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Definitions

• This invention relates to compounds, or pharmaceutically acceptable salts thereof, which possess anti-angiogenic activity and are accordingly useful in methods of treatment of disease states associated with angiogenesis in the animal or human body.
• the invention also concerns processes for the preparation of the compounds, pharmaceutical compositions containing the compounds as active ingredient, and methods for the use of the compounds in the manufacture of medicaments for use in the production of anti-angiogenic effects in warmblooded animals such as humans.
• the Tie2 receptor tyrosine kinase also known as TEK
• TEK Tie2 receptor tyrosine kinase
• Angiogenesis is a fundamental process defined as the generation of new blood vessels from existing vasculature. It is a vital yet complex biological process required for the formation and physiological functions of virtually all the organs. Normally it is transient in nature and is controlled by the local balance of angiogenic and angiostatic factors in a multi- step process involving vessel sprouting, branching and tubule formation by endothelial cells (involving processes such as activation of endothelial cells (ECs), vessel destabilisation, synthesis and release of degradative enzymes, EC migration, EC proliferation, EC organisation and differentiation and vessel maturation).
• ECs endothelial cells
• angiogenesis In the adult, physiological angiogenesis is largely confined to wound healing and several components of female reproductive function and embryonic development. In undesirable or pathological angiogenesis, the local balance between angiogenic and angiostatic factors is dysregulated leading to inappropriate and/or structurally abnormal blood vessel formation. Pathological angiogenesis has been associated with disease states including diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi's sarcoma and haemangioma (Fan et al, 1995, Trends Pharmacology. Science. 16: 57-66; Folkman, 1995, Nature Medicine 1: 27-31). In cancer, growth of primary and secondary tumours beyond 1-2 mm 3 requires angiogenesis (Folkman, J.
• NEGF vascular endothelial growth factor
• angiopoietins Two major classes of angiogenic factors are the vascular endothelial growth factor (NEGF) and the angiopoietins. These polypeptide moieties interact with their respective receptors (transmembrane tyrosine kinases wliich are predominantly endothelial cell specific) and induce cellular responses via ligand mediated signal transduction.
• RTKs Receptor tyrosine kinases
• NEGF and the angiopoietins cooperate to regulate various aspects of the angiogenic process during both normal and pathological angiogenesis via signalling through their respective receptors.
• Receptor tyrosine kinases are important in the transmission of biochemical signals across the plasma membrane of cells. These transmembrane molecules characteristically consist of an extracellular ligand-binding domain connected through a segment in the plasma membrane to an intracellular tyrosine kinase domain. Binding of ligand to the receptor results in stimulation of the receptor-associated tyrosine kinase activity that leads to phosphorylation of tyrosine residues on both the receptor and other intracellular molecules.
• RTK subfamilies defined by amino acid sequence homology.
• One of these subfamilies is presently comprised by the fms-like tyrosine kinase receptor, Fit or Fltl, the kinase insert domain-containing receptor, KDR (also referred to as Flk-1), and another fms-like tyrosine kinase receptor, Flt4.
• the transmembrane receptors Tiel and Tie2 constitute a family of endothelial cell specific tyrosine kinase receptors involved in maintenance of blood vessel integrity and which are involved in angiogenic outgrowth and vessel remodelling. Structurally Tiel and Tie2 share a number of features (e.g. the intracellular domains of both these receptors each contain a tyrosine kinase domain interrupted by a kinase insert region) and thus constitute a distinct RTK subfamily. Overall sequence identity between Tiel and Tie2 receptors at the amino acid level is 44% while their intracellular domains exhibit 76% homology.
• Targeted disruption of the Tiel gene results in a lethal phenotype characterised by extensive haemorrhage and poor microvessel integrity (Puri, M. et al. 1995 EMBO Journal:14: 5884-5891).
• Transgenic mice deficient in Tie2 display defects in vessel sprouting and remodelling and display a lethal phenotype in mid gestation (E9.5-10.5) caused by severe defects in embryonic vasculature (Sato, T. et al. 1995 Nature 370: 70-74). To date no ligands have been identified for Tiel and little is known regarding its signalling abilities.
