Field of the Invention

The present invention relates to the use of compounds as angiogenesis inhibitors in the manufacture of a medicament for use in the treatment of disease states associated with angiogenesis, and anti-angiogenic pharmaceutical compositions containing them as active ingredient.

Backeround of the invention

Angiogenesis, the process of new blood vessels formation, plays an important role in numerous physiological events, both normal and pathological. Under normal conditions, angiogenesis is associated with such events as wound healing, several components of female reproductive function, formation of the corpus luteum, endometrium, placenta and embryonic development, as discussed by Folkman, et al., Science 243, 1490-1493 (1989). However, a number of serious diseases are also dominated by abnormal neovascularization including solid tumor growth and metastases, some types of eye disorders, and rheumatoid arthritis, reviewed by Auerbach, et al., J. Microvasc. Res. 29, 401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, pp. 175-203 (Academic Press, New York 1985); Patz, Am. J. Opthalmol. 94, 715-743 (1982); and Folkman, et al., Science 221, 719-725 (1983). For example, there are a number of eye diseases, many of which lead to blindness, in which ocular neovascularization occurs in response to the diseased state. There are a number of other eye diseases which are also associated with neovascularization, including retrolental fibroplasia, uveitis, approximately twenty eye diseases associated with choroidal neovascularization and approximately forty eye diseases which are associated with iris neovascularization.

Growth of neoplasms is very often associated with angiogenesis. In cancers, the cellular heterogeneity of malignant cell populations contributes to the complex expression of multiple angiogenic factors (Cao Y. Nat Rev Cancer. 2005 Sep; 5(9):735-43). The vascular endothelial growth factor (VEGF) is a key angiogenic factor that exerts multiple functions, including regulation of angiogenesis, vasculogenesis, inflammation, and vascular permeability. A significant portion of research on ocular and tumor angiogenesis has focused on VEGF and its blockade. The success of clinical trials based on the use of anti- VEGF agents (bevacizumab, commercially known as Avastin ^® , a humanized monoclonal antibody) for the treatment of colorectal cancer patients has set a milestone in the area of antiangiogenic cancer therapy (Cao Y. Front Biosci. 2009; 14:3962-73; Cao Y, Liu Q. Adv. Cancer Res. 2007;97:203-24; Fernando NH, Hurwitz HI. Oncologist. 2004; 9 Suppl 1:11- 8). Angiogenesis inhibitor stops tumor growth by preventing the formation of new blood vessels by targeting and inhibiting the function of the VEGF that stimulates new blood vessel formation. Non-oncologic applications of anti-VEGF agents are also known, as it is the case for the treatment of the exudative form of Age-related Macular Degeneration (AMD) with the a monoclonal antibody ranibizumab (Lucentis ^® ).

Current therapeutic approaches targeting angiogenic pathways are not only focused on the development of anti-VEGF agents, which include VEGF neutralizing antibodies, soluble VEGF receptors (VEGFR), anti-VEGF receptor neutralizing antibodies, and anti- intracellular signaling components (Ferrara N, Kerbel RS. Nature. 2005 Dec 15, 438(7070): 967-74; Griffioen AW. Trends Cardiovasc Med. 2007 Jul, 17(5): 171-6). Small molecule angiogenesis inhibitors are also clinically approved, for instance, tyrosine kinase inhitibors (TKIs): erlotinib for non-small cell lung cancer and pancreatic cancer; sorafenib for hepatocellular and renal cell carcinoma; sunitinib for renal cell carcinoma. Recently, carboxyamidotriazole (CAI) has been described as a potential angiogenesis inhibitor for proliferative retinopaties in humans. Low molecular weight therapeutic agents offer several advantages over protein or peptide compounds. They have better tissue penetration capacity. They can be administrated at dose higher than peptides, giving more exposure of tissue to the compound.

