatic cancer cells to apoptosis and PIM-1 levels correlate to clinicopathological parameters in pancreatic cancer.

Leukaemia/ly m phoma : Adam (2006), Hammerman (2005), Cohen (2004), Hogan

(2008), Lin (2010), Kim (2005), Chen (2008) and Brault (2010) have shown PIM-2 expression is increased in leukaemia/lymphoma, expression of PIM-1 and PIM-2 is dependent on Abl kinase activity and PIM-1 mediates homing and migration of malignant haematopoietic cells.

Oral cancer: Chiang (2006) and Choi (2010) have shown PIM-1 expression to be high in squamous cell carcinoma. Prostrate cancer: Chen (2005), umenthaler (2009), He (2007), Xu (2005), Dai (2005) and Roh (2008) have shown PIM-1 overexpression in prostatic carcinoma.

Breast cancer: Roh (2008) has shown PIM-1 overexpression to convert mammary epithelia cells to become tumourgenic.

Adipocyte tumours: Nga (2010) has shown benign and malignant adipocytic tumours to have strong PIM-1 expression.

PIM kinases are constitutively active and their activity as shown above and in Amaravadi (2005) and Shah (2008) supports in vitro and in vivo human cell growth and survival. ROCK II kinase

The pathology of a number of human and animal diseases including hypertension, erectile dysfunction, coronary cerebral circulatory impairments, neurodegenerative disorders and cancer can be linked directly to changes in the actin cytoskeleton. These diseases pose a serious unmet medical need. The actin cytoskeleton is composed of a meshwork of actin filaments and actin-binding proteins found in all eukaryotic cells. In smooth muscle cells the assembly and disassembly of the actin cytoskeleton is the primary motor force responsible for smooth muscle contraction and relaxation. In non- muscle cells, dynamic rearrangements of the actin cytoskeleton are responsible for regulating cell morphology, cell motility, actin stress fiber formation, cell adhesion and specialized cellular functions such as neurite retraction, phagocytosis or cytokinesis (Van Aelst, et al. Genes Dev 1997,11 ,2295).

The actin cytoskeleton is controlled by a family of proteins that are a subset of the Ras superfamily of GTPases. This subset currently consists of RhoA through E and RhoG (refereed to collectively as Rho), Rac 1 and 2, Cdc42Hs and G25K and TC10 isoforms (Mackay, et al. JBiol Chem 1998,273,20685). These proteins are GTP (guanine nucleotide triphosphate) binding proteins with intrinsic GTPase activity. They act as molecular switches and cycles between inactive GDP (guanine nucleotide diphosphate) bound and active GTP bound states. Using biochemical and genetic manipulations, it has been possible to assign functions to each family member. Upon activation the Rho proteins controls the formation of actin stress fibers, thick bundles of actin filaments, and the clustering of integrins at focal adhesion complexes. When activated the Rac proteins control the formation of lamellopodia or membrane ruffles on the cell surface and Cdc42 controls filopodia formation. Together this family of proteins plays a critical part in the control of key cellular functions including cell movement, axonal guidance, cytokinesis, and changes in cell morphology, shape and polarity.

Depending on the cell type and the activating receptor, the Rho proteins can control different biological responses. In smooth muscle cells, Rho proteins are responsible for the calcium sensitization during smooth muscle contraction. In non-smooth muscle cells the Rho GTPases are responsible for the cellular responses to agonist such as lysophosphatidic acid (LPA), thrombin and thromboxane A2 (Fukata, et al. Trends Pharcol Sci 2001 , 22,32).

Agonist response is coupled through heterotrimeric G proteins Galphal2 or Galphal3 (Goetzl, et al. Cancer Res 1999,59,4732; Buhl, et al. J Biol Chem 1995,270,24631) though other receptors may be involved. Upon activation Rho GTPases activate a number of downstream effectors including PIP5-kinase, Rhothekin, Rhophilin, PKN and Rho-Kinase isoforms ROCK-1/ROKbeta and ROCK-2/ROKalpha (Mackay and Hall J Biol Chem 1998,273, 20685; Aspenstrom Curr Opin Cell Biol 1999, 11 , 95; Amano, et al. Exp Cell Res 2000,261 , 44). Rho-kinase was identified as a RhoA interacting protein isolated from bovine brain (Matsui, et al. Embo J 1996,15,2208). It is a member of the myotonic dystrophy family of protein kinase and contains a serine/threonine kinase domain at the amino terminus, a coiled- coil domain in the central region and a Rho interaction domain at the carboxy terminus (Amano, et al. Exp Cell Res 2000,261 ,44). Its kinase activity is enhanced upon binding to GTP-bound RhoA and when introduced into cells, it can reproduce many of the activities of activated RhoA. In smooth muscle cells Rho-Kinase mediates calcium sensitization and smooth muscle contraction and inhibition of Rho-kinase blocks 5-HT and phenylephrine agonist induced muscle contraction. When introduced into non- smooth muscle cells, Rho- kinase induces stress fiber formation and is required for the cellular transformation mediated by RhoA (Sahai, et al. Curr Biol 1999, 9,136). Rho- kinase regulates a number of downstream proteins through phosphorylation, including myosin light chain (Somlyo, et al. J Physiol (Lond) 2000,522 Pt 2,177), the myosin light chain phosphatase binding subunit (Fukata, et al. J Cell Biol 1998, 141 , 409) and LIM- kinase 2 (Sumi, et al. J Bio Chem 2001 , 276,670). ROCK kinases are effectors of the small GTPase Rho and belong to the AGC family of kinases. The Rho-associated kinases (ROCKs) are serine/threonine protein kinases that serve as key downstream effectors of the Rho GTPase, RhoA, and play an important role in cytoskeletal function. The Rho/ROCK signal transduction participates in signalling pathways via rearrangement of the actin cytoskeleton that leads to various cellular reactions. The ROCKs impact cytoskeletal function through their ability to phosphorylate a range of proteins that directly regulate cytoskeletal proteins, including myosin light chain kinase, myosin light chain phosphatase, beta-catenin, cofilin, FAK and LIMK, and thus the ROCKs are positioned to play essential roles in the physiological and pathophysiological processes of various cell types. The ROCK enzymes, ROCK1 and ROCK2, share 65% overall homology. Gene knockout and siRNA studies suggest that each isoform plays distinct roles in mammalian biology. Despite having similar kinase domains, ROCK1 and ROCK2 may serve different functions and may have different downstream targets. ROCK1 expression tends to be ubiquitous, while ROCK2 is most highly expressed in cardiac and brain tissues. Clinically, inhibition of the ROCK pathway is believed to contribute to some of the cardiovascular benefits of statin therapy that are independent of lipid lowering (Liao et al 2010, J Cardiovascular Pharmacol 2007, 50(1):17-24).

Rho kinase is involved in a wide range of diseases such as glaucoma, vasospasm, pulmonary hypertension multiple sclerosis and nerve injury. Pharmacology studies of ROCK kinase inhibitors have shown that compounds of this activity are effective in animal models of glaucoma (for example at reducing intraocular pressure (IOP) in animal models by improving aqueous hour drainage through the trabecular pathway). Inhibition of Rho-kinase activity in animal models has demonstrated a number of benefits of Rho-kinase inhibitors for the treatment of human diseases. Several patents have appeared claiming (+)-trans-4- (1-aminoethyl)-1- (pyridin-4-ylaminocarbonyl) cyclohexane dihydrochloride monohydrate (WO-00078351 , WO-00057913) and substituted isoquinolinesulfonyl (EP-00187371) compounds as Rho-kinase inhibitors with activity in animal models. These include models of cardiovascular diseases such as hypertension (Uehata, et al. Nature 1997,389,990), atherosclerosis (Retzer, et al. FEBS Lett 2000, 466, 70), restenosis (Eto, et al. Am JPhysiol Heart Circ Physiol 2000, 278, H1744 ; Negoro, et al.

Biochem Biophys Res Commun 1999,262,211), cerebral ischemia (Uehata, et al. Nature 1997,389,990; Seasholtz, et al. Circ Res 1999, 84, 1186; Hitomi, et al. Life Sci 2000,67, 1929; Yamamoto, et al. J Cardiovasc Pharmacol 2000, 35, 203), cerebral vasospasm (Sato, et al. Circ Res 2000,87,195; Kim, et al. Neurosurgery 2000,46,440), penile erectile dysfunction (Chitaley, et al. Nat Med 2001,7,119), central nervous system disorders such as neuronal degeneration and spinal cord injury (Hara, et al. J Neurosurg 2000,93,94; Toshima, et al. Stroke 2000,31 ,2245) and in neoplasias where inhibition of Rho-kinase has been shown to inhibit tumor cell growth and metastasis (Itoh, et al. Nat Med 1999,5,221; Somlyo, et al. Biochem Biophys Res Commun 2000,269,652), angiogenesis (Uchida, et al. Biochem Biophys Res Commun 2000,269,633; Gingras, et al. Biochem J 2000,348 Pt 2, 273), arterial thrombotic disorders such as platelet aggregation (Klages, et al. I Cell Biol 1999,144,745; Retzer, et al. Cell Signal 2000, 12, 645) and leukocyte aggregation (Kawaguchi, et al. Eur JPharmacol 2000, 403, 203; Sanchez-Madrid, et al. Embo J 1999, 18, 501), asthma (Setoguchi, et al. Br J Pharmacol 2001, 132, 111; Nakahara, et al. Eur J Pharmacol 2000, 389, 103), regulation of intraoccular pressure (Honjo, et al. Invest Ophthalmol Vis Sci 2001, 42, 137) and bone resorption (Chellaiah, et al. JBiol Chem 2000, 275, 11993; Zhang, et al. J Cell Sci 1995, 108, 2285).

The inhibition of Rho-kinase activity in patients has benefits for controlling cerebral vasospasms and ischemia following subarachnoid hemorrhage (Pharma Japan 1995,1470, 16).

Non-isoform selective ROCK inhibitors such as fasudil have been shown to prevent cerebral vasospasm after subarachnoid hemorrhage. Despite the potential clinical importance of ROCK inhibition, fasudil is the only ROCK inhibitor approved for clinical use. Fasudil was approved in 1995 in Japan and China for prevention and treatment of cerebral vasospasm following surgery for subarachnoid hemorrhage and has since been used in over 124,000 patients in Japan. Many pharmaceutical and biotechnology companies are developing other selective and non-selective ROCK inhibitors (see table below modified and updated from Liao et al 2007). The proven safety and efficacy of fasudil suggests that ROCK inhibition will not have any safety limiting factors and that ROCK is a significant and genuine drug target. However even the relatively selective ROCK inhibitors such as Y27632 have been shown to inhibit additional kinases. Compounds selective for ROCK represent a significant advancement over those described currently as ROCK inhibitors.

ROCK inhibitors in development Compound Therapeutic Area Company Development status

(intravenous and vasospasm, acute Pharmaceuticals for cerebral oral formulation) stroke, angina, Inc (licensed to vasospasm and (also known as HA- , pulmonary CoTherix in USA ischemia. Phase 2 1077) hypertension and Europe for use for other

in pulmonary indications hypertension)

Y27632 Reperfusion injury, Mitsubishi Discontinued, hypertension, Pharmaceuticals research tool stroke, asthma,

cancer

Y39983 Glaucoma Mitsubishi Phase 1

(Ophthalmic liquid Pharmaceuticals,

formulation) Senju

Pharmaceuticals,

Novartis AG

Wf-536 Cancer Mitsubishi Discovery Phase

Pharmaceuticals

SLx-2119 Artherosclerosis, Surface Logix Inc Discovery Phase fibrosis, solid

tumours

Azabenzimidazole- Hypertension antiGlaxo Smithkline Discovery Phase aminofurazans inflammatory

DE-104, Olefins, Glaucoma Santern Discovery Phase

Isoquinolines, Pharmaceuticals,

Indazoles, UBE Industries

pyridinealkene

derivatives

H-1152P Glaucoma Kowa Discovery Phase

Pharmaceuticals

ROCK inhibitor Asthma, BioFocus pic Discovery Phase cardiovascular

disease, erectile

dysfunction,

glaucoma, HIV

infection,

osteoporosis

XD-400 Atherosclerosis, Xcellsyz Ltd Discovery Phase fibrosis, solid

tumours

HMN-1152 Hearing loss, Nagoya University Discovery Phase tinnitus,

hypertension,

cerebrovascular

disease

AR-12286 Glaucoma Aerie Phase 2

Pharmaceuticals

Rhostatin Solid tumours, BioAxone Discovery Phase brain tumours Therapeutics

BA-210 Macular BioAxone Phase 2

degeneration, Therapeutics

spinal cord injury, Compound Therapeutic Area Company Development

status

glaucoma, cancer

Ki-23095 Cancer VasGene Discovery Phase

Therapeutics

ROCK inhibitor Cancer Bayer AG Discovery Phase (quinazoline)

Advantages of a combination PIM kinase inhibitor and ROCK kinase inhibitor

In some situations it may be advantageous for a compound to inhibit more than one or multiple protein kinases for maximal biological effect. Compounds that inhibit both PIM kinase and ROCK kinase may have a particular advantage over and above those inhibiting these kinases individually because the PIM kinase and ROCK kinase pathways intersect through p21Cip1/WAF1 (hereafter referred to as p21). In particular PIM kinase phosphorylates p21 on Thr 145 and thus regulates its stability and cellular localization in cells (Zhang et al., 2007, Mol Cancer Res 5(9): 909-922). When p21 is phosphorylated on Thr 145 it localizes to the nucleus and results in the disruption of the association between proliferating cell nuclear antigen (PCNA) and p21. Furthermore phosphorylation of Thr 145 promotes stabilization of p21. Inhibition of PIM and the consequent reduction in phosphorylation of Thr 145 of p21 would result in reduced stability of p21, and a reduction in cell proliferation. P21 also intersects with the ROCK/LIMK/Cofilin pathway. P21 forms a physical complex with ROCK and inhibits its activity (Lee et al., 2004, Journal Biological Chemistry 279(3): 1885-1891).