• Tiel is believed to influence Tie2 signalling via heterodimerisation with the Tie2 receptor (hence potentially modulating the ability of Tie2 to autophosphorylate (Marron, M. et al. 2000 Journal of Biological Chemistry: 275, 39741- 39746) and recent chimaeric Tiel receptor studies have indicated that Tie-1 may inhibit apoptosis via the PI 3 kinase/Akt signal transduction pathway (Kontos, CD., et al., 2002 Molecular and Cellular Biology: 22, 1704-1713).
• angiopoietins a number of ligands, designated the angiopoietins have been identified for Tie2 of which Angiopoietin 1 (Angl) is the best characterised. Binding of Angl induces tyrosine phosphorylation of the Tie2 receptor via autophosphorylation and subsequently activation of its signalling pathways via signal transduction. Ang2 has been reported to antagonise these effects in endothelial cells (Maisonpierre, P. et al. 1997 Science: 277, 55-60). The knock-out and transgenic manipulation of Tie2 and its ligands suggest that stringent spatial and temporal control of Tie2 signalling is imperative for the correct development of new vasculature.
• Activation of the Tie2 receptor by Angl inhibits apoptosis (Papapetropoulos, A., et al, 2000 Journal of Biological Chemistry: 275 9102-9105), promotes sprouting in vascular endothelial cells (Witzenbicher, B., et al., 1998 Journal of Biological Chemistry: 273, 18514-18521) and in vivo promotes blood vessel maturation during angiogenesis and reduces the permeability and consequent leakage from adult microvessels (Thurston, G. et al., 2000 Nature Medicine: 6, 460-463).
• Tie2 receptor is reported to be involved in the branching, sprouting and outgrowth of new vessels and recruitment and interaction of periendothelial support cells important in maintaining vessel integrity and overall appears to be consistent with promoting microvessel stability. Absence of Tie2 activation or inl ibition of Tie2 auto phosphorylation may lead to a loss of vascular structure and matrix/cell contacts (Brindle, N., in press, 2002) and in turn may trigger endothelial cell death, especially in the absence of survival or growth stimuli. On the basis of the above reported effects due to Tie2 kinase activity, inhibiting Tie2 kinase may provide an anti-angiogenic effect and thus have application in the therapy of disease states associated with pathological angiogenesis.
• Tie2 expression has been shown to be up-regulated in the neovasculature of a variety of tumours (e.g. Peters, K.G. et al, (British Journal of Cancer 1998; 77,51-56) suggesting that inhibiting Tie2 kinase activity will result in anti-angiogenic activity.
• studies with soluble Tie2 receptor (extracellular domain) (Pengnian, L. et al., 1997, Journal of Clinical Investigation 1997: 100, 2072-2078 and Pengnian, L. et al., 1998, Proceedings of the National Academy of Sciences 1998: 95, 8829-8834) have shown anti-rumour activity in in vivo tumour models.
• NEGF A and Angl reported to collaborate to induce angiogenesis and produce non-leaky mature vessels Thurston, G, et al., 1999 Science: 286,2511-2514). It is quite possible that a mix of such angiogenic processes even exist within a single tumour. Tie2 has also been shown to play a role in the vascular abnormality called venous malformation (VM) (Mulliken, J.B. & Young, A.E. 1998, Nascular birthmarks: W. B. Saunders, Philadelphia). Such defects can either be inherited or can arise sporadically. NM's are commonly found in the skin or mucosal membranes but can affect any organ.
• VM venous malformation
• Upregulation of Tie2 expression has also been found within the vascular synovial pannus of arthritic joints in humans, which is consistent with the role of inappropriate neovascularisation. Such examples provide further indications that inhibition of Tie2 phosphorylation and subsequent signal transduction will be useful in treating disorders and other occurrences of inappropriate neovascularisation. To date only a few inhibitors of Tie2 are known in the art.
• Z is selected from N and CH
• W is selected from N and CH;
• X is selected from NH and CH 2 ;
• Y is selected from F, Cl, Br and I; and n is 1, 2 or 3; and salts or solvates thereof (particularly pharmaceutically acceptable salts thereof).
• Z is N; W is selected from N and CH;
• X is selected from NH and CH 2 ;
• Y is selected from F, Cl, Br and I; and n is i, 2 or 3; and salts or solvates thereof (particularly pharmaceutically acceptable salts thereof).