Several angiogenesis inhibitors are known, but there are disadvantages associated with several of these compound. For example, suramin is a potent angiogenesis inhibitor, but causes (at doses required to reach antitumor activity) severe systemic toxicity in humans. Other compounds, may be difficult or costly to make, e.g. Avastin ^® is one of the most expensive drugs widely marketed. Despite the efforts made until now, it is still necessary to develop angiogenesis inhibitor compounds useful as therapeutic agents for treating or preventing pathologies occurring with angiogenesis development.

EP 2091531 discloses the use of phenyl substituted maleimides inhibiting the opening of the mitochondrial permeability transition pore (MPTP) in cells for the preparation of medicaments for the prevention and/or treatment of diseases resulting from opening of the MPTP which are characterized by degenerative tissue damages. There is however no disclosure, no teaching whatsoever or any suggestion that any of the compounds disclosed may be used as angiogenesis inhibitors.

An object of the present invention is to provide angiogenesis inhibitors for the treatment of diseases, including cancers, associated with abnormal angiogenesis.

Summary of the Invention

Surprisingly, now we have found that compounds of formula (I)

(I)

wherein Ri and R ₂ , each independently, are hydrogen; halo; hydroxy; (C ₁ -C ₃ ) alkyl; (C ₁ -C ₃ ) haloalkyl; (C ₁ -C ₃ ) alkoxy; or (C ₁ -C ₃ ) haloalkoxy;

as well as its stereoisomers, tautomers, polymorphs, solvates, mixtures and metabolites thereof, are active as angiogenesis inhibitors.

Accordingly, the present invention relates to the use of a compound of formula (I) as above defined in the manufacture of a medicament for use in the treatment of disease states associated with angiogenesis. Such treatment comprises administering to the patient a therapeutically effective amount of a compound of formula (I) as above defined.

A further object of the invention relates to a compound of formula (I) as above defined for use in the treatment of disease states associated with angiogenesis. A further object of the invention relates to an anti-angiogenic pharmaceutical composition comprising as an active principle at least a compound of formula (I) as above defined. Preferred compounds of formula (I) are those wherein Ri and R ₂ , each independently, are hydrogen; halo; preferably Ri is chloro and R ₂ is hydrogen.

Detailed Description of the Invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

The term "halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

The term "(C ₁ -C ₃ ) alkyl" refers to a fully saturated straight or branched saturated hydrocarbon chain having one to three carbon atoms. Examples include methyl, ethyl, n- propyl, isopropyl.

The term "(C ₁ -C ₃ ) haloalkyl" refers to a C ₁ -C ₃ alkyl substituted with one or more halogens which may be the same or different. Examples of haloalkyl include, without limitation, monofluoromethyl, difluoromethyl, trifluoromethyl, monochloromethyl, dichloromethyl, trichloromethyl, monobromomethyl, dibromomethyl, tribromomethyl, bromodifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 1-chloroethyl, 2-chloroethyl, 2,2-dichloroethyl, 2,2,2-trichloroethyl, 1 -bromoethyl, 2- bromoethyl, 2,2-dibromoethyl, 2,2,2-tribromoethyl, 2-iodoethyl, pentafluoroethyl, 3-fluoro- n-propyl, 3-chloro-n-propyl, 3-bromo-n-propyl, l,3-difluoro-2-propyl, l,3-dichloro-2- propyl, l,l,l-trifluoro-2-propyl, l-chloro-3-fluoro-2-propyl, l,l,l,3,3,3-hexafluoro-2- propyl, l,l,l,3,3,3-hexafluoro-2-chloro-2-propyl, 2,2,3,3,3-pentafluoro-n-propyl, heptafluoro-i-propyl, heptafluoro-n-propyl or the like.

The term "(C ₁ -C ₃ ) alkoxy" refers to the group C ₁ -C ₃ alkyl-O-.