Ras activates p21 transcription and promotes p21 protein stability and it has been suggested that p21 is an essential factor in the signalling pathways that contribute to Ras-induced actin cytoskeletal re-modelling. The inhibition of PIM and ROCK together may therefore be particularly significant in tumours dependent upon or transformed through Ras mutation.

Pyrrolnitrin derivatives of formula I and salts thereof are referred to herein as "the compounds of the invention":

Pharmaceutically-acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

By the term "ester" is included those formed with an alcohol of formula R ¹ OH, wherein R ¹ represents aryl or alkyl; and those formed with a thiol of formula R SH, wherein R ¹ is as hereinbefore defined (i.e. a thioester). It is preferred that the ester is not a thioester. In embodiments R ¹ represents C _1-6 alkyl, for example Ci alkyl (eg methyl).

Compounds of the invention may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

Compounds of the invention may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of the .invention may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a 'chiral pool' method), by reaction of the appropriate starting material with a 'chiral auxiliary' which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

Unless otherwise specified, C _1-q alkyl groups (where q is the upper limit of the range) defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of two or three, as appropriate) of carbon atoms, be branched-chain, and/or cyclic (so forming a Ca-q-cycloalkyl group). Such cycloalkyl groups may be monocyclic or bicyclic and may further be bridged. Further, when there is a sufficient number (i.e. a minimum of four) of carbon atoms, such groups may also be part cyclic. Such alkyl groups may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms, be unsaturated (forming, for example, a C ₂ . _q alkenyl or a C _2-q alkynyl group). Where the number of carbon atoms permits, C _1-q alkyl groups may also be spiro-groups (i.e. two cycloalkyi rings linked together by a single common carbon atom), although they are preferably not so.

The term "halo", when used herein, includes fluoro, chloro, bromo and iodo.

Heterocycloalkyi groups that may be mentioned include non-aromatic monocyclic and bicyclic heterocycloalkyi groups (which groups may further be bridged) in which at least one (e.g. one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom), and in which the total number of atoms in the ring system is between three and twelve (e.g. between five and ten). Further, such heterocycloalkyi groups may be saturated or unsaturated containing one or more double and/or triple bonds, forming for example a C _2-q heterocycloalkenyl (where q is the upper limit of the range) or a C _7-q heterocycloalkynyl group. C _2-q heterocycloalkyi groups that may be mentioned include 7- azabicyclo-[2.2.1]heptanyl, 6-azabicyck>[3.1.1]heptanyl, 6-azabicyclo[3.2.1]-octanyl, 8- azabicyclo[3.2.1]octanyl, aziridinyl, azetidinyl, dihydropyranyl, dihydropyridyl, dihydropyrrolyl (including 2,5-dihydropyrrolyl), dioxolanyl (including 1 ,3-dioxolanyl), dioxanyl (including 1 ,3-dioxanyl and 1 ,4-dioxanyl), dithianyl (including 1,4-dithianyl), dithiolanyl (including 1 ,3-dithiolanyl), imidazolidinyl, imidazolinyl, morpholinyl, 7- oxabicyclo[2.2.1]heptanyl, 6-oxabicyclo[3.2.1]-octanyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolidinonyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, sulfolanyl, 3-sulfolenyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydropyridyl (such as 1 ,2,3,4-tetrahydropyridyl and 1 ,2,3,6-tetrahydropyridyl), thietanyl, thiiranyl, thiolanyl, thiomorpholinyl, trithianyl (including 1 ,3,5-trithianyl), tropanyl and the like. Substituents on heterocycloalkyi groups may, where appropriate, be located on any atom in the ring system including a heteroatom. Further, in the case where the substituent is another cyclic compound, then the cyclic compound may be attached through a single atom on the heterocycloalkyi group, forming a so-called "spiro"-compound. The point of attachment of heterocycloalkyi groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system. Heterocycloalkyi groups may also be in the N- or S- oxidised form.

For the avoidance of doubt, the term "bicyclic" (e.g. when employed in the context of heterocycloalkyi groups) refers to groups in which the second ring of a two-ring system is formed between two adjacent atoms of the first ring. The term "bridged" (e.g. when employed in the context of heterocycloalkyi groups) refers to monocyclic or bicyclic groups in which two non-adjacent atoms are linked by either an alkylene or heteroalkylene chain (as appropriate).

Aryl groups that may be mentioned include C _6- 14 (such as Ce.^ (e.g. C _6-10 )) aryl groups. Such groups may be monocyclic or bicyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. C _6- i ₄ aryl groups include phenyl, naphthyl and the like, such as 1,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are preferably linked to the rest of the molecule via an aromatic ring.

Heteroaryl groups that may be mentioned include those which have between 5 and 14 (e.g. 10) members. Such groups may be monocyclic, bicyclic or tricyclic, provided that at least one of the rings is aromatic and wherein at least one (e.g. one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom). Heteroaryl groups that may be mentioned include acridinyl, benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzodioxolyl (including 1,3-benzodioxolyl), benzofuranyl, benzofurazanyl, benzothiazolyl, benzoxadiazolyl (including 2,1 ,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro-2H-1,4-benzoxazinyl), benzoxazolyl, benzomorpholinyl, benzoselenadiazolyl (including 2,1 ,3-benzoselenadiazolyl), benzothiadiazolyl (including 2,1 ,3-benzothiadiazolyl), benzothienyl, carbazolyl, chromanyl, cinnolinyl, furanyl, imidazolyl, imidazopyridyl (including imidazo[4,5-fo]pyridyl, imidazo[5,4- ?]pyridyl and imidazo[1 ,2-a]pyridyl), indazolyl, indolinyl, indolyl, isobenzofuranyl, isochromanyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiaziolyl, isothiochromanyl, isoxazolyl, naphthyridinyl (including 1 ,6-naphthyridinyl or, preferably, 1 ,5-naphthyridinyl and 1 ,8- naphthyridinyl), oxadiazolyl (including 1,3,4-oxadiazolyl), oxazolyl, phenazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolizinyl, quinoxalinyl, tetrahydroiso- quinolinyl (including 1,2,3,4-tetrahydroisoquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl), tetrahydroquinolinyl (including 1,2,3,4-tetrahydroquinolinyl and 5,6,7,8- tetrahydroquinolinyl), tetrazolyl, thiadiazolyl (including 1 ,3,4-thiadiazolyl), thiazolyl, oxazolopyridyl (including oxazolo[4,5-0]pyridyl, oxazolo[5,4- )]pyridyl and, in particular, oxazolo[4,5-c]pyridyl and oxazolo[5,4-c]pyridyl), thiazolopyridyl (including thiazolo[4,5- b] pyridyl, thiazolo[5,4-/>]pyridyl and, in particular, thiazolo[4,5-c]pyridyl and thiazolo[5,4- c] pyridyl), thiochromanyl, thienyl, triazolyl (including 1 ,2,3-triazolyl and 1 ,2,4-triazolyl) and the like. Substituents on heteroaryl groups may, where appropriate, be located on any atom in the ring system including a heteroatom. The point of attachment of heteroaryl groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system. However, when heteroaryl groups are polycyclic, they are preferably linked to the rest of the molecule via an aromatic ring. Heteroaryl groups may also be in the N- or S- oxidised form.

Heteroatoms that may be mentioned include phosphorus, silicon, boron, tellurium, selenium and, preferably, oxygen, nitrogen and sulphur.

For the avoidance of doubt, in cases in which the identity of two or more substituents in a compound of the invention may be the same, the actual identities of the respective substituents are not in any way interdependent. For example, in the situation in which two X ¹ groups are present, which both represent R ^5a , i.e. a C _1-6 alkyl group optionally substituted as hereinbefore defined, the alkyl groups in question may be the same or different. Similarly, when groups are substituted by more than one substituent as defined herein, the identities of those individual substituents are not to be regarded as being interdependent. For example, when there are two X ¹ substituents present, which represent -R ^5a and -C(0)R ^5b in which R ^5b represents R ^5a , then the identities of the two R ⁵³ groups are not to be regarded as being interdependent. Likewise, when Y ² or Y ³ represent e.g. an aryl group substituted by G ¹ in addition to, for example, C _1-8 alkyl, which latter group is substituted by G , the identities of the two G groups are not to be regarded as being interdependent. In a second aspect of the invention, there is provided a pharmaceutical composition comprising a pyrrolnitrin derivative of formula I or a salt or ester thereof and a pharmaceutically acceptable excipient, diluent or carrier.

In one embodiment of the invention there is provided a composition comprising between 10mg and 2000mg of an active ingredient per dosage unit, wherein the active ingredient is a compound of the invention or a derivative, salt or variant thereof.

By dosage unit we mean the unit of medicament administered to a patient at one time. For example, the dosage unit, or single dose may be administered by a single capsule/tablet, single injection, or single intravenous infusion, a single subcutaneous injection, or by a single procedure using other routes of administration, as discussed below. Alternatively, the single dose may be administered to the patient by two or more capsules/tablets or injections given simultaneously or sequentially to deliver the entire dose to the patient in the continuous, single and defined treatment period; by two or more intravenous infusions given simultaneously or sequentially to deliver the entire dose to the patient in the continuous, single and defined treatment; or by multiple procedures using other routes of administration as discussed below.

Alternatively, the single dose to be administered to the patient can be delivered by a combination of routes to deliver the entire dose to the patient in the continuous, single and defined treatment.

The dosage unit may then be repeated at intervals of time such as a few hours, days, weeks, or months later.

Dosage units can be administered to patients in such a way that the patient receives a loading dose followed by one or more maintenance doses. For example the loading dose may be a high dose in order to quickly reach a desired plasma concentration and then subsequent maintenance doses are a lower dose than the loading dose in order to maintain the required plasma concentration. By active ingredient we mean the molecule having the desired effect. In this case of this invention we primarily mean the compounds of the invention and derivatives, salts or variants thereof.

By variants and derivatives we mean any molecules of substantially identical chemical structure but including minor modifications that do not alter activity but may offer improved or alternative properties for formulation, such as formation into a salt.

In human therapy, the compound of the invention containing composition, and medicaments of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the compound of the invention containing composition, and medicaments of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compound of the invention containing composition, and medicaments of the invention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compound of the invention containing composition, medicaments and pharmaceutical compositions of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol,. propylene glycol and glycerin, and combinations thereof. The compound of the invention containing composition, and medicaments of the invention can also be administered parenterally, for example, intravenously, intra- arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, 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. The medicaments and pharmaceutical compositions 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. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The compound of the invention containing composition, and medicaments of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active agent, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant,, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention containing composition, of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff contains an effective amount of an agent or polynucleotide of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the compound of the invention containing composition, and medicaments of the invention 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, gel, ointment or dusting powder. The compound of the invention containing composition, and medicaments of the invention may also be transdermal^ administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the compound of the invention containing composition, and medicaments of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum. For application topically to the skin, the compound of the invention containing composition, and medicaments of the invention can be formulated as a suitable ointment containing the active agent suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Generally, in humans, oral or parenteral administration of the compound of the invention containing composition, medicaments and pharmaceutical compositions of the invention is the preferred route, being the most convenient. For veterinary use, the compound of the invention containing composition, and medicaments of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

The compound of the invention containing composition, as defined herein may be formulated as described in the accompanying Examples.

The composition may comprise any effective amount of active ingredient, this may be between 10mg and 2000mg of active ingredient per dosage unit, and preferably is between 50mg and lOOOmg. Advantageously it is lOOOmg. Conveniently, the dosage unit contains an amount of active ingredient per dosage unit selected from 10mg, 20mg, 25mg, 50mg, 100mg, 200mg, 300mg, 400mg, 450mg, 600mg, 750mg, 950mg, 10OOmg and 1200mg.

Alternatively, the composition may comprise between 10-50mg, 10-75mg, 10-100mg, 10- 200mg, 10-300mg, 10-400mg, 10-600mg, 10-750mg, 10-950mg, 10-1 OOOmg, 10- 1200mg, 50-75mg, 50-100mg, 50-200mg, 50-300mg, 50-450mg, 50-600mg, 50-750mg, 50-950mg, 50-1 OOOmg, 50-1200mg, 75-1 OOmg, 75-200mg, 75-300mg, 75-450mg, 75- 600mg, 75-750mg, 75-950mg, 75-1000mg, 75-1200mg, 100-200mg, 100-300mg, 100- 450mg, 100-600mg, 100-750mg, 100-950mg, 100-1 OOOmg, 100-1200mg, 200-300mg, 200-450mg, 200-600mg, 200-750mg, 200-950mg, 200-1 OOOmg, 200-1200mg, 300- 450mg, 300-600mg, 300-750mg, 300-950mg, 300-1 OOOmg, 300-1200mg, 400-600 mg, 400-750mg, 400-950mg, 400-1 OOOmg, 400-1200mg, 450-600mg, 450-750mg, 450- 950mg, 450-1 OOOmg, 450-1200mg, 600-750mg, 600-950mg, 600-1 OOOmg, 600-1200mg, 700-950mg, 700-IOOOmg, 700-1200mg, 950-1000mg, 950-1200mg and 1000-1200mg

Preferably there is 400-600mg of active ingredient. More preferably there is 400-1200mg of active ingredient. Most preferably there is 10OOmg of active ingredient.