• Particular compounds of the present invention include, for example, compounds of the formula I, or salts or solvates thereof (particularly pharmaceutically acceptable salts thereof), wherein Z is N, W is N and X, Y and n are as hereinbefore defined.
• Further particular compounds of the present invention include, for example, compounds of the formula I, or salts or solvates thereof (particularly pharmaceutically acceptable salts thereof), wherein Z is N, W is CH and X, Y and n are as hereinbefore defined.
• Z is selected from N and CH; W is N; X is selected from NH and CH 2 ; Y is selected from F, Cl, Br and I; and n is 1, 2 or 3; and salts or solvates thereof (particularly pharmaceutically acceptable salts thereof).
• Particular compounds of the present invention include, for example, compounds of the formula I, or salts or solvates thereof (particularly pharmaceutically acceptable salts thereof), wherein Z is CH, W is N and X, Y and n are as hereinbefore defined.
• Z is selected from N and CH
• W is CH
• X is selected from NH and CH 2 ; Y is selected from F, Cl, Br and I; and n is 1, 2 or 3; and salts or solvates thereof (particularly pharmaceutically acceptable salts thereof).
• Particular compounds of the present invention include, for example, compounds of the formula I, or salts or solvates thereof (particularly pharmaceutically acceptable salts thereof), wherein Z is CH, W is CH and X, Y and n are as hereinbefore defined.
• Y is selected from fluoro, chloro, bromo and iodo, particularly selected from fluoro and chloro. It should be understood that when n is 2 or 3 that Y may be the same or different.
• Specific compounds of the present invention are one or more of the following:
• N-(3 -fluorophenyl)urea N- ⁇ 3-[5-(4-aminothieno[2,3-fl ⁇ pyrirmdin-6-yl)- -methyl-lH-imidazol-4-yl]phenyl ⁇ - N-(2-fluorophenyl)urea N- ⁇ 3-[5-(4-aminothieno[2,3- ⁇ pyrimidin-6-yl)- -methyl-lH-imidazol-4-yl]phenyl ⁇ - N-(3 -chlorophenyl)urea N- ⁇ 3-[5-(4-aminothieno[2,3- ⁇ 7 ]pyrimidin-6-yl)- -methy 1- 1 H-imidazol-4-yl]pheny 1 ⁇ - N-(2-chlorophenyl)urea N- ⁇ 3-[5-(4-aminothieno[2,3-(i]pyrimidin-6-yl
• N-(3-chlorophenyl)urea N- ⁇ 3 - [5 -(4-aminothiazolo [2,3 -oHpyrimidin-6-y I 1 -methyl- lH-imidazol-4-yl]phenyl ⁇ - N-(2-chlorophenyl)urea N- ⁇ 3-[5-(4-aminothiazolo[2,3- ⁇ 3 pyrimidin-6-yr - 1 -methyl- lH-imidazol-4-yl]phenyl ⁇ - 2-(3-fluorophenyl)acetamide N- ⁇ 3-[5-(4-aminothiazolo[2,3-(J
• a suitable pharmaceutically-acceptable salt of a compound of the formula I is, for example, an acid-addition salt of a compound of the formula I, for example an acid-addition salt with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, trifluoroacetic, citric or maleic acid; or, for example, a salt of a compound of the formula I which is sufficiently acidic, for example an alkali or alkaline earth metal salt such as a calcium or magnesium salt, or an ammonium salt, or a salt with an organic base such as methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2- hydroxyethyl)amine.
• an acid-addition salt of a compound of the formula I for example an acid-addition salt with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, trifluoroacetic, citric or maleic acid
• the invention includes in its definition any such optically active or racemic form which possesses the above-mentioned activity.
• the synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form.
• the above-mentioned activity may be evaluated using the standard laboratory techniques referred to hereinafter.
• certain compounds of the formula I may exist in solvated as well as unsolvated forms such as, for example, hydrated forms.
• the invention encompasses all such solvated forms which exhibit an inhibitory effect on a Tie2 receptor tyrosine kinase. It is also to be understood that certain compounds of the foraiula I may exhibit polymorphism, and that the invention encompasses all such forms which exhibit an inhibitory effect on a Tie2 receptor tyrosine kinase.