The term "(C ₁ -C ₃ ) haloalkoxy" refers to a straight or branched haloalkoxy group having 1 to 3 carbon atoms which is substituted with one or more halogen atoms which may be the same or different. Examples of haloalkoxy include, without limitation, trifluoromethoxy, difluoromethoxy, monofluoromethoxy, pentafluoroethoxy, l,l,l,3,3,3-hexafluoro-2- propyloxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, 2-chloroethoxy, 2,2- dichloroethoxy, 2,2,2-trichloroethoxy, 3-fluoro-n-propyloxy, 2,2,3, 3-tetrafluoro-n- propyloxy, 2,2,3,3,3-pentafluoro-n-propyloxy, l,3-difluoro-2-propyloxy, l-chloro-3-fluoro- 2-propyloxy, 1,1,2,3,3,3-hexafluoropropyloxy, 2,2,2-trifluoro-l-trifluoromethylethoxy, 2- trifluoromethoxy- 1 , 1 ,2-trifluoroethoxy, 1 , 1 ,2,2-tetrafluoroethoxy or the like.

The branched alkyl moieties of the above radicals may contain one or more asymmetric carbon atoms which may give rise to enantiomers and/or diastereoisomers.

The term "stereoisomers" refers to all isomers of individual molecules that differ only in the configuration of their carbon atoms in the space. This term includes enantiomers and diastereoisomers. The present invention includes all the possible stereoisomers of the compounds of formula (I) both as single isomers and their mixtures, including racemates. The invention also includes within its scope all the possible isomers, in particular Z- and E- isomers and their mixtures.

In cases when compounds may exist in tautomeric forms, each form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.

Some crystalline forms of the compounds may exist as polymorphs, which are also included in the present invention. Some of the compounds are hydrates or solvates, and as such they are also intended to be encompassed within the scope of the invention.

The term "hydrate" refers to a solvate comprising a disclosed or claimed compound and a stoichiometric or non-stoichiometric amount of water.

The term "solvate" refers to a molecular complex comprising a disclosed or claimed compound and a stoichiometric or non-stoichiometric amount of one or more solvent molecules (e.g., EtOH).

The invention also includes within its scope the metabolites of the compounds of formula (I). The term "metabolite" refers to the break-down or end product of a compound of formula (I) or its salt produced by metabolism or biotransformation in the animal or human body; e. g., biotransformation to a more polar molecule such as by oxidation, reduction, or hydrolysis, or to a conjugate (see Goodman and Gilman, "The Pharmacological Basis of Therapeutics" 10th Ed., Pergamon Press, Gilman et al. (eds.), 2001 for a discussion of biotransformation. Preferred metabolites are compounds wherein the maleimide group is hydrolyzed.

The term "disease states associated with angiogenesis" is used herein, for purposes of the specification and claims, to mean certain pathological processes in mammals, including humans, where angiogenesis is abnormally prolonged.

An angiogenesis inhibitor is an agent capable of suppressing the growth of new blood vessels and would allow modulation in such angiogenesis-associated disease having excessive vascularization. The compounds of the invention, including but not limited to those specified in the examples, possess antiangiogenic activity. Antiangiogenic therapy would allow modulation in such angiogenesis associated diseases having excessive vascularization. Therefore, the compounds of the present invention are useful in treating several pathological conditions in mammals, including humans, where the growth of new blood vessels is detrimental, for example, cancer. As angiogenesis inhibitors, such compounds are useful in the treatment of both primary and metastatic solid tumors, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gall, bladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract, (including vulva, cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as haemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma) and tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas). Such compounds may also be useful in treating solid tumors arising from hematopoietic malignancies such as leukemias (i.e. chloromas, plasmacytomas and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia) as well as in the treatment of lymphomas (both Hodgkin's and non- Hodgkin's lymphomas), multiple myeloma. In addition, these compounds may be useful in the prevention of metastases from the tumors described. In particular such compounds of the invention are expected to slow advantageously the growth of primary and recurrent solid tumours. Further uses include the treatment and prophylaxis of autoimmune diseases, such as rheumatoid, immune and degenerative arthritis; various ocular diseases, many of which lead to blindness, in which ocular neovascularization occurs in response to the diseased state such as diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, age-related macular degeneration, uveitis, ocular diseases with retinal vessel proliferation, proliferating retinitis, diabetic retinitis, hypoxia, angiogenesis in the eye associated with infection or surgical intervention, eye diseases associated with choroidal neovascularization and iris neovascularization and other abnormal neovascularization conditions of the eye; blood vessel, vascular diseases such as capillary proliferation within atherosclerotic plaques, atheroma; Osier- Webber Syndrome; myocardial angiogenesis, myocardial infarction; obstructive arteriosclerosis, plaque neovascularization, diabetic angiopathy, vascular malformation, hypertonia, angina pectoris, cerebral infarction; telangiectasia; hemophiliac joints; angiofibroma, arterial restenosis; and wound granulation; reproductive system diseases such as uterus dysfunction, endometriosis, dysfunctional uterine bleeding, placental dysfunction, ovarian hyperergasia or follicle cyst; transplanted organ rejection. Another use is as a birth control agent, by inhibiting ovulation and establishment of the placenta. The compounds of the invention are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylori). The compounds of the invention are also useful to reduce bleeding by administration prior to surgery, especially for the treatment of resectable tumors.