Conveniently, the composition is pharmaceutically acceptable, and may optionally contain a pharmaceutically acceptable excipient, diluent, carrier or filler.

The examples describe some methods of producing pharmaceutical formulations, however the skilled person will appreciate that the most appropriate formulation will depend on a number of factors including route of administration, patient type (e.g. patient age, weight/size).

A further aspect of the invention provides a method of treating cancer (such as cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases) comprising administering to the patient an effective amount of a pyrrolnitrin derivative of formula I or a salt or an ester thereof.

A further aspect of the invention provides a method of treating diseases and conditions selected from vasospasm, pulmonary hypertension, post operative scar formation, multiple sclerosis, hypertension (such as pulmonary hypertension), atherosclerosis, restenosis, cerebral ischemia, neuronal degeneration, nerve (such as spinal cord) injury, thrombotic disorders, asthma, glaucoma osteoporosis, reperfusion injury, asthma, fibrosis, anti-inflammatory diseases, HIV infection, hearing loss, tinnitus, macular degeneration, and other indications mediated by Rho-kinase, e.g., coronary heart disease. In addition, the compounds of the invention are useful to treat erectile dysfunction, i.e. erectile dysfunction mediated by Rho-kinase. Erectile dysfunction can be defined as an inability to obtain or sustain an erection adequate for intercourse.

The term "patient" includes all animals including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, and pigs. The term "patient" means an animal having a disorder in need of treatment. Preferably, the patient is selected from humans, cows, dogs, cats, goats, sheep, and pigs. More preferably, the patient is a human.

The compounds of the invention may be used in cancer treatment either alone or in combination with well known anti-cancer agents.

Cancer treatments promote tumour regression by inhibiting tumour cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumour growth) and/or prohibiting- metastasis by reducing tumour cell motility or invasiveness. Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumours/malignancies, locally advanced tumours, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumour in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumours, neuroblastoma, astrocytic brain tumours, gliomas, metastatic tumour cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumour progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma. Compounds of the invention may be administered to treat cancer. The cancer to be treated is preferably selected from pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, liver cancer, chondrosarcoma, lung cancer, head and neck cancer, colon cancer, sarcoma, leukaemia, myeloma, lymphoma, kidney cancer, thyroid cancer and brain cancers such as glioblastoma.

Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumour size, slowing rate of tumour growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.

The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of the compound or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine. Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the compounds of the invention include: 17AAG, 17DMAG, 5FU, 7-hydroxystaurosporine (UCN-01), ABT888, Actinomycin D, Alsterpaullone, Axitinib, Aminoglutethimide, Amsacrine, Asparaginase, Azacitidine, AZD7762, Bay 11-7082, Belinostat, Bendamustine, Bexarotene, BIBW 2992, Bisindolylmaleimide I, Bleomycin, Bortezomib, Bosutinib, Busulfan, Canertinib , Capecitabine, Carboplatin, Carmustine, CDK4/6 IV, Chelerythrine Chloride, Chlorambucil, CHR-2863 (CHROMA), CHR-3531 (CHROMA), Chromomycin A3, CI-994, Cisplatin, Cladribine, Clofarabine, Clofibrate, CP690550, CXR1002 (APFO), Cyclopamine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin, DBZ, Decitabine, dk2 inhibitor II, DMFO, Docetaxel, Doramapimod, Dovitinib, Doxorubicin, Entinostat, Epirubicin, Erlotinib, Estramustine, Etoposide (V16- 213), Everolimus, Flavopiridol, fludarabine phosphate, Flutamide, GDC-0449, Gefitanib, Geldanamycin, Go 6976, GSK-1904529A, GW2580, GW501516, HA14-1 , Hexamethylmelamine, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha- 2a, Interferon Alpha-2b, lnterleukin-2, Leuprolide acetate (LHRH-releasing factor analog), Idarubicin, Imatinib, Ispinesib mesilate, Kenpaullone, K0143, KU-0058948, Lapatinib, Lenalidomide, Lestaurtinib, Lomustine, LY 2157299, LY294002, Masitinib, Mechlorethamine HCI (nitrogen mustard), Melphalan, Mercaptopurine (6-MP), Mesna, MTX, Mitoguazone, Mitomycin C, MK 1775, Mocetinostat, Mitoxantrone HCI, Nelarabine, Nilotinib, Nobiletin, Nogalamycin, NSC 625987, Nutlin-3, NVP-AEW541 , NVP-TAE684 - TAE 684, Obatoclax mesylate, Octreotide, Olaparib (KU0059436), Oxaliplatin, Pentostatin, Plicamycin, Procarbazine HCI, Paclitaxel, Panobinostat, Pazopanib, PD 98059, PD173074, Pemetrexed, Perifosine, perillic acid, PLX4032, PLX4720, PPP (Picropodophyllin), Puromycin, Radicicol, Raltitrexed, Rapamycin, Ridaforolimus, Semustine, Streptozocin, Saracatinib, SB 431542, SB202190, SB203580, Sorafenib, SP600125, Sunitinib, Suramin, Tamoxifen CITRATE, Teniposide, Tandutinib, Tegafur , Temodal, Thalidomide, Thioguanine (6-TG), Thiotepa, Topotecan hydrochloride hydrate, Tozasertib, Trichostatin A, Tyrphostin AG 490, Tyrphostin AG 538, Tyrphostin AG879, U0126, Valproic acid, Vandetanib, Vatalanib, Vinblastine sulfate salt, Vincristine, Vindesine sulphate, Vinolrelbine ditartrate salt hydrate, Wortmannin, XAV 939, YM155, ZSTK474,4HC,6-[4-(2-Piperidin-1-ylethoxy)phenyl]-3-pyridin-4 -ylpyrazolo[1 ,5- ajpyrimidine (Dorsomorphin), Etoposide, Gemcitabine, Mitoxanthrone and Vorinostat.

In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer. There are hereditary conditions and/or environmental situations (e.g. exposure to carcinogens) known in the art that predispose an individual to developing cancers. Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the compounds of the invention to reduce the risk of developing cancers.

In vitro models can be used to determine the effective doses of the compounds of the invention as a potential cancer treatment. These in vitro models include proliferation assays of cultured tumour cells, growth of cultured tumour cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY Ch 18 and Ch 21), tumour systems in nude mice as described in Giovanella et a/., J. Natl. Can. Inst, 52: 921-30 (1974), mobility and invasive potential of tumour cells in Boyden Chamber assays as described in Pilkington et at., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et a/., Intl. J. Dev. Biol., 40: 1189-97 (1999) and Li ef a/., Clin. Exp. Metastasis, 17:423-9 (1999), respectively. Suitable tumour cells lines are available, e.g. from American Type Tissue Culture Collection catalogues.

In one embodiment, the method, use or composition of the invention additionally comprises a further chemotherapeutic agent. Preferably, the further chemotherapeutic agent is selected from 17AAG, 17DMAG, 5FU, 7-hydroxystaurosporine (UCN-01), ABT888, Actinomycin D, Aisterpaullone, Axitinib, Aminoglutethimide, Amsacrine, Asparaginase, Azacitidine, AZD7762, Bay 11-7082, Belinostat, Bendamustine, Bexarotene, BIBW 2992, Bisindolylmaleimide I, Bleomycin, Bortezomib, Bosutinib, Busulfan, Canertinib , Capecitabine, Carboplatin, Carmustine, CDK4/6 IV, Chelerythrine Chloride, Chlorambucil, CHR-2863 (CHROMA), CHR-3531 (CHROMA), Chromomycin A3, CI-994, Cisplatin, Cladribine , Clofarabine, Clofibrate, CP690550, CXR1002 (APFO), Cyclopamine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin, DBZ, Decitabine, dk2 inhibitor II, DMFO, Docetaxel, Doramapimod, Dovitinib, Doxorubicin, Entinostat, Epirubicin, Erlotinib, Estramustine, Etoposide (V16-213), Everolimus, Flavopiridol, fludarabine phosphate, Flutamide, GDC-0449, Gefitanib, Geldanamycin, Go 6976, GSK-1904529A, GW2580, GW501516, HA14-1 , Hexamethylmelamine, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, lnterleukin-2, Leuprolide acetate (LHRH-releasing factor analog), Idarubicin, Imatinib, Ispinesib mesilate, Kenpaullone, K0143, KU-0058948, Lapatinib, Lenalidomide, Lestaurtinib, Lomustine, LY 2157299, LY294002, Masitinib, Mechlorethamine HCI (nitrogen mustard), Melphalan, Mercaptopurine (6-MP), Mesna, MTX, Mitoguazone, Mitomycin C, MK 1775, Mocetinostat, Mitoxantrone HCI, Nelarabine, Nilotinib, Nobiletin, Nogalamycin, NSC 625987, Nutlin-3, NVP-AEW541 , NVP-TAE684 - TAE 684, Obatoclax mesylate, Octreotide, Olaparib (KU0059436), Oxaliplatin, Pentostatin, Plicamycin, Procarbazine HCI, Paclitaxel, Panobinostat, Pazopanib, PD 98059, PD173074, Pemetrexed, Perifosine, perillic acid, PLX4032, PLX4720, PPP (Picropodophyllin), Puromycin, Radicicol, Raltitrexed, Rapamycin, Ridaforolimus, Semustine, Streptozocin, Saracatinib, SB 431542, SB202190, SB203580, Sorafenib, SP600125, Sunitinib, Suramin, Tamoxifen CITRATE, Teniposide, Tandutinib, Tegafur , Temodal, Thalidomide, Thioguanine (6-TG), Thiotepa, Topotecan hydrochloride hydrate, Tozasertib, Trichostatin A, Tyrphostin AG 490, Tyrphostin AG 538, Tyrphostin AG879, U0126, Valproic acid, Vandetanib, Vatalanib, Vinblastine sulfate salt, Vincristine, Vindesine sulphate, Vinolrelbine ditartrate salt hydrate, Wortmannin, XAV 939, YM155, ZSTK474,4HC,6-[4-(2-Piperidin-1- ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1 ,5-a]pyrimidine (Dorsomorphin), Etoposide, Gemcitabine, Mitoxanthrone and Vorinostat.

Due to the favourable toxicity and safety profiles of the compounds of the invention, the compounds may be used to treat patients who may not be amenable to conventional chemotherapeutic agents. Conventional chemotherapies are generally immunosuppressive and so are withdrawn in certain situations, such as for patients who are pre-operative, post-operative, receiving radiation, terminally ill, elderly, or receiving adjuvant or neo-adjuvant thereapy. However, the compounds of the invention may still be used in such situations.

Therefore, in a further aspect of the invention the compounds of the invention are used to treat patients who may not be amenable to conventional chemotherapeutic agents, particularly those from whom conventional chemotherapeutic agents have been withdrawn.

A further aspect of the invention provides a kit of parts comprising:

(i) pyrrolnitrin derivative of formula I or a salt or ester thereof or a pharmaceutical composition comprising a pyrrolnitrin derivative of formula I or a salt or ester thereof and a pharmaceutically acceptable excipient, diluent or carrier;

(ii) apparatus for administering the compound or pharmaceutical composition; and

(iii) instructions for use.

In one embodiment, the kit of parts additionally comprises a further chemotherapeutic agent. Preferably the further chemotherapeutic agent is selected from 17AAG, 17D AG, 5FU, 7-hydroxystaurosporine (UCN-01), ABT888, Actinomycin D, Alsterpaullone, Axitinib, Aminoglutethimide, Amsacrine, Asparaginase, Azacitidine, AZD7762, Bay 11-7082, Belinostat, Bendamustine, Bexarotene, BIBW 2992, Bisindolylmaleimide I, Bleomycin, Bortezomib, Bosutinib, Busulfan, Canertinib , Capecitabine, Carboplatin, Carmustine, CDK4/6 IV, Chelerythrine Chloride, Chlorambucil, CHR-2863 (CHROMA), CHR-3531 (CHROMA), Chromomycin A3, CI-994, Cisplatin, Cladribine, Clofarabine, Clofibrate, CP690550, CXR1002 (APFO), Cyclopamine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin, DBZ, Decitabine, dk2 inhibitor II, DMFO, Docetaxel, Doramapimod, Dovitinib, Doxorubicin, Entinostat, Epirubicin, Erlotinib, Estramustine, Etoposide (V16- 213), Everolimus, Flavopiridol, fludarabine phosphate, Flutamide, GDC-0449, Gefitanib, Geldanamycin, Go 6976, GSK-1904529A, GW2580, GW501516, HA14-1 , Hexamethylmelamine, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha- 2a, Interferon Alpha-2b, lnterleukin-2, Leuprolide acetate (LHRH-releasing factor analog), Idarubicin, Imatinib, Ispinesib mesilate, Kenpaullone, K0143, KU-0058948, Lapatinib, Lenalidomide, Lestaurtinib, Lomustine, LY 2157299, LY294002, Masitinib, Mechlorethamine HCI (nitrogen mustard), Melphalan, Mercaptopurine (6-MP), Mesna, MTX, Mitoguazone, Mitomycin C, MK 1775, Mocetinostat, Mitoxantrone HCI, Nelarabine, Nilotinib, Nobiletin, Nogalamycin, NSC 625987, Nutlin-3, NVP-AEW541 , NVP-TAE684 - TAE 684, Obatoclax mesylate, Octreotide, Olaparib (KU0059436), Oxaliplatin, Pentostatin, Plicamycin, Procarbazine HCI, Paclitaxel, Panobinostat, Pazopanib, PD 98059, PD173074, Pemetrexed, Perifosine, perillic acid, PLX4032, PLX4720, PPP (Picropodophyllin), Puromycin, Radicicol, Raltitrexed, Rapamycin, Ridaforolimus, Semustine, Streptozocin, Saracatinib, SB 431542, SB202190, SB203580, Sorafenib, SP600125, Sunitinib, Suramin, Tamoxifen CITRATE, Teniposide, Tandutinib, Tegafur , Temodal, Thalidomide, Thioguanine (6-TG), Thiotepa, Topotecan hydrochloride hydrate, Tozasertib, Trichostatin A, Tyrphostin AG 490, Tyrphostin AG 538, Tyrphostin AG879, U0126, Valproic acid, Vandetanib, Vatalanib, Vinblastine sulfate salt, Vincristine, Vindesine sulphate, Vinolrelbine ditartrate salt hydrate, Wortmannin, XAV 939, YM155, ZSTK474,4HC,6-[4-(2-Piperidin-1-ylethoxy)phenyl]-3-pyridin-4 -ylpyrazolo[1 ,5- a]pyrimidine (Dorsomorphin),Etoposide,Gemcitabine,Mitoxanthrone and Vorinostat.