• the invention relates to all tautomeric forms of the compounds of the formula I forms which exhibit an inhibitory effect on a Tie2 receptor tyrosine kinase.
• pharmaceutically-acceptable salts of compounds of the invention are preferred, other non-pharmaceutically-acceptable salts of compounds of the invention may also be useful, for example in the preparation of pharmaceutically-acceptable salts of compounds of the invention.
• pro-drugs of compounds of the invention as herein before or herein after defined. Compounds of the invention may be administered in the form of a pro-drug which is broken down in the human or animal body to give a compound of the Formula (I).
• pro-drugs include in- ivo hydrolysable amides of a compound of the Formula (I).
• Various forms of pro-drugs are known in the art. For examples of such pro-drug derivatives, see: a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 "Design and Application of Prodrugs", by H. Bundgaard p.
• a compound of formula I, or a pharmaceutically acceptable salt thereof may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Such processes, when used to prepare a compound of formula I are provided as a further feature of the invention and are illustrated by the following representative process variants. Necessary starting materials may be obtained by standard procedures of organic chemistry.
• the present invention provides a process for preparing a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein X is NH and Z, W, Y and n are as defined in formula I, which process comprises of reacting an amine of the formula II with an isocyanate of the formula III Formula II Formula III and thereafter if necessary: i) converting a compound of the formula (I) into another compound of the formula (I); ii) forming a salt or solvate.
• the aforementioned process is conveniently carried out in a suitable inert solvent or diluent such as an ether, for example tetraliydrofuran, DMF, DCM, DMA or pyridine.
• a suitable inert solvent or diluent such as an ether, for example tetraliydrofuran, DMF, DCM, DMA or pyridine.
• the process is preferably carried out at a temperature of less than 50°C, for example 0 to 30°C, preferably at ambient temperature.
• the amine of the formula II may be prepared by the reaction scheme (i) below: Scheme (i) wherein: (a) Piperazine in THF (b) m-CPBA in DCM (c) Concentrated aqueous ammonia in 1,4-dioxane (d) Benzophenone imine, l, -bis(diphenylphosphino)ferrocene, bis(benzylideneacetone)palladium and sodium tert-butoxide in dioxane (e) HCl in THF
• the sulfone of the formula lib may be prepared by the reaction of trimethylsilylchloride, 3-iodobenzaldehyde, formamide and toluene sulpliinc acid to produce ⁇ (3-iodophenyl)[(4-methylphenyl)sulphonyl]metliyl ⁇ formamide and then the reaction of the ⁇ (3-iodophenyl)[(4-methylphenyl)sulphonyl]methyl ⁇ formamide with phosphorus oxychloride to produce the sulfone of the formula lib.
• the present invention provides a process for preparing a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein X is CH 2 and Z, W, Y and n are as defined in formula I, which process comprises of reacting an amine of the formula II with an appropriate acyl chloride or acyl ester, as the skilled person would appreciate.
• a pharmaceutically acceptable salt of a compound of formula I is required, for example an acid addition salt, it may be obtained by, for example reaction of a compound of the formula I with a suitable acid such as hydrochloric acid, using a conventional procedure.
• an acid addition salt of the compound of formula I may be treated with a suitable base, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine, N-methylmorpholine or diazabicyclo[5.4.0]undec-7-ene, or, for example, an alkali or alkaline earth metal carbonate or hydroxide, for example sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide.
• a suitable base for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine, N-methylmorpholine or diazabicyclo[5.4.0]undec-7-ene, or, for example, an alkali or alkaline earth metal carbonate or hydroxide, for example sodium carbonate, potassium carbonate, calcium carbonate
• the invention encompasses all geometric and optical isomers of the compounds of formula I and mixtures thereof including racemates. Tautomers and mixtures thereof also form an aspect of the present invention.
• Isomers may be resolved or separated by conventional techniques, e.g. chromatography or f actional crystallisation.
• Enantiomers may be isolated by separation of a racemic or other mixture of the compounds using conventional techniques (e.g. chiral High Performance Liquid Chromatography (HPLC)).