The compounds of formula (I) as above defined are active as angiogenesis inhibitors.

According to another aspect of the invention, there is provided the use of a compound of formula (I) as above defined, as well as its stereoisomers, tautomers, polymorphs, solvates, mixtures and metabolites thereof, in the manufacture of a medicament for use in the treatment of disease states associated with angiogenesis.

According to another aspect of the invention, there is provided a compound of formula (I) as above defined, as well as its stereoisomers, tautomers, polymorphs, solvates, mixtures and metabolites thereof, for use in the treatment of disease states associated with angiogenesis.

Preferred compounds are compound of formula (I) wherein Ri and R ₂ , each independently, are hydrogen; halo; preferably, Ri is chloro and R ₂ is hydrogen. The most preferred compound is l-(3-chlorophenyl)-3-phenyl-pyrrole-2,5-dione (GNX 686).

The compounds of formula (I) are known compounds and may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Such processes include, for example, those illustrated in EP 2091531.

The biological activity of the compounds of formula (I) above as angiogenesis inhibitors and their use for the manufacture of medicaments in the treatment of disease states associated with angiogenesis have been demonstrated through a series of in vitro and in vivo assays including an anti proliferation assay, a human umbilical vein endothelial cells (HUVEC) angiogenic tube formation assay and two in vivo models: the chorioallantoic membrane (CAM) model of embryonic angiogenesis and the retinopathy of prematurity (ROP) model of proliferative diabetic retinopathy.

The angiogenesis inhibitor activity of the compounds of the present invention is shown, e.g., by the fact that they have been found to be active in the chorioallantoic membrane (CAM) test according to the Folkman's method (Nature. 297, 307, 1982). For instance, the representative compound of the invention GNX 686, when tested in the CAM assay reduces capillary branching, the mean area of the capillary network meshes and the 3 rd quartile of the mesh area histogram, an explanation of these descriptors is detailed in Nowak- Sliwinska et al (Microvasc Res. 79(1): 21-8, 2010).

The compounds of formula (I) which are used for the manufacture of the medicaments according to the present invention are usually incorporated into pharmaceutical compositions which are suitable for achieving the intended purpose.

According to another aspect of the invention, there is provided an anti-angiogenic pharmaceutical composition comprising as an active principle at least a compound of formula (I) as above defined.

Thus the invention provides a pharmaceutical preparation for use in the treatment of disease states associated with angiogenesis characterized in that it contains at least a compound of formula (I) as above defined. All the pharmaceutical preparations used in the treatment of disease states associated with angiogenesis can be used as pharmaceuticals in human or veterinary medicine.

The compounds of formula (I) of the present invention, suitable for administration to a mammal, e.g., to humans, can be administered by the usual routes in a variety of dosage forms containing the active ingredient along with pharmaceutically acceptable carriers and excipients. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Accordingly, the compounds are contained in the above said pharmaceutical compositions in an amount which is effective for the disease to be prevented or treated. Typically, the compound may be administered to mammals, e.g. human at a dose varying from 0,1 to 300 mg per kilogram of body weight per day, a determination of the optimal range for each individual being however within the skill of the art. The dosage depends upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, conditions of the patient, general health, sex and diet of the individual being treated; the time and route of administration, the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; the severity of the illness being treated and like factors well known by those skilled in the art.