EXAMPLES The following examples embody various aspects of the invention. It will be appreciated that the specific compounds used in the examples serve to illustrate the principles of the invention and are not intended to limit its scope.

The following examples are described with reference to the accompanying figures in which:

Figure 1 - Kinase specificity of fludioxonil and fenpiclonil

Figure 1 shows the % inhibition of Pim1, 2 and 3 kinase with increasing concentration (μΜ) of fludioxonil (1A) or fenpiclonil (1B). Figure 2 - Kinase specificity of fludioxonil and fenpiclonil

Figure 2 shows by western blot PIM 1 kinase and cleaved PARP levels in response to increasing concentrations if fludioxonil and fenpiclonil.

Figure 3 - Rock kinase specificity of fludioxonil and fenpiclonil

Figure 3 shows the % inhibition of Rock I and Rock II kinase with increasing concentration of fludioxonil or fenpiclonil. Figure 4 - Bodyweights and liver weights from a PC3 xenograft study in mice

Figure 4 shows (a) bodyweight over duration of study (b) terminal bodyweight and (c) liver weight of mice from a PC3 xenograft study with fludioxonil (CXR6032) and fenpiclonil (CXR6069). Figure 5 - Tumour volume and weight from a PC3 xenograft study in mice

Figure 5 shows (a) ALT, AST and ALP levels, (b) tumour volume and (c) tumour weight over the duration of the PC3 xenograft study with fludioxonil (CXR6032) and fenpiclonil (CXR6069).

Figure 6 - Fludioxonil whole blood pK results

Figure 6 shows the Log10 of fludioxonil concentration in ng/mL over time, both with a single dose and with 4 daily repeat doses.

Figure 7 - Fenpiclonil whole blood pK results

Figure 7 shows the Log10 of fenpiclonil concentration in ng/mL over time, both with a single dose and with 4 daily repeat doses. Figure 8 - Diagram of GM-CSF signalling pathway overlaid with fludioxonil (CXR6032) EC50 signature list

Figure 8 shows the fold change data from fluduioxonil (CXR6032) EC50 signature list superimposed on the genes shown in the above pathway using IPA software (Ingenuity Systems Inc.). Fold changes (black lettering under gene icons) greater than 1.5 fold causes in the gene icons to be coloured either dark grey = up-regulated or light grey = down-regulated. Icons in medium grey signify fold changes of less than 1.5. Icons in white signify genes were un-changed by the treatment or not significantly changed (p>0.01).

Figure 9 - Diagram of GM-CSF signalling pathway overlaid with fenpiclonil (CXR6069) EC50 signature list

Figure 9 shows the fold change data from fenpiclonil (CXR6032) EC50 signature list superimposed on the genes shown in the above pathway using IPA software (Ingenuity Systems Inc.). Fold changes (black lettering under gene icons) greater than 1.5 fold causes in the gene icons to be coloured either dark grey = up-regulated or light grey = down-regulated. Icons in medium grey signify fold changes of less than 1.5. Icons in white signify genes were un-changed by the treatment or not significantly changed (p>0.01). Figure 10 - Diagram of acute myeloid leukaemia signalling pathway overlaid with fludioxonil (CXR6032) EC50 signature list

Figure 10 shoes the fold change data from fluduioxonil (CXR6032) EC50 signature list superimposed on the genes shown in the above pathway using I PA software (Ingenuity Systems Inc.). Fold changes (black lettering under gene icons) greater than 1.5 fold causes in the gene icons to be coloured either dark grey = up-regulated or light grey = down-regulated. Icons in medium grey signify fold changes of less than 1.5. Icons in white signify genes were un-changed by the treatment or not significantly changed (p>0.01). Figure 11 - Diagram of acute myeloid leukemia signalling pathway overlaid with fenpiclonil (CXR6069) EC50 signature list

Figure 11 shows the fold change data from fenpiclonil (CXR6032) EC50 signature Rst superimposed on the genes shown in the above pathway using IPA software (Ingenuity Systems Inc.). Fold changes (black lettering under gene icons) greater than 1.5 fold causes in the gene icons to be coloured either dark grey = up-regulated or light grey = down-regulated. Icons in medium grey signify fold changes of less than 1.5. Icons in white signify genes were un-changed by the treatment or not significantly changed (p>0.01).

Figure 12 - Diagram of RhoA signalling pathway overlaid with fludioxonil (CXR6032) EC50 signature list

Figure 12 shows the fold change data from fludioxonil (CXR6032) EC50 signature list superimposed on the genes shown in the above pathway using IPA software (Ingenuity Systems Inc.). Fold changes (black lettering under gene icons) greater than 1.5 fold causes in the gene icons to be coloured either dark grey = up-regulated or light grey = down-regulated. Icons in medium grey signify fold changes of less than 1.5. Icons in white signify genes were un-changed by the treatment or not significantly changed (p>0.01).

Figure 13 - Diagram of RhoA signalling pathway overlaid with fenpiclonil (CXR6069) EC50 signature list Figure 13 shows fold change data from fenpiclonil (CXR6032) EC50 signature list superimposed on the genes shown in the above pathway using IPA software (Ingenuity Systems Inc.). Fold changes (black lettering under gene icons) greater than 1.5 fold causes in the gene icons to be coloured either dark grey = up-regulated or light grey = down-regulated. Icons in medium grey signify fold changes of less than 1.5. Icons in white signify genes were un-changed by the treatment or not significantly changed (p>0.01).

Figure 14 - RNA microarray for ftudioxonil and fenpiclonil experimental structure Figure 14 shows the fludioxonil (CXR6032) and fenpiclonil (CXR6069) RNA microarray experimental structure using triplicates of each sample.

Example 1: selection of the compounds of the invention based on specific properties

The following method details how the compounds of the invention were selected to arrive at those to be used for the treatment of diseases such as cancer.

The compounds of the invention were selected using the following method:

Step 1 : A database was produced of some 182 compounds registered for use as agro- or industrial chemicals with available dossiers regarding bioactivity and toxicity collated from sources including the UK's Advisory Committee on Pesticides (ACP), the US Environmental Protection Agency (EPA), the European Food Safety Authority (EFSA), the US Department of Health & Human Services' Agency for Toxic Substances and Disease Registry (ATSDR), Canada's Bureau of Chemical Safety and the International Programme on Chemical Safety (IPCS).

Table 13 details the 182 compounds contained in the database.

Step 2: A panel of 144 chemically diverse compounds were selected as being readily available from the database produced in (1).

Table 14 details the 144 chemically diverse compounds.

Step 3: The compounds from (2) were tested against 15 human tumour cell lines for cytotoxicity. Effects on cell survival at 72 hours and at 7 days at a single compound concentration (100μΜ) were measured. Tables 11 and 12 show cell proliferation data for the 144 compounds at 72 hours and 7 day exposure, respectively. The data is expressed as a percentage of untreated control cells, so 100 % means that there are the same number of cells as the control, less than 100 % indicates there are fewer cells than the control and greather than 100 % indicates that more cells than the control are present. Cell assay methods

Cell Culture

Test compounds were dissolved in DMSO. Cells were seeded at a density of 500 cells per well in 75 μΙ of DMEM (10% FBS/2 mM Glutamine) media for 7 day incubation in 96- well plates or 5000 cells per well for 72 hrs incubations. The following day, 25 μΙ of test compounds, diluted 1:25 in DMEM (10% FBS), were added to the wells to give a final compound concentrations of 100μΜ. Duplicate plates were assembled and then incubated for either 72 hrs or 7 days at 37°C/5% C0 ₂ . No media/drug replacement during the incubation period was undertaken.

WST-1 Assay

Cell number was estimated using WST-1 reagent from Roche Applied Bioscience according to the manufacturer's instructions. Briefly 10μΙ of reagent was added to 100 μΙ of media and incubated until colour developed. The plates were read at wavelength 450nm on an appropriate absorbance plate reader.

Data Analysis

Data from the plate reader in text format was entered into an excel spreadsheet. The data was converted into a percentage value of the untreated control wells using the following formula:

Step 4: The cytotoxicity data from (3) was used to rank compounds with respect to (a) global cytotoxicity; (b) selective cytotoxicity; and (c) published literature (i.e. a lack of published literature linking the compounds with cytotoxicity).

Step 5: Panel of 48 compounds chosen as the top-ranked compounds using the results of (4). Step 6: Compounds selected under (5) were tested for cytotoxicity with compound dose response (see table 10) and the published toxicology data reviewed for (a) bioavailability (see table 3); (b) rat LD50 (see table 5); (c) Therapeutic Index (NOEL (No Observable Effects Limit): IC50 ratio) (table 2).

Step 7: Panel from (6) screened for effects on protein kinase activities. The results are shown in Table 8.

Step 8: Compounds selected for further development on basis of meeting at least criteria (1) and (2) (but preferably more) of the 7 criteria listed below:

Step 9: (optional) Kinase inhibition (such as PIM-Kinase) by dose response results can be used to select specific compounds for further development (see Examples 3 and 4) Step 10: Compounds selected under (8) and (9) will be tested further in xenograft studies (see Example 6).

The seven criteria for compound selection 1. Available Toxicoloqv/Toxicitv Data must be available

Toxicological testing is a significant barrier in the pre-clinical development of pharmaceuticals, both in terms of cost and risk. Selection of molecules already tested for toxicity is therefore an attractive alternative.

Sufficient publically available safety pharmacology, toxicology and toxicity data should be available, as outlined in the ICH Safety Guidelines S9 available from http:/ www.ich.org/products/guidelines/safety/article/safety-guidel ines, to support the compound's use in clinical trials.

2. Favourable Therapeutic Index - NOELICsn Ratio must be greater than 5

Current anticancer drugs give rise to significant side-effects at the dose levels given to patients. This is due to the fact that these traditional molecules function as cellular toxins, their anti-cancer properties arising from the toxic nature. The methodology described herein of selecting compounds shown to be of low toxicity will overcome this limitation and give rise to anti-cancer agents with lower toxicology liabilities. An ideal pharmaceutical would exhibit efficacy in the absence of any toxicological or pharmacological side-effects at the therapeutic dose. A compound with these desirable properties is described as having a favourable therapeutic index or therapeutic window.

As an indicator of compounds with a potentially favourable Therapeutic Index we use the ratio of the rat "No Observed Effect Level" (NOEL) from toxicology studies and the lowest IC ₅₀ value of the compound on our cell line panel as a proxy. Compounds which have a larger ratio are more likely to inhibit the growth of tumour cells whilst having no observable side-effects on the animal.

Table 2 shows which of a selection of 48 compounds (as selected according to step 5 of the screening method) have an IC50/NOEL ratio of greater than five. Table 2

Rat Rat

Min IC50 Ratio

Compound NOEL/NOAEL NOEL/NOAEL

(M) IC50/NOEL

(mg/kg) (M)

Benfuracarb 1.3 3.2 10.00 0.3

Flusilazole 1.5 4.8 10.00 0.5

fenpropathrin 1.5 4.3 15.00 0.3

Fenbuconazol 5.2 15.4 15.00 1.0

Transfluthrin 15 40.4 22.00 1.8

Fenazaquin 5 16.3 0.26 62.8

Fenoxycarb 80 265.8 10.20 26.1

Metconazole 2.5 7.8 17.00 0.5

Folicur 10.8 35.1 26.00 1.3

ethiocarb 1.3 5.8 2.60 2.2

Myclobutanil 15 51.9 25.00 2.1

cyfluthrin 10 23.1 17.00 1.4

Bromuconazol 14 37.1 30.00 1.2

Dimethomorph 3.75 9.7 15.00 0.6

Triazophos 0.29 0.9 22.00 0.0

phorate 0.05 0.2 26.00 0.0

irgarol 9.7 38.3 22.00 1.7

Pyriproxifen 2 74.8 15.70 4.8

tau-fluvalinate 1 2.0 14.00 0.1

Fluoroglycofen-ethyl 84 200.0 10.00 20.0

Esfenvalerate 15 35.7 9.58 3.7

Fenoxaprop-ethyl 6 16.6 43.00 0.4

cyhalofop-butyl 3 8.4 35.00 0.2

Flufenoxuron 12.5 25.6 8.00 3.2

Dimefuron 20 59.0 27.00 2.2

Isoproturon 50 242.7 20.00 12.1

Ethoprophos 0.1 0.4 43.31 0.0

3. Oral bioavailability preferred to be greater than 20% Oral bioavailability is an attractive property for pharmaceutical compounds. Oral dosing will mean patients do not have to travel to the hospital situation regularly during their treatment. This will not only save considerable resources at the treatment centre but also reduce patient comfort by reducing travel and in-patient time. Oral bioavailability is calculated by measuring the plasma concentration of the compound over time after administering the drug both orally (po) and intravenously (IV). The area under the curve (AUC) is measured for both modes of administration and is corrected for different dose levels for the different routes. The following formula is used for calculating oral bioavailability (F):

[AUC] _po * Dose,

(AUC] _IV * Dose,

Table 3 shows which of a selection of compounds has an oral bioavailability of >20%:

Table 3

Compound Bioavailability

Metconazole >80%

Folicur 90%

Methiocarb 0%

Myclobutanil -50%

cyfluthrin >60%

Bromuconazole -25%

Dimethomorph 90%

Triazophos -75%

Phorate 30%

irgarol 0%

Pyriproxifen 7%

tau-fluvalinate 20-40%

Fluoroglycofen-ethyl 20%

Esfenvalerate -50%

Fenoxaprop-P-ethyl -50%

cyhalofop-butyl -90%

Flufenoxuron 50%

Dimefuron 30%

Isoproturon 85%

Ethoprophos 95%

4. When data is available it is preferred that the LP™ be greater than 500 mq/ka

The selection approach described is to select compounds which have undergone extensive toxicological testing and have been deemed of low toxicity and safe for release to the environment as either agrochemicals or for use in other industries.