• HPLC High Performance Liquid Chromatography
• the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g.
• Protecting groups may be removed by any convenient method as described in the literature or known to tlie skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
• Specific examples of protecting groups are given below for the sake of convenience, in which "lower", as in, for example, lower alkyl, signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned are, of course, within the scope of the invention.
• a carboxy protecting group may be the residue of an ester-forming aliphatic or arylaliphatic alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
• carboxy protecting groups include straight or branched chain (l-12C)alkyl groups (for example isopropyl, and tert-butyl); lower alkoxy- lower alkyl groups (for example memoxymethyl, ethoxymethyl and isobutoxymethyl); lower acyloxy-lower alkyl groups, (for example acetoxymethyl, propionyloxymethyl, butyryloxymethyl and pivaloyloxymethyl); lower alkoxycarbonyloxy-lower alkyl groups (for example -methoxycarbonyloxyethyl and 1-ethoxycarbonyloxy ethyl); aryl-lower alkyl groups (for example benzyl, 4-methoxybenzylj 2-nitrobenzyl, 4-nitrobenzyl
• Methods particularly appropriate for the removal of carboxyl protecting groups include for example acid-, base-, metal- or enzymically-catalysed cleavage.
• hydroxy protecting groups include lower alkyl groups (for example tert- butyl), lower alkenyl groups (for example allyl); lower alkanoyl groups (for example acetyl); lower alkoxycarbonyl groups (for example tert-butoxy carbonyl ; lower alkenyloxy carbonyl groups (for example allyloxycarbonyl); aryl-lower alkoxycarbonyl groups (for example benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
• tri(lower alkyl)silyl for example trimethylsilyl and tert-butyldimethylsilyl
• aryl-lower alkyl for example benzyl
• amino protecting groups include formyl, aryl-lower alkyl groups (for example benzyl and substituted benzyl, 4-methoxybenzyl, 2-nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-4-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (for example tert-butoxy carbonyl); lower alkenyloxy carbonyl (for example allyloxycarbonyl); aryl-lower alkoxycarbonyl groups (for example benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl and 4-nitrobenzyloxy carbonyl); trialkylsilyl (for example trimethylsilyl and tert-butyldimethylsilyl); alkylidene (for example methylidene) and benzylidene and substituted benzylidene groups.
• aryl-lower alkyl groups for example benzy
• Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis for groups such as 2-nitrobenzyloxycarbonyl, hydrogenation for groups such as benzyl and photolytically for groups such as 2-nitrobenzyloxycarbonyl.
• groups such as 2-nitrobenzyloxycarbonyl
• hydrogenation for groups such as benzyl
• photolytically for groups such as 2-nitrobenzyloxycarbonyl.
• certain of the various ring substituents in the compounds of the present invention may be introduced by standard aromatic substitution reactions or generated by conventional functional group . modifications either prior to or immediately following the processes mentioned above, and as such are included in the process aspect of the invention. Such reactions and modifications include, for example, introduction of a substituent by means of an aromatic substitution reaction, reduction of substituents, alkylation of substituents and oxidation of substituents.
• reagents and reaction conditions for such procedures are well known in the chemical art.
• a particular example of an aromatic substitution reaction includes the introduction of a halogeno group.
• Biological Assays The following assays can be used to measure the effects of the compounds of the present invention as Tie2 inhibitors in vitro and as inhibitors of Tie2 autophosphorylation in whole cells. a.
• In vitro receptor tyrosine kinase inhibition assay To test for inhibition of Tie2 receptor tyrosine kinase, compounds are evaluated in a non-cell based protein kinase assay by their ability to inhibit the protein kinase enzyme phosphorylation of a tyrosine containing polypeptide substrate in an ELISA based microtitre plate assay. In this particular case, the assay was to determine the IC 50 , for three different recombinant human tyrosine kinases Tie2, KDR and Fit. To facilitate production of the tyrosine kinases, recombinant receptor genes were produced using standard molecular biology cloning and mutagenesis techniques.
• recombinant proteins fragments encoded within these genes consist of only the intracellular portion C-terminal portion of the respective receptor, within which is found the kinase domain.