If desired, the effective daily dose may be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. Of course, these dosage regimens may be adjusted to provide the optimal therapeutic response by the practitioner who is treating any particular patient.

The pharmaceutical compositions, which are useful for administering the compounds of formula (I) according to this invention, can be administered by a variety of routes including oral, parenteral (including intramuscular, intravenous, subcutaneous, intra-arterial, intraperitoneal, intrasternal, intraarticular, by injection or infusion), transmucosal (including buccal, sublingual, nasal, transurethral, intravaginal and rectal), topical, transdermal, ocular or intravitreal route, by inhalation, permucous or percutaneous or using any other route of administration.

They will thus be presented in the form of both solid forms such as tablets, plain or coated tablets, sugar or film coated tablets, capsules, wafer capsules, gel capsules, pills, cachets, sachets, lozenges, dragees, powders, granules, bolus; liquid forms such as solutions, syrups, elisirs, emulsions or suspensions; in the form of aerosol, suppositories, rectal capsules, past, creams, ointments, gels, drops, transdermal patch, multi-dose bottles, electuary, ocular implants, reservoir implants, directly into tumor masses or using any other route of administration.

The compositions of the invention may be obtained by conventional procedure using conventional pharmaceutical excipient, well known in the art. A person skilled in the art is aware of a whole variety of such excipients suitable to formulate a pharmaceutical composition. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art. Excipients include, by way of illustration and not limitation, diluents, fillers, agglutinants, disintegrants, disintegration inhibitors, absorption accelerators, adjuvant, binders, carriers, suspensing/dispersing agents, film formers/coatings, adhesives, antiadherents, wetting agents, lubricants, glidants, preservatives, sorbents, surface active agents, substances added to mask or counteract a disagreeable taste or odor, flavorings, colorants, fragrances, aromatising agents, sweeteners and substances added to improve appearance of the composition. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

The pharmaceutical compositions of the invention may contain a compound of formula (I) as the active substance, in association with one or more pharmaceutically acceptable excipients and/or carrier, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. A sustained-release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid- base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids, such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.

The solid oral forms, e.g. tablets, dragees, capsules, granules, may contain, together with the active compound, diluents, e.g. alkaline-earth metal carbonates, lactose, dextrose, saccharose, calcium phosphates, magnesium phosphate, cellulose, starches, corn starch or potato starch, modified starches, white sugar, glucose, sucrose, sorbitol, crystalline cellulose, microcrystalline cellulose derivatives, silicon dioxide and the like; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium salts, and/or polyethylene glycols, sodium oleate, sodium stearate, sodium benzoate, sodium acetate, sodium chloride and the like; binding agents, e.g., starches, gelatin, polyvinyl pyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, arabic gums, tragacanth or sodium alginate, cellulose derivatives such as methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, waxes, gelatine methylcellulose, and the like; disaggregating agents, e.g. a starch, alginic acid, alginates, sodium starch glycolate; disintegrating agents, e.g. starch, agar, alginic acid or its salts, sodium starch glycolate, cross-linked polyvinyl pyrrolidone, methyl cellulose, bentonite, xanthan gum, and the like; effervescing mixtures; dyestuffs; sweeteners; flavoring (e.g. cocoa powder, menthol, aromatic powder, peppermint oil, borneol, cinnamon powder), preservative (e.g. sorbic acid, potassium sorbate, benzalkonium chloride, benzethonium chloride, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, chlorobutanol), dispersing, coloring agent (the coloring agent includes e.g. those coloring agents approved to be added to pharmaceutical preparations); wetting agents, for instance, lecithin, polysorbates, laurylsulphates and the like; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Said pharmaceutical preparations may be manufactured in a known manner, for example by means of mixing, filling or compression, granulating, grinding