One measure of toxicity is the Lethal Dose 50 (LD ₅₀ ). This is the concentration of compounds which, when dosed to a test population, causes the death of 50 % of the test population within a set time period. LD ₅₀ was introduced in the 1920's by Trevan (The error of determination of toxicity. Proc Roy Soc 1927;101 B:483-514) as a measure of toxicity but has been largely replace the Maximum Tolerated Dose or MTD. MTD is the highest concentration at which the death rate in a dosed population is no greater than the death rate in a control population

According to the classification of Hodge and Sterner (J Ind Hyg Toxicol. 1949 Mar;31(2):79-92), the toxicity of molecules can be classified according to the table below. In contrast with current anti-cancer agents which are known toxic agents, the compounds selected are of no greater than "low toxicity", with oral LD50 values in the rat no lower than 500 mg kg. Table 4 details the different measures of toxicity of compounds. Table 5 details which of a selection of compounds have an oral LD50 in the rat of over 500mg/kg. Table 4

Table 5

Fenpiclonil 5000

Cyprodinil >2000

brodifacoum 1

flocoumafen 0

Difenoconazole 1450

kresoxim-methyl 5000

Azamethiphos 830

Benfuracarb 120 (rats),250 (dogs)

Flusilazole 674

fenpropathrin 870

Fenbuconazole 2000

Transfluthrin 590 - 870

Fenazaquin 134

Fenoxycarb 10000

Metconazole 595

Folicur 1700

Methiocarb 22

Myclobutanil 2000

cyfluthrin 600-1200 (r), >100 (d)

Bromuconazole 300-400

Dimethomorph 3900

Triazophos 65

Phorate 3

irgarol 2000

Pyriproxifen >5000

tau-fluvalinate >3000

Fluoroglycofen-ethyl 1480

Esfenvalerate -200

Fenoxaprop-P-ethyl -2500

cyhalofop-butyl >5000

Flufenoxuron 3000

Dimefuron 2000

Isoproturon 1826

Ethoprophos 33 5. Preferred that in vivo half-life greater than 4 hours

For a compound to exert its biological effect, it is clear that the compound must be exposed to its molecular target. Should a compound be metabolised or excreted rapidly then compound concentration and so pharmacological effect would naturally fall too. In order to circumvent these issues, medicinal chemistry manipulates the chemical structure of the molecule in order to improve resistance to metabolism and excretion.

Compounds are selected from a library partly on the basis of in vivo half-life measured during development of the compounds as agro-industrial chemicals. In vivo half-life is a measure of the rate at which a compound is excreted or metabolised. The change in plasma concentration of a compound following dosing is described by the equation:

Where C _t is the concentration at a time t, C ₀ represents the concentration at t = 0, Ke is the rate constant of elimination and t is the chosen time point. The half-life can then be calculated using the relationship between rate constant and half-life using the formula:

It is clearly desirable not to have the compound eliminated rapidly from the plasma in order to increase the likely efficacy of the compound in vivo. Our threshold of 4 hours means that if peak dosing were 4 fold of the IC _∞ level, then compound levels would be at least the IC50 level for 8 hours out of 24 and therefore dosing would be a convenient 3 times per day. Table 6 details which of a selection of compounds have a half-life of >4 hours.

Table 6

Compound Half-Life

Thiodicarb Rapid

Buprofezin Rats 13hrs and 60 hrs

Dinocap 3 hrs

Thiophanate-methyl < 24hrs

Tolylfluanid 3hrs, 3 days

Triazoxid 2.6 - 5.2 hrs + slow phase

Tebufenpyrad <72hrs

Triflusulfuron-methyl 15 hrs

Fenpiclonil -160

Cyprodinil Rats ~24hrs

brodifacoum Rat 24days, Dog 120 days flocoumafen > 7 days

Difenoconazol <72hrs

kresoxim-methyl 16.9-30.5

Azamethiphos <6hrs

Benfuracarb ~24hrs

Flusilazole 30hrs

fenpropathrin 24hrs

Fenbuconazol 15 hrs

Transfluthrin < 48 hrs

Fenazaquin 25 hrs

Fenoxycarb ~24hrs

Metconazole ND

Folicur 40hrs

Methiocarb ND

Myclobutanil 5.3hrs, 25.7 hrs cyfluthrin <24hrs

Bromuconazol 9 - 90 hrs

Dimethomorph 3 hrs

Triazophos ND

phorate >7 days

irgarol ND

Pyriproxifen 2-8hrs, 23-35hrs tau-fluvalinate ~24hrs

Fluoroglycofen-ethyl 12-37hrs

Esfenvalerate ~12hrs

Fenoxaprop-ethyl 10 hrs, 3 days

cyhalofop-butyl ~3h

Flufenoxuron ND

Dimefuron < 72hrs

Isoproturon 8h

Ethoprophos < 6 hrs 6. Preferred that compounds exhibit activity on Kinase or other molecular target

In order to direct pre-clinical development it is useful to have an idea of which tumour types may be best targeted with the molecule. Some of this information can be learned from testing the compound for effects on cellular proliferation on a panel of tumour ceH lines. An alternative approach is to test the molecules for activity, inhibitory or activatory, on to use in vitro assays of molecular targets such as protein kinases, GPCRs, enzymes or other cellular machinery. It is preferable that the compounds have a known mode of action. One aspect of this could be inhibitory activity of a compound against a member(s) of a panel of protein kinases known to be implicated in cancer biology. Such a panel could be comprised of the protein kinases shown in table 7: Table 7: A panel of kinases known to be implicated in cancer biology

Kinase assay methods

Kinase assays were performed as described below for each individual kinase except the test compound was dissolved in DMSO and added to reaction mix to give final concentrations of 10μΜ and 100μ

MAPK2/ERK2 assay MAPK/ERK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na ₃ V0 ₄ , 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5 μΙ in 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.05 mM [33 ^ρ -γ-ΑΤΡ](500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. MKK1 assay

This is a two-step assay where inactive MAPK (0.06 mg/ml) is activated by MKK1 (diluted in 25 mM Tris, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.01% Brij35, 1 mg/ml BSA) in 25.5 μΙ containing 25 mM Tris, 0.1 mM EGTA, 0.01% Brij35, 10 mM magnesium acetate and 0.005 mM ATP. After incubating at room temperature for 30 min, 5 μΙ from the first reaction is pipetted into 20 μΙ of the second reaction mix containing (final concentration) 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na ₃ V0 ₄ , 0.66 mg/ml myelin basic protein (MBP), 10 mM magnesium acetate and 0.05 mM [33 ^ρ -γ-ΑΤΡ] (500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

PKCa assay

PKCa (5-20 mU diluted in 20 mM Hepes pH 7.4, 0.03% Triton X-100) is assayed against Histone H1 in the presence of PtdSerine and DAG (0.1 mg/ml. and 10 pg/ml) and 0.1 mM CaCI2. The assay is carried out in a final volume of 25.5 μΙ containing 20 mM Hepes pH 7.4, 0.03% Triton X-100, 0.1 mg/ml Histone H1 , 10 mM magnesium acetate and 0.02 ητ)Μ[33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

PtdSer/DAG preparation:- PtdSer stock = 10 mg/ml in MeOH/Chloroform (1 :2). Dry down required amount. Resuspend in appropriate volume of 10 mM Hepes pH 7.4. Vortex and briefly sonicate. (2 x 10-15 seconds at 10-15 seconds apart). DAG stock = 10 mg/ml in MeOH/chloroform (1 :2). Dry down required amount. Add sonicated PtdSer solution. Vortex and sonicate. PDK1 assay

PDK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 1 mg/ml BSA) is assayed against PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 100 μΜ substrate peptide, 10mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

APH-PKBa-S473D assay

APH-PKBa-S473D (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β- mercaptoethanol, 1 mg/ml BSA) is assayed against a modified Crosstide peptide GRPRTSSFAEGKK in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% β- mercaptoethanol, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM

[33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

SGK assay

SGK (5-20mU diluted in 20 mM MOPS pH 7.5, 1mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against a modified Crosstide peptide GRPRTSSFAEGKK in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^p -y-ATPJ (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

S6K1/ P70 S6K assay

S6K1/P70 S6K (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against substrate peptide (KKRNRTLTV) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.1 mM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^p -v- ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

ROCK-II (ROKa) assay

ROCK-II (ROKa) (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β- mercaptoethanol, 1 mg/ml BSA) is assayed against Long S6 substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 μΜ Long S6 substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

CHK1 assay CHK1 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1% β-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA) is assayed against CHKtide substrate peptide (KKKVSRSGLYRSPSMPENLNRPR) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μΜ CHKtide, 10 mM magnesium acetate and 0.02 mM [33 ^p - γ-ΑΤΡ](50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

LCK assay LCK (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% B-mercaptoethanol, 1 mg/ml BSA) is assayed against Cdc2 peptide (KVEKIGEGTYGWYK) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3Vo4, Cdc2 peptide (0.25 mM), 10 mM magnesium acetate and 0.05mM [33 ^ρ -γ-ΑΤΡ](500-1000 cpm/pmole) and incubated for 15 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

CDK2/cyclin A assay

CDK2/cyclin A (5-20 mU diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCI) is assayed against Histone H1 in a final volume of 25.5 μΙ containing 50 mM Hepes pH7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCI, Histone H1 (1 mg/ml), 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ](500-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. NEK2a assay

5-20mU of NEK2a (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1mg ml BSA, 0.1%, β- mercaptoethanol) is assayed against NEK2a peptide (RFRRSRRMI) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.01% Brij, 0.1%, β- mercaptoethanol, 300μΜ NEK2a peptide, 10 mM magnesium acetate and 0.05 mM [33 ^p - γ-ΑΤΡ]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

MAPKAP-K1 b/RSK2 assay

MAPKAP-K1b (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% β-mercaptoethanol, 1mg/ml BSA) is assayed against substrate peptide (KKLNRTLSVA) in a final volume of 25.51 containing 50 mM Na- β-glycerophosphate (pH 7.5), 0.5 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

IKKb assay

5-20mU of IKKb (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1mg/ml BSA, 0.1% β- mercaptoethanol) is assayed against substrate peptide (LDDRHDSGLDSMKDEEY) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1%, ^mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33 ^ρ -γ-ΑΤΡ]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

Aurora B assay

Aurora B (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide (LRRLSLGLRRLSLGLRRLSLGLRRLSLG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

CHK2 assay

CHK2 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1% β-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA) is assayed against CHKtide substrate peptide (KKKVSRSGLYRSPSMPENLNRPR) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μΜ CHKtide, 10 mM magnesium acetate and 0.02 mM [33 ^p - γ-ΑΤΡ](50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

Src assay

Src (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide (KVEKIGEGTYGWYK) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

PLK1 assay

PLK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 100μΜ Vanadate) is assayed against a substrate peptide (ISDELMDATFADQEAKKK) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% β-mercaptoethanol, 10μΜ Vanadate, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid

PIM1 assay PIM1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide (RSRHSSYPAGT) in a final volume of 25.5 pi containing 50mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. PIM3 assay

PIM3 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide (RSRHSSYPAGT) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. FGF-R1 assay

FGF-R1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ- ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

EPH-A2 assay

EPH-A2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33 ^p - γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. IGF-1R assay

IGF-1R (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KKKSPGEYVNIEFG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. VEG-FR assay

VEG-FR (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KKKSPGEYVNIEFG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

TBK1 (DU12569) assay

TBK1 (DU12569) (5-20mll diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KKKKERLLDDRHDSGLDSMKDEE) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

IKKepsilon (DU14231) assay

5-20mU of IKKepsilon (DU14231)(diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.05 mM [33 ^ρ -γ-ΑΤΡ]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid HER4 assay

HER4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against Poly Glut Tyr in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 1mg/ml Poly Glut Tyr, 10 mM magnesium acetate and 0.005mM [33 ^p -y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

Aurora A assay

Aurora A (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β- mercaptoethanol, 1mg/ml BSA) is assayed against LRRLSLGLRRLSLGLRRLSLGLRRLSLG in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM LRRLSLGLRRLSLGLRRLSLGLRRLSLG, 10 mM magnesium acetate and 0.005mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

JAK2 assay

JAK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β-mercaptoethanol, 1 mg/ml BSA) is assayed against PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ- EMFRDFDYIADWC) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β-mercaptoethanol, 100 μΜ substrate peptide, 10mM magnesium acetate and 0.005 mM [33 ^p -y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

TrkA assay TrkA (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10mM MnCI, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33 ^ρ -γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped- by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.-

Kinase Data Analysis

Raw data was converted into percent inhibition by using the following equation:

( P ^m test background

Percent Inhibition = 100 - (— — *

CP ^m control ~ ^C P ^m b ckgro nd

The percent inhibition data was then modeled to the 4-parameter logistic equation:

{Compound Concentration

The values of Bottom, Top, Hill and EC ₅₀ were changed in order to give the best values to fit the experimentally obtained data.