• the recombinant genes encoding the kinase domain containing fragments were cloned and expressed in standard baculovirus/Sf21 system (or alternative equivalent). Lysates were prepared from the host insect cells following protein expression by treatment with ice-cold lysis buffer (20mM N-2-hydroxyethylpiperizine-
• HEPES N'-2-ethanesulphonic acid
• Reactions were terminated by the removal of the liquid components of the assay followed by washing of the plates with PBS-T (phosphate buffered saline with 0.5% Tween 20) or an alternative equivalent wash buffer.
• PBS-T phosphate buffered saline with 0.5% Tween 20
• the immobilised phospho-peptide product of the reaction was detected by immunological methods. Firstly, plates were incubated for 4 hours at room temperature with murine monoclonal anti-phosphotyrosin -HRP (Horseradish Peroxidase) conjugated antibodies (4G10 from Upstate Biotechnology UBI 16-105). Following extensive washing with PBS-T, HRP activity in each well of the plate was measured colorimetrically using murine monoclonal anti-phosphotyrosin -HRP (Horseradish Peroxidase) conjugated antibodies (4G10 from Upstate Biotechnology UBI 16-105). Following extensive washing with PBS-T, HRP activity in each well of the plate was measured colori
• Cellular Tie2 autophosphorylation assay This assay is based on measuring the ability of compounds to inhibit autophosphorylation of the Tie2 receptor which normally leads to the production of "activated" receptor that in turn initiates the particular signal transduction pathways associated with the receptor function. Autophosphorylation can be achieved by a number of means. It is known that expression of recombinant kinase domains in baculoviral systems can lead to the production of phosphorylated and activated receptor. It is also reported that over expression of receptors in recombinant cell lines can itself lead to receptor autophosphorylation in the absence of the ligand (Heldin C-H. 1995 Cell: 80, 213-223; Blume-J. P, Hunter T. 2001 Nature: 411, 355- 65).
• chimaeric receptors have been constructed.
• the natural, external cell surface domain of the receptor has been replaced with that of a domain which is known to be readily dimerised via the addition of the appropriate ligand (e.g. TrkA-Tie2/NGF ligand (Marron, M.B., et al., 2000 Journal of Biological Chemistry: 275:39741-39746) or C-fms-Tie-l/CSF-1 ligand (Kontos, CD., et al., 2002 Molecular and Cellular Biology: 22, 1704-1713).
• TrkA-Tie2/NGF ligand Marron, M.B., et al., 2000 Journal of Biological Chemistry: 275:39741-39746
• C-fms-Tie-l/CSF-1 ligand Kontos, CD., et al., 2002 Molecular and Cellular Biology: 22, 1704-1713.
• Natural Angl ligand can be isolated using standard purification technology from either tumour cell supernatants or alternatively the Angl gene can be cloned and expressed recombinantly using stand molecular biology techniques and expression systems. In this case one can either attempt to produce the ligand either in its native state or as recombinant protein which for example may have been genetically engineered to contain additional of purification tags (eg. polyhistidine peptides, antibody Fc domains) to facilitate the process.
• purification tags eg. polyhistidine peptides, antibody Fc domains
• EA.hy926/B3 or HUVEC cellular Tie2 receptor a Angl ligand stimulated cellular receptor phosphorylation assay can be constructed which can be used to analyse to determine the potential of compounds to inhibit this process.
• EA.hy926/B3 cells were grown in the appropriate tissue culture media plus 10% foetal calf serum (FCS) for two days in 6 well plates starting with an initial seeding density of 5x10 5 cells/well. On the third day the cells were serum starved for a total of 2 hours by replacmg the previous media with media containing only 1% FCS.
• FCS foetal calf serum
• the media was removed and replace with 1 ml of the test compound dilutions (compound dilutions made in serum starvation media yet keeping the DMSO concentration below 0.8%).
• orthovanidate was added to a final concentration of 0.1 mM for the final 10 minutes of serum starvation.
• the ligand plus orthovandiate was added to stimulate autophosphorylation of the cellular Tie2 receptor (ligand can be added either as purified material diluted in serum starvation media or non-purified cell supernatant containing ligand e.g. when recombinantly expressed mammalian cells).