Compounds were tested for activity at concentrations of 100μΜ and 10μΜ. Compounds were declared active if the activity at 100 μΜ exceeded a threshold of 50% above or below control level. Table 8 indicates which kinases are activated or inhibited by certain compounds.

Table 8

Compound Activated Kinases Inhibited Kinases

SGK, S6K1, CHK1,

Thiodicarb VEGFR, FGF R1

Buprofezin

PKBAph, Aurora B, PIM1 , SRC, TRKA,

Dinocap CHK1

RSK2, IKKb, PIM3, FGF R1

Thiop anate-methyl RSK2

NEK2a, TBK1 (DU12569), IKKepsilon (DU14231), Aurora B, VEGFR, PKCa,

Tolylfluanid Aurora A, TRKA, RSK2, IKKb, PLK1

(okadaic acid), FGF R1, HER4

Triazoxid

Tebufenpyrad S6K1

Triflusulfuron-methyl SGK, S6K1

SGK, ROCK-II, CDK2/cyclin A, IKKepsilon (DU14231), Aurora B, PIM1,

Fenpiclonil IGF-1R, TRKA, RSK2, IKKb, PIM3,

PLK1 (okadaic acid)

Cyprodinil PIM3

PDK1, PKBAph, MKK1, Aurora B, EPH brodifacoum S6K1, CHK1 A2 VEGFR, SRC, JAK2, TRKA, RSK2,

IKKb PLK1 (okadaic acid), HER4

PKBAph, SGK, KK1, Aurora B, flocoumafen S6K1, CHK1 VEGFR, PKCa, SRC, TRKA, RSK2,

IKKb, PLK1 (okadaic acid), HER4

Difenoconazol Aurora B, RSK2

kresoxim-methyl

Azamethiphos

Benfuracarb SGK, S6K1

Flusilazole

CBK1, VEGFR, PIM3,

fenpropathrin FGF R1

Fenbuconazol S6K1

Transfluthrin CHK1 MKK1

Fenazaquin

Fenoxycarb MKK1

Metconazole

Folicur

Methiocarb

yclobutanil

cyfluthrin CHK1, CHK2

Bromuconazol MKK1

Dimethomorph

Triazophos SGK VEGFR

phorate SGK RSK2

irgarol Compound Activated Kinases Inhibited Kinases

CHK1, VEGFR,

Pyriproxifen FGF R1

tau-fluvalinate CHK1, VEGFR

Fluoroglycofen-ethyl CHK1 , VEGFR

CHK1,

Esfenvalerate

FGF R1

Fenoxaprop-ethyl

PDK1 , SGK, S6K1,

cyhalofop-butyl CHK1, Aurora B,

PKCa, RSK2, PIM3

Flufenoxuron VEGFR

Dimefuron

Isoproturon

Ethoprophos SGK

7. In vitro Cytotoxic/Cytostatic Activity - Preferred that Potency be less than 200 uM on at least one cell line

Compounds must exhibit broad activity against a panel of cell lines from tumour types of varying origins. Effects on cellular proliferation were measured against 14 cell lines representing carcinomas of the lung, prostate, breast, liver, colon, pancreas as well as sarcomas. Cells were treated with 8 concentrations of compound 300, 100, 30, 10, 3, 1 , 0.3, 0.1, 0.03 and 0.01 μΜ and cell number estimated at each concentration. Cell number was calculated as a percentage survival relative to untreated cells. EC ₅₀ was calculated by fitting the data to the 4-parameter logistic model:

Table 9 details the transformed cell lines.

Table 9 Table 10 shows the cytotoxicity data (μΜ EC50s) for selected compounds against 14 tumour cell lines over a 7 day exposure

Table 10: Cytotoxicity data (μΜ) for selected compounds against 14 tumour cell lines

Table 11- 72 hour Cell Proliferation Data at 100 μΜ Compound Concentration (data shown as cell number as % of untreated control)

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Table 12 - 7 day Cell Proliferation Data at 100 μΜ Compound Concentration (data shown as cell number as % of untreated control)

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Table 13-182 compounds contained in the database according to screening step 1

Table 14 - A panel of 144 compounds (chemically diverse) from screening step 2

Example 2 - Kinase screening

Table 15 shows a summary of the kinase screening data for fludioxonil and fenpiclonil. Results are represented as % inhibition relative to a positive control. Negative values represent compounds which activate kinase activity. Where (-) is shown there was no activity of the compound. Inhibition of the kinases SGK, ROCKII, PIM1 , TRKA, RSK2, PIM3 and FGFR1 was common to both fludioxonil and fenpiclonil.

Table15: Summary table of kinase inhibition for selected compounds

Example 3: Inhibition ofPIM Kinase activity PIM kinase inhibition has been investigated for each of PIM-1 , PIM-2 and PIM-3 kinase molecules.

PIM 1 (hr

The PIM-1 assay is performed using the Upstate IC ₅₀ Profiler Express™ service. In a final reaction volume of 25 μΙ, human recombinant PIM-1 (5-1 OmU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 100 μΜ KKRNRTLTV, 10mM MgAcetate and [v- ³³ P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μΙ of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.

PIM-2 (h)

The PIM2 assay is performed using the Upstate IC ₅₀ Profiler Express™ service. In a final reaction volume of 25 μΙ, human recombinant PIM-2 (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 300 μΜ RSRHSSYPAGT, 10 mM MgAcetate and [?- ³³ P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μΙ of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in menthanol prior to drying and scintillation counting.

PIM-3 (h)

The PIM-3 assay is performed using the Upstate IC ₅₀ Profiler Express™ service. In a final reaction volume of 25 μΙ, human recombinant PIM-3 (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 300 μΜ RSRHSSYPAGT, 10 mM MgAcetate and p-^P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μΙ of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in menthanol prior to drying and scintillation counting.

Results

Dose response against PIM kinase family members gave low μΜ IC50 (see figure 1). Figure 1a and 1b shows the inhibition of PI 1, PIM2 and PIM3 for fludioxonil and fenpiclonil respectively. The plots show % inhibition in an in vitro kinase assay. Table 16 details the IC50 and EC50 data for each compound against the PIM kinases.

Table 16

K562 cells express high levels of PIM1 protein. It is known that use of a PIM inhibitor can result in downregulation of PIM1 protein as shown in Swords et al. (SGI-1776: A novel PIM kinase inhibitor with potent preclinical activity against Acute Myeloid Leukemia (AML), Swords, R et al, Abstracts of the AACR Annual Meeting, 2009, April 18-22, Denver, CO, USA, Abstract umber 3743).

To test whether fenpiclonil and fludioxonil could down-regulate PIM, an assay was established using K562 cells. Test compounds are dissolved in 100% dimethylsulfoxide, diluted to the appropriated concentration and added to the culture medium of K562 cells. DMSO treated cells were used as a negative control. Compound CXR6099 was used as a positive control. The results are shown in figure 2 where fludioxonil and fenpiclonil can be seen to cause a reduction in PIM1 levels with increasing concentration.

Precise Method for Western Blot:

After trypsinisation and dilution in RPMI1640 medium, 1.4 x 10 ⁶ K562 cells were transferred to a 60 mm culture dish, and cultured overnight. Following 24 h of treatment with different concentrations of compound, cells were collected and lysed. Protein level of PIM1 was evaluated by immunoblot analysis.

Briefly, proteins were transferred to a PVDF membrane and incubated for 1 hour at room temperature in blocking solution. Then the membranes were incubated overnight at 4°C in antibody solution containing anti-PIM1 (12H8) antibody (Santa Cruz Biotechnology) or containing anti-cleaved PARP antibody (Abeam pic). After washing, the membrans were incubated for 1 hour at room temperature in solution containing appropriate dilution of HRP-conjugated secondary antibody (GE Healthcare, UK).

ECL plus (GE Healthcare, UK) was used to detect PI 1 and cleaved PARP protein levels.

Example 4: Inhibition of ROCK activity

ROCK-II (ROKa) assay

ROCK-II (ROKa) (2.6 - 10.5 ng active Rock-ll diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) is assayed against Long S6 substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 μΜ Long S6 substrate peptide, 10 mM magnesium acetate and 0.1 mM ATP (spiked with sufficient [33 ^p -v- ATP] to give approximately 500-800 cpm/pmole) and incubated for 10 min at 30°C. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then 10 μΙ harvested onto P30 filter-mat and washed 3 times with 75 mM orthophosphoric acid, and once with methanol.

ROCK-I (ROKb) assay

ROCK-I (ROK) (72.7 - 200.5ng active Rock-I diluted in 20mM MOPS/NaOH pH 7.0, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1% β -mercaptoethanol, 1 mg/ml BSA) is assayed against 30μΜ Long S6 substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) in a final volume of 25 μΙ containing 8mM MOPS/NaOH pH 7.0, 0.2 mM EDTA, 30 μΜ Long S6 substrate peptide, 10 mM magnesium acetate and 0.1 mM ATP (spiked with sufficient [33 ^p - γ - ATP] to give approximately 500-800 cpm/pmole) and incubated for 10 min at 30°C. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then 10 μΙ harvested onto P30 filter-mat and washed 3 times with 75 mM orthophosphoric acid, and once with methanol.

Results

Figure 3 show the increase in % inhibition of Rock I and Rock II with increasing does of fludioxonil or fenpiclonil. Table 17 details the IC50 and EC50 data for ROCK-I and ROCK-II using fludioxonil, fenpiclonil and pyrollnitrin. Table 17

Example 5: Cytotoxic effects of fludioxonil and fenpiclonil

Table 18 shows the 7 day IC50 (μΜ) of fludioxonil and fenpiclonal on a selection of cancer cell models.

Table 18

Cytotoxicity assay methods Cell Culture

Test compounds were dissolved in DMSO and dilution series performed. Cells were seeded at a density of 500 cells per well in 75 μΙ of DMEM (10% FBS/2 mM

Glutamine) media for 7 day incubation in 96-well plates. The following day, 25 μΙ of test compounds, diluted 1 :25 in DMEM (10% FBS), were added to the wells to give final compound concentrations as detailed above. Duplicate plates were assembled and then incubated for 7 days at 37°C/5% C0 ₂ . No media/drug replacement during the 7 day period was undertaken.

For drug combination experiments and 72 hour cytoxicity testing on day one cells were seeded at a density of 5000 cells per well in 75 μΙ of DMEM (10% FBS/2 mM Glutamine) media in 96-well plates. The following day, 12.5 μΙ of each test compound, diluted 1 :50 in DMEM (10% FBS), was added to the wells to give final compound concentrations. Duplicate plates were assembled and then incubated for 3 days at 37°C/5% C0 ₂ .

WST-1 Assay

Cell number was estimated using WST-1 reagent from Roche Applied Bioscience according to the manufacturer's instructions. Briefly 10μΙ of reagent was added to 100 μΙ of media and incubated until colour developed. The plates were read at wavelength 450nm on an appropriate absorbance plate reader.

Data Analysis

Data from the plate reader in text format was entered into an excel spreadsheet. The data was converted into a percentage value of the untreated control wells using the following formula:

The %Survival data was then modelled to the 4-parameter logistic equation:

Compound Concentration ¹

The values of Bottom, Top, Hill and EC ₅₀ were changed manually in order to give the best values to fit the experimentally obtained data.

Example 6 - Data from Xenograft study CXR0929

The aim of this study was to assess the efficacy of fludioxonil and fenpiclonil in vivo in nude mouse xenografts using mice harbouring tumours derived from human prostate (PC3).

The PC3 cell line was grown in culture and implanted into female nu-nu mice; dosing was initiated 72 hours later and tumour growth was monitored by calliper measurements. Mice harbouring PC3 tumour cells were dosed with ethanol/PEG200/water only or CXR6032 (Fludioxonil), or CXR6069 (Fenpiclonil). Compounds were to be administered daily for 28 days by oral gavage.

At termination, blood was harvested by cardiac puncture and mouse tissues and tumour pieces were fixed in NBF and also flash frozen.

Materials and methods Cell Line

The PC3 (ECACC catalogue number 90112714) cell line was resuscitated from stocks maintained at CXR Biosciences.

Safety Precautions

The normal safety precautions as detailed in the relevant SOPs and COSHH assessments applied, no additional precautions were considered necessary.

Animals Adult (6 - 8 weeks) female, athymic nude (nu/nu) mice were obtained from Charles River, UK.

Animal Accommodation and Husbandry

On arrival in the animal unit the mice were housed, up to 8 per cage, on sterile sawdust in sterile, solid-bottom, polypropylene cages.

The cages were individually vented units attached to a Techniplast Slimline Air Handling Unit. This unit maintains 70-80 air changes per cage, per hour, through HEPA air filters. Bedding was changed once weekly in a laminar flow unit. The temperature was maintained within a target range of 19-23° C and relative humidity of the IVC within a range of 40-70%. Twelve-hour periods of light were cycled with twelve-hour periods of darkness.

Diet

Sterile RM1 diet (Special Diet Services Ltd., Stepfield, Witham, Essex, UK) was used. The animal unit held the specification of the diet. Deionised water was autoclaved prior to use and changed at least once a week. Mice were allowed water and diet ad libitum and were acclimatised for at least 5 days prior to the study start.

Animal Health and Welfare

The mice have specific pathogen free (SPF)-status and the housing and changing system assured that the SPF-status was preserved during the study. Trained personnel under supervision handled the mice.

Experimental Design

Identification of Animals and Allocation ofnu-nu Mice into Experimental Groups

The animals were randomly allocated to groups on arrival. The mice were numbered appropriately and weighed prior to the start of the experiment. The first mouse assigned to a cage was individually identified by tail tattooing with the lowest number for that cage; the second mouse was assigned the second (lowest) number and so on. An experimental card was placed on each cage and showed the project licence code, test group, study number, sex and individual numbers of the mice within, and identified the Home Office Licensee. In addition, these cards were colour coded to correlate with the coding for the group.

Growth and Preparation of Cell Lines for Subcutaneous Injection into nu-nu Mice

PC3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat inactivated foetal calf serum (FCS) supplemented with 2mM L-glutamine, penicillin (50 lU/ml) and streptomycin (50μ9/πιΙ). Cultures were incubated in a humidified incubator at 37°C, 5% C0 ₂ , until sufficient cells were available to implant the mice. Cells were harvested, pooled, centrifuged, and re-suspended in cold medium. This wwe mixed with an equal volume of cold Matrigel, so that the tumour cell injection solution was a 50:50 mixture of tumour cells/medium and Matrigel. Tumour cells were injected in a volume of 100μΙ in a single flank only. PC3 cells were injected at a density of 2.5 x 10 ⁶ cells per flank. The cell/Matrigel suspension was aliquoted into pre-chilled Eppendorf tubes and kept on ice prior to injection. The period between preparation of the cell/Matrigel suspension and injection of tumour cells did not exceed two hours.

Implantation of Cell Lines

Cells were harvested as detailed above and 100μΙ of cell suspension was injected (s.c.) into a single flank of each mouse.

Repeat Dosing of Compounds

Mice were administered Test Compounds 72 hours following implantation of tumour cells. Mice harbouring PC3 tumour cells were dosed with CXR6032 (Fludioxonil), or CXR6069 (Fenpiclonil) in an ethanol/PEG200/water vehicle. Refer to Table 1 for the experimental design.

Compounds were administered daily for 28 days by oral gavage in a dose volume of 10ml/kg.

Table 19: Experimental Design

Experimental Procedures Bodyweight

The bodyweight of each mouse was recorded at the time of tail tattooing, before implantation and daily prior to dosing. Clinical Observations

Prior to the start of the study, all mice were observed to ensure that they were physically normal and that they exhibited normal activity. Only normal mice were allocated to the study. Following cell inoculation, each mouse was observed twice weekly and a general assessment of condition recorded in the study diary. Animals were terminated if tumours became ulcerated or if the Home Office Project License moderate severity limit was exceeded.

Tumour Measurement

Tumour growth was measured twice weekly for the duration of the study following cell implantation once tumours became palpable. Tumour diameters were measured at four different sites - two lengths and two widths - using a digital slide gauge. This was increased to three lengths and three widths if the tumour was an irregular shape.

Tumour volumes were calculated using the formula: 4/ ₃ π

Where d = mean diameter (n

Animals were terminated if tumour volumes exceeded 1.44cm ³ or if they became ulcerated.

Terminal Procedures

On the day of termination tumour volumes were recorded and the mice were weighed and transferred to the post mortem room. The animals were killed by exposure to a rising concentration of C0 ₂ .

Blood was harvested by cardiac puncture and transferred to heparinised tubes for plasma preparation. Tumours and livers were processed as follows:

Tumours

• Each tumour was removed and the weights and gross morphology recorded. • The tumour was cut in half and one half placed in 10 % neutral buffered formalin (NBF) then prepared for paraffin embedding for possible future histochemical analysis.

• The remaining half was cut in two and each piece was placed in a separate cryovial and flash frozen. Cryovial 1 was labeled microarray and cryovial 2 was labeled MS. These samples were stored at approximately - 80°C for possible future analysis.

Livers

• The liver was removed and the weight recorded.

• 2 slices of liver (approximately 2 mm strips) were taken, one from the left lobe and one from the median lobe. These were placed in 10 % neutral buffered formalin (NBF) then prepared for paraffin embedding (section 6.4) for possible future histochemical analysis.

• 2 pieces of liver were placed in separate cryovials and flash frozen. Cryovial 1 was labeled 'Microarray' and cryovial 2 was labeled 'MS'. These samples were stored at approximately - 80°C for possible future analysis.

Laboratory Procedures Plasma Preparation

Following removal into tubes suitable for plasma preparation, venous blood samples were mixed on a roller for 10 minutes. Red blood cells were removed by centrifugation at 2,000 - 3000 rpm for 10 minutes at 8 - 10 °C. The supernatant (plasma) was transferred to a second tube and stored at approximately -70 °C until required for analysis.

Clinical Chemistry

Plasma was analyzed using a Cobas Integra 400 analyzer. Kits purchased from Roche Diagnostics GmbH were used to measure ALT (cat # 20764957322), AST (cat # 20764949322) and ALP (cat # 03333752190).

Plasma Levels of Test Compounds Plasma samples were stored appropriately and were analysed only with the agreement of the study leader.

Preparation of tissues for paraffin embedding

Tumour and liver samples were placed immediately into NBF for 36-45 hours. Tissues were processed according to internal standard operating procedures. The tissues were embedded in paraffin wax for possible future histochemical analysis.

Results

Figure 4 (a) shows the bodyweight over the duration of study in PC3 xenograft mice treated with fludioxonil (CXR6032), fenpiclonil (CXR6069) and vehicle control. Figure 4 (b) and (c) shows the terminal body and liver weights respectively from the same study.

Figure 5 shows (a) the Alanine transaminase (ALT), Aspartate transaminase (AST) and Alkaline phosphatase (ALP) activity levels and (c) tumour weight from the same study. Figure 5 (b) shows the tumour volume over time.

Example 7: Metabolism and pharmacokinetic properties of fludioxonil and fenpiclonil

P450 Inhibition Study (ref CXR0979) - Determination of P450 inhibition and IC ₅₀ using human microsomes

Microsomal incubations

Microsomal incubation mixtures (500pL) were prepared in HEPES buffer (50mM HEPES, 15mM MgCI ₂ , 0.1 mM EDTA, pH 7.6) containing a NADPH regenerating system (1 mM NADPH, 10mM glucose-6-phosphate 1 IU glucose-6-phosphate dehydrogenase), pooled human liver microsomes (0.5mg protein), and 5pL of 7 concentrations and positive control inhibitors (see Table 21).

Table 20. Microsomal incubation conditions HEPES buffer HEPES 57mM 50mM 440 PH7.6 MgCI ₂ 17mM 15mM

EDTA 114μΜ 100μΜ

NADPH NADPH 20mM 1mM 25 regenerating

glucose-6- 200mM 10mM

system

phosphate

glucose-6- 20 IU 1 IU

phosphate

dehydrogenase

Microsomes pooled human liver 20mg/mL 1mg/mL 25 microsomes

Substrate Phenacetin 1mM 10μΜ 5 cocktail* in 50/50

Tolbutamide 10mM 100μΜ

(v/v)

Omeprazole 1mM 10μΜ

acetonitrile/water

Bufuralol 1mM 10μΜ

Midazolam 1mM 10μΜ

* CYP1A2 activity was monitored by phenacetin O-deethylation, CYP2C9 by tolbutamide hydroxylation, CYP2C19 by omeprazole hydroxylation, CYP2D6 by bufuralol l'-hydroxylation, and CYP3A4 by midazolam hydroxylation. Table 21. Positive inhibitor controls and Test Item concentrations

Serial dilutions of each Test Item were performed in the appropriate solvents as in Table 22 below:

MeCN = Acetonitrile

After addition of the Test Items or positive control inhibitors, the samples were pre- incubated at 37°C for approximately 5 minutes. Subsequently, the NADPH regenerating system was added to the reaction mixture and incubated for 20 minutes at 37°C. Reactions were terminated by the addition of 250μΙ_ of methanol containing 5μΜ dextrorphan as an internal standard.

Samples were vortexed briefly, placed on ice, and then centrifuged at approximately 3500 x g for 10 minutes to remove debris. The supernatant from each sample was then transferred to a separate vial for immediate LC-MS/MS analysis.

Mass Spectrometric analysis

Analysis to determine the concentration of the substrate metabolites was carried out using reverse phase HPLC with tandem mass spectrometric detection (LC-MS/MS). Multiple Reaction Monitoring (MRM) of the parent compound ion and specific fragment ion was monitored using a Micromass Quattro Micro Mass Spectrometer with Micromass MassLynx software version 4.0.

Data analysis

Analysis of the data was carried out using Micromass QuanLynx software Version 4.0 to determine the amount of the specific substrate metabolites produced in each sample (relative to the control samples). The results produced were plotted versus the different concentrations of the Test Items or positive inhibitor control compound, and the IC ₅₀ values calculated using Prism non-linear curve fitting software (version 4.03).

Table 23 - Test Item Results Summary

Data of potential relevance (<10μΜ) are highlighted in bold italics.

No inhibition over concentration range tested.

Conclusion

Three of the five Test Items inhibited (IC ₅₀ values below 10μΜ) one or more of the P450 enzymes tested. For this inhibition to be significant in the clinic the affected drug must have an appreciable proportion of its clearance via the P450 enzyme being inhibited i.e. fraction metabolised greater than 30%.

• Test Items Cyprodinil and Fludioxonil inhibited CYP1A2 with IC ₅₀ values of 0.621 μΜ and 1.23μΜ, respectively.

• Test Item Fluazinam inhibited CYP2C9 with an IC ₅₀ value of 0.967μΜ.

• Test Items Fludioxonil and Fenpiclonil show a similar trend in their P450 inhibition profiles reflecting their close structural relationship.

PK study (ref CXR 0966) Aim

The aim of this study was to examine the pharmacokinetics of fenpiclonil and fludioxonil administered by oral dosing to nude mice. In one treatment group, sampling took place over a 1 day period after a single oral dose of compound to obtain a 24hr post dose profile. In a second group the mice were repeat dosed, daily for 4 days, prior to PK sampling.

Method

Sufficient adult (6 - 8 weeks) female, athymic nude (nu/nu) mice were obtained from Charles River, UK. The mice were housed, up to 7 per cage, on sterile sawdust in sterile, solid-bottom, polypropylene cages. The cages were individually vented units attached to a Techniplast Slimline Air Handling Unit. This unit maintains 70-80 air changes per cage, per hour, through HEPA air filters. Bedding was changed once weekly in a laminar flow unit. The temperature was maintained within a target range of 19-23° C and relative humidity of the IVC within a range of 40-70%. Twelve-hour periods of light were cycled with twelve-hour periods of darkness.

The mice were fed with sterile RM1 diet (Special Diet Services Ltd., Stepfield, Witham, Essex, UK). Deionised water was autoclaved prior to use and changed at least once a week. Mice were allowed water and diet ad libitum and were acclimatised for at least 5 days prior to the study start.

Identification of Animals and Allocation ofnu-nu Mice into Experimental Groups

The animals were randomly allocated to groups on arrival. The mice were numbered appropriately and weighed prior to the start of the experiment. The first mouse assigned to a cage was individually identified by tail tattooing with the lowest number for that cage; the second mouse will be assigned the second (lowest) number and so on. An experimental card was placed on each cage and showed the project licence code, test group, study number, sex and individual numbers of the mice within, ans identified the Home Office Licensee. In addition, these cards were colour coded to correlate with the coding for the group.

Oral dosing

Oral dosing solutions of the Test Items were prepared at concentrations of 12mg/mL (for Fludioxonil and Fenpiclonil) in the dosing vehicle ethanol:PEG-200:water in the ratio 3:5:2 v/v. The Test Items were orally administered at a dose of 120mg/kg (for Fludioxonil and Fenpiclonil) all with a dosing volume of lOmlJkg. The bodyweight of each mouse was recorded immediately before each daily dose.

Oral pharmacokinetic samples from the 4 daily repeat dosed animals (not including control animals receiving a vehicle-only solution) were taken pre-dose (on day 4) and at 0.5, 1, 1.5, 2, 3, 4, 6 and 24 hours after administration of the final daily dose.

Oral pharmacokinetic samples from the single dosed animals were taken at 0.5, 1 , 1.5, 2, 3, 4, 6 and 24 hours after administration of the dose.

Whole blood pharmacokinetic samples

A pre-dose bleed was taken from the mice in the repeat dose groups prior to the fourth and final repeat dosing of each Test Item. Following the fourth daily oral administration of each Test Item the mice in the repeat dose groups were bled at approximately 0.5, 1 , 1.5, 2, 3, 4, 6 and 24 hours. Whole blood (0.020ml_) was taken from the tail vein of each animal and added to plastic sample vials containing milli Q water (0.020ml_) at each of the 9 timepoints. The whole blood:water samples were collected onto dry ice and stored at approximately -70°C prior to analysis.

Following a single oral administration of each Test Item, the mice were bled at approximately 0.5, 1 , 1.5, 2, 3, 4, 6 and 24 hours. Whole blood (0.020mL) was taken from the tail vein of each animal and added to plastic sample vials containing milli Q water (0.020ml_) at each of the 8 timepoints. The whole blood:water samples were collected onto dry ice and stored at approximately -70°C prior to analysis.

Terminal Procedures

At the time of termination the mice were transferred to the post mortem room or designated Laboratory. The animals were killed by exposure to a rising concentration of C0 ₂ .

Blood was harvested by cardiac puncture and transferred to heparinised tubes for plasma preparation.