• the cells were cooled on ice washed with approximately 5mls with cold PBS containing 1 mM orthovanadate, after which 1 ml of ice cold lysis buffer ((20 mM Tris pH 7.6, 150 mM NaCl, 50 mM NaF, 0.1 % SDS, 1% NP40, 0.5 % DOC, 1 mM orthovanadate, 1 mM EDTA, 1 mM PMSF, 30 ⁇ l / ml Aprotinin, 10 ⁇ g/ml Pepstatin, 10 ⁇ g/ml Leupeptin) was added the cells and left on ice for 10- 20 minutes.
• ice cold lysis buffer ((20 mM Tris pH 7.6, 150 mM NaCl, 50 mM NaF, 0.1 % SDS, 1% NP40, 0.5 % DOC, 1 mM orthovanadate, 1 mM EDTA, 1 mM PMSF, 30 ⁇ l / ml
• the lysate was removed and transferred to a 1.5 ml Eppendorf tube and centrifuged for 3 minutes at 13000 rpm at 4 °C. 800 ⁇ l of each lysate was transferred to fresh 2 ml Eppendorf tubes for the immuno-precipitation.
• 3 mg 15 ⁇ l of anti-phospho-tyrosine antibody (Santa Cruz PY99 -sc-7020) was added to the lysates and left to incubate for 2 hours at 4 °C 600 ⁇ l washed
• MagnaBind beads (goat anti-mouse IgG, Pierce 21354) were added to the lysates and the tubes left to rotate over night at 4 °C.
• the western blots of the various immuno-precipitated samples were developed the blots with LumiGLO (NEB 7003) and transferred to an X-Ray cassette and films exposed for 15 sec / 30 sec and 60 sec.
• the relative strength of the protein band which pertains to the phosphorylated Tie2 receptor was evaluated using a FluorS BioRad image analyser system. The percentage phosphorylation for each test compound dilution series was determined from which IC 50 values were calculated by standard methods using the appropriate control samples as reference.
• compositions of the invention which comprises a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore in association with a pharmaceutically acceptable diluent or carrier.
• the compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing
• compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.
• compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
• the amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the. host treated and the particular route of administration.
• a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent (more suitably from 0.5 to 100 mg, for example from 1 to 30 mg) compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.
• the size of the dose for therapeutic or prophylactic purposes of a compound of the formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.
• a compound of the formula I for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.1 mg/kg to 75 mg/kg body weight is received, given if required in divided doses.
• a parenteral route is employed.
• a dose in the range for example, 0.1 mg/kg to 30 mg/kg body weight will generally be used.
• a dose in the range for example, 0.05 mg kg to 25 mg/kg body weight will be used.
• Oral administration is however preferred, particularly in tablet form.
• unit dosage forms will contain about 0.5 mg to 0.5 g of a compound of this invention.
• the compounds according to the present invention as defined herein are of interest for, amongst other things, their antiangiogenic effect.
• the compounds of the invention are expected to be useful in the treatment or prophylaxis of a wide range of disease states associated with undesirable or pathological angiogenesis, including cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi's sarcoma, haemangioma, lymphoedema, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, acute inflammation, excessive scar formation and adhesions, endometriosis, dysfunctional uterine bleeding and ocular diseases with retinal vessel proliferation.
• Cancer may affect any tissue and includes leukaemia, multiple myeloma and lymphoma.
• such compounds of the invention are expected to slow advantageously the growth of primary and recurrent solid tumours of, for example, the colon, breast, prostate, lungs and skin.
• the antiangiogenic properties of the compounds according to the present invention arise from their Tie2 receptor tyrosine kinase inhibitory properties.
• the compounds of the present invention are expected be useful to produce a Tie2 inhibitory effect in a warm-blooded animal in need of such treatment.
• the compounds of the present invention may be used to produce an antiangiogenic effect mediated alone or in part by the inhibition of Tie2 receptor tyrosine kinase. More particularly the compounds of the invention are expected to inhibit any form of cancer associated with Tie2.
• a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore for use as a medicament.
• a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore in the manufacture of a medicament for use as a Tie2 receptor tyrosine kinase inhibitor in a warm-blooded animal such as man.