Plasma preparation

Following removal into tubes suitable for plasma preparation, venous blood samples were mixed on a roller for 10 minutes. Red blood cells were removed by centrifugation at 2,000 - 3000 rpm for 10 minutes at 8 - 10 °C. The supernatant (plasma) was transferred to a second tube and stored at approximately -70 °C until required for analysis.

LC-MS/MS analysis

Analysis to quantify the concentrations of each Test Item in the whole blood:water samples was carried out using reverse phase HPLC followed by mass spectrometric detection. Single analysis was performed.

Data Analysis

Blood concentration time data was analysed, where possible, using Pharsight WinNonLin software Version 5.2, for appropriate pharmacokinetic parameters such as half-life, bioavailability, Cmax, Tmax, AUC and clearance.

Result

Table 24: Plasma concentrations of fludioxonil (ng/ml) per animal for repeat dose and single dose groups

90.00 4016.30 5439.21 7147.80 5534.43 1567.92

120.00 4267.42 5344.50 8379.26 5997.06 2132.18

180.00 1612.34 1335.58 2165.80 1704.57 422.73

240.00 1426.10 2227.90 1866.42 1840.14 401.54

360.00 1621.26 1315.79 2886.68 1941.24 832.90

1440.00 43.31 45.84 45.19 44.78 1.31

Figure 6 shows graphically the changes in fludioxonil concentration over time for both single dose and for four daily repeat doses.

Table 25 - pharmacokinetic parameters following oral administration of fludioxonil

(repeat dose)

Table 26 - pharmacokinetic parameters following oral administration of fludioxonil

(single dose)

Number of point T1/2 7 6 5 6 1

Table 27: Plasma concentrations of fenpiclonil (ng/ml) per animal for repeat dose and single dose groups

Figure 7 shows graphically the changes in fenpiclonil concentration over time for both single dose and for four daily repeat doses. Table 28 - pharmacokinetic parameters following oral administration of fenpiclonil

(repeat dose)

Table 29 - pharmacokinetic parameters following oral administration of fenpiclonil (single dose)

Table 30 details the metabolism and pharmacokinetic properties of fludioxonil and fenpiclonil.

Table 30

mice

Example 8 - Microarray study:

A microarray study was undertaken with the 2 phenylpyrrole compounds. The aim of the study was to examine the effect of CXR6032 (Fludioxonil) and CXR6069 (Fenpiclonil) on the human tumour cell line PC3. PC3 had been chosen for this experiment because in xenograft studies, PC3 tumours showed a delayed growth response to CXR6032 and CXR6069.

Agilent Whole Genome Human Microarray 4x44K (G4122F) and Agilent 4x44K gasket slides G2534-60015 were used for the study.

Experimental Procedures Cell Culture experimental work Cell Line Culture Conditions

Cell lines were cultured according to the suppliers' specifications in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat inactivated foetal calf serum (FCS) supplemented with 2mM L-glutamine, penicillin (50 lU/ml) and streptomycin ^g/ml) at 37°C and 5% C0 ₂ .

Plating Densities of PC3 Cells

Cells were plated into sterile, clear 6 well plates at densities of 3 x 10 ⁵ cells per well, in DMEM medium containing 10% Foetal Calf Serum (FCS) and 2mM Glutamine. 4 plates, giving a total of 24 wells were plated. Dosing of Cells

Cells were incubated at 37°C and 5% C0 ₂ for 48 hours to allow the cells to attach. Once cells had reached ~75% confluency the tissue culture medium was changed as follows:

The tissue culture medium in all 6 wells on plate 1 was replaced with tissue culture medium containing 0.1% DMSO. CXR6032 and CXR6069 at the EC ₂₀ in tissue culture medium was added to 2 wells. CXR6032 and CXR6069 at the EC ₅₀ was added to the remaining 2 wells on each plate. The cells were cultured at 37°C and 5% C0 ₂ for a further 24 hours.

The EC ₂ o and EC ₅₀ values to be used are specified in the Table 31 below:

Table 31: IC ₂₀ and IC ₅₀ concentrations for test items

Cell harvest

The medium was aspirated from all the wells and the cells washed in PBS. 700μΙ RLT buffer containing 10μΙ β-mercaptoethanol per 1ml buffer (Qiagen) was added to each well. The cells were lysed for 10 minutes on a gyro-rotator. The wells were scraped with a sterile cell scraper and lysed cells removed to labelled microtubes. RNA was isolated from cell samples according to CXR Method Sheet entitled "RNA isolation from cell lines".

Microarray experimental structure

The RNA microarray analysis involved labelled (one colour) RNA from triplicate samples of PC3 cell line treated vehicle, CXR6032, CXR6069 at EC ₂₀ and CXR6032, CXR6069 at EC ₅ o as shown in figure 14.

Microarray experimental work Quality control ofRNA using the Agilent Bioanalyser

RNA integrity was checked using the Agilent bioanalyser and the RNA nano lab chip kit according to the CXR method sheet 'Set-up and Running of Nanochip Assay for RNA Quality Control'.

A potential problem was flagged at this point with sample 4-1 (Fenpiclonil EC20 replicate 1). The bioanalyser failed to produce a RIN for this sample. Visual examination of the electropherogram showed that the peaks were of similar appearance to all the samples that had produced a RIN of 10. The RNA concentration of this sample was approximately a third of the other samples, on the limit of the nanochip detection range. It was hypothesised that the low concentration may be why the bioanalyser program had been unable to calculate a RIN for this sample. It was decided to proceed with the labelling of all samples while remaining aware that this sample may fail to label.

Probe labelling

500ng RNA was labelled prior to microarray hybridisation using the Quick Amp Labelling Kit - Two Color (Agilent* 5190-0442), according to the CXR method sheet entitled "Transcriptional Profiling using Standard Agilent 1 Colour Protocol" v2.

Microarray Hybridisation and Scanning

Agilent whole genome human microarray 4x44K (G4122F were hybridised, washed and then scanned on an Agilent microarray scanner according to the CXR method sheet entitled, "Microarray hybridisation and scanning".

Microarray data processing to generate signature lists

Images from the scanner were processed using Agilent feature extraction software version 10.1.7. The data files were loaded into Rosetta resolver 6 software for tertiary analysis. The data were analysed to generate a "signature" gene list comprising the genes that are significantly regulated (p<0.001) according to the CXR method sheet entitled, "Microarray Data Entry and Signature List Production for One colour and Two-colour data in Resolver 6 Software". Signature lists of significantly altered genes were generated for the comparisons shown in Table 32. The lists were not filtered by fold change. The lists were named with the denominator name first followed by the test name and the number of genes in the list.

Table 32: Details signature lists generated. Denominator (D) Test (T) Number of lists

Vehicle vs. CXR6032 EC ₂₀ 1

Vehicle vs. CXR6032 EC ₅₀ 1

Vehicle vs. CXR6069 EC ₂₀ 1

Vehicle vs. CXR6069 EC ₅₀ 1

Bioinformatics

Bioinformatic analysis using Ingenuity pathways analysis™ (IPA) software was used to identify gene changes in pathways involving PIM kinase and the RhoA signalling pathway. A search was carried out in the ingenuity database under Genes and Chemicals for PIM and RhoA. Results for these searches included a list of all reported pathways that both genes are members of. Pathways were overlayed with the signature list data and tables of gene changes were produced.

Results

Treatment of cells with the test compounds at EC20 and EC50 doses caused ~500- ~4500 gene expression changes, Table 33. The top 25 up- and down-regulated genes in these 'signature' lists are shown in Appendices. Complete signature gene- list tables, available on an accompanying DVD, have not been included in this report due to their size.

Table 33: Data table showing the number of 'signature' genes changes for each treatment

Fold change data from the signature lists was overlaid on two PIM-associated pathways: granulocyte-macrophage colony stimulating factor signalling pathway (GM-CSF), Figures 8 and 9; Table 34, and acute myeloid leukemia signalling pathway (AML), Figures 10 and 11 ; Table 35. Fold change data was also overlaid on the RhoA signalling pathway, Figures 12 and 13; Table 36.

Data overlaid on the GM-CSF pathway for fludioxonil (FLU) and fenpiclonil (FEN) (EC50) show down-regulation of signal transducer and activator of transcription 1 , (STAT1). STAT1 regulates pim-1 oncogene (PIM1) at the level of transcription, (Yip- Schneider et al, 1995), although more recent data suggests that PIM1 regulation may be STAT1 independent (Ramana et al, 2002). Hence our results suggest that signalling via PIM1 may be suppressed by these compounds. It had been suggested that treatment with anti-cancer drugs can lead to the activation of survival pathways via PIM as a result of STAT3 activation, (Zemskova et al 2008). FLU (but not FEN), showed up-regulation of cyclin D1 and BCL2-like 11 apoptosis facilitator (Bcl-XL), which could indicate a compensatory effect to PIM inhibition.

Despite differences in the inhibitory effects of FLU and FEN at the level of PIM kinase activity there was no indication of large differences between the effects of the three compounds on the AML signalling pathway that features PIM (Figures 10 and 11). CCAAT/enhancer binding protein alpha, (C/EBPa), an inflammatory signalling transcription factor in the AML pathway, (Mackey et al, 2004), was significantly up- regulated by both compounds in a dose-dependent manner, (Figure 10 and 11 , Table 35).

FLU and FEN (EC50) microarray data (fold change values) overlaid on the RhoA Signalling pathway showed down-regulation of multiple genes. Both compounds led to the down-regulation of Rho associated kinase, (ROCK), spetin and actin-related protein complex (ARP2/3). Some of these genes represent interaction molecules comprising of several family members as reflected by a double-circle symbol in the IPA pathway diagrams. Not all members have been affected by the drug treatments in the same manner; hence a fold change is not shown in Table 36. Treatment with FLU also led to the down-regulation of p190 RhoGAP (RhoGAP), Rho guanine nucleotide exchange factor GEF 12 (LARG), phospholipase D1 (PLD1), citron rho- interacting, serine/threonine kinase 21 (Cit) and myosin light chain (MLC). Treatment with FEN led to down-regulation of lysophosphatidic acid receptor (LPAR), focal adhesion kinase (FAK), actin binding protein (ANLN) and ezrin (EZR). The majority of the genes down-regulated by FLU or FEN are involved in either cytokinesis or actin nucleation and polymerisation.

Conclusions

Both FLU and FEN led to a down-regulation of STAT1 , which regulates PIM1. This finding supports a previous study which suggested FLU and FEN inhibit PIM. The AML pathway, which also features PIM, was not greatly affected by either of the compounds with the exception that both compounds caused an increase in C/EBP expression that was dose-dependent.

FLU and FEN both caused the down regulation of multiple genes in the RhoA signalling pathway. All of the genes down-regulated are down-stream effectors of RhoA signalling, consistent with inhibition of Rho associated kinase.

The microarray data suggests that FLU and FEN have a similar mode of action.

Table 34: Data table showing gene changes in GM-CSF signalling pathway in CXR6032 and CXR6069 EC50 and EC20 signature lists.

Table 35: Data table showing gene changes in acute myeloid leukaemia signalling pathway in CXR6032 and CXR6069 EC50 and EC20 signature lists.

Table 36: Data table showing gene changes in RhoA signalling pathway in CXR6032 and CXR6069 EC50 and EC20 signature lists.

Table 37: Vehicle vs FLU CXR6032 EC ₂₍ >

Table 38: Vehicle vs FEN CXR6069 EC ₂₀

Table 39: Vehicle vs FLU CXR6032 EC ₅₀

Table 40: Vehicle vs FEN CXR6069 EC ₅₀

Example 9 - Preferred pharmaceutical formulations and modes and doses of administration.

The compounds of the present invention may be delivered using an injectable sustained- release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period. The compounds of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for administration. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.

Compounds of the invention can also be delivered by electroincorporation (El). El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative method of administration is the ReGel injectable system that is thermosensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

Compounds of the invention can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of compounds to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient (Derossi et al., 1998, Trends Cell Biol., 8, 84-87).

Preferably, the pharmaceutical formulation of the present invention is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.

The compounds of the invention can be administered by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the compounds of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical exipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Formulations suitable 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. 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. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For ophthalmic use, the compounds of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum. For application topically to the skin, the compounds of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Generally, in humans, oral or parenteral administration of the compounds of the invention is the preferred route, being the most convenient.

For veterinary use, the compounds of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

The formulations of the pharmaceutical compositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of an active ingredient. A preferred delivery system of the invention may comprise a hydrogel impregnated with a polypeptides, polynucleotides and antibodies of the invention, which is preferably carried on a tampon which can be inserted into the cervix and withdrawn once an appropriate cervical ripening or other desirable affect on the female reproductive system†ias been produced.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question.

Example 10- Exemplary pharmaceutical formulations

Whilst it is possible for compounds of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen-free.

The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a compound of the invention.

Example 10A: Injectable Formulation

Active ingredient 0.200 g

Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer (35-40 ^° C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Example 10B: Intramuscular injection

Active ingredient 0.20 g

Benzyl Alcohol 0.10 g

Glucofurol 75 ^® 1.45 g

Water for Injection q.s. to 3.00 ml The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1). Example 10C: Tablet

Active ingredient 100 mg

Lactose 200 mg

Starch 50 mg

Polyvinylpyrrolidone 5 mg

Magnesium stearate 4 mg

359 mg

Tablets are prepared from the foregoing ingredients by wet granulation followed by compression.

Example 10D: Ophthalmic Solution

Active ingredient 0.5 g

Sodium chloride, analytical grade 0.9 g

Thiomersal 0.001 g

Purified water to 100 ml

pH adjusted to7.5

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