• a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore in the manufacture of a medicament for use in the production of an anti-angiogenic effect in a warm-blooded animal such as man.
• a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore in the manufacture of a medicament for use in the treatment of cancers in a warm-blooded animal such as man.
• a cancer selected from leukaemia, breast, lung, colon, rectal, stomach, prostate, bladder, pancreas, ovarian, lymphoma, testicular, neuroblastoma, hepatic, bile duct, renal cell, uterine, thyroid and skin cancer in a warm-blooded animal such as man.
• a method of inliibiting Tie2 receptor tyrosine kinase in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
• a method for producing an anti-angiogenic effect in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
• a method of treating cancers in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound of the formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
• a compound of tl e formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore for use in the treatment of cancer.
• a cancer selected from leukaemia, breast, lung, colon, rectal, stomach, prostate, bladder, pancreas, ovarian, lymphoma, testicular, neuroblastoma, hepatic, bile duct, renal cell, uterine, thyroid or skin cancer.
• a compound of the present invention will possess activity against other diseases mediated by undesirable or pathological angiogenesis including psoriasis, rheumatoid arthritis, Kaposi's sarcoma, haemangioma, lymphoedema, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, acute inflammation, excessive scar formation and adhesions, endometriosis, dysfunctional uterine bleeding and ocular diseases with retinal vessel proliferation.
• the anti-angiogenic activity defined herein may be applied as a sole therapy or may involve, in addition to a compound of the invention, one or more other substances and/or treatments.
• Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment.
• the field of medical oncology it is normal practice to use a combination of different forms of treatment to treat each patient with cancer.
• the other component(s) of such conjoint treatment in addition to the cell cycle inhibitory treatment defined hereinbefore may be: surgery, radiotherapy or chemotherapy.
• Such chemotherapy may include one or more of the following categories of anti-rumour agents: (i) anti-invasion agents (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function); (ii) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea, or, for example, one of the preferred antimetabolites disclosed in European Patent Application No.
• anti-invasion agents for example metalloproteinase inhibitors like marimastat and inhibitor
• 562734 such as 5 (2S)-2- ⁇ o-fluoro-p_-[N- ⁇ 2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)- N-(prop-2-ynyl)amino]benzamido ⁇ -4-(tetrazol-5-yl)butyric acid); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol
• topoisomerase inliibitors for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin
• cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example
• inhibitors of growth factor function include growth factor antibodies, growth factor receptor antibodies, famesyl transferase inhibitors,
• tyrosine kinase inhibitors and serine/threonine kinase inhibitors for example inhibitors of the epidermal growth factor family (for example the EGFR tyrosine kinase inhibitors N-(3- chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (ZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (CP 358774) and 6- acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine (Cl
• biotherapeutic therapeutic approaches for example those which use peptides or proteins (such as antibodies or soluble external receptor domain constructions) which either sequest receptor ligands, block ligand binding to receptor or decrease receptor signalling (e.g.
• antisense therapies for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense
• gene therapy approaches including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy
• immunotherapy approaches including for example ex- vivo and in- vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected
• Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.
• Such combination products employ the compounds of this invention within the dosage range described hereinbefore and the other pharmaceutically active agent witiiin its approved dosage range.
• a pharmaceutical product comprising a compound of the formula I as defined hereinbefore and an additional anti-tumour substance as defined hereinbefore for the conjoint treatment of cancer.
• the compounds of formula I and their pharmaceutically acceptable salts are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of inhibitors of cell cycle activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.
• DMTMM 4-(4,6-Dimethoxy- 1 ,3 ,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride dppf 1 , 1 '-Bis(diphenylphosphino)ferrocene
• the starting material was prepared as follows:- Intermediate 1 ⁇ (3-iodophenyI)[(4-methylphenyl)sulfonvn methyl) formamide Trimethylsilylchloride (9.1 mL) was added to a stirred solution of 3-iodobenzaldehyde (15.1 g) and formamide (6.5 mL) in MeCN (34 mL) and toluene (34 mL) under an inert atmosphere. The reaction was then heated at 50°C for 5 hours. Toluene sulfinic acid (15.3 g) was added and the reaction mixture was heated at 50°C for a further 5 hours.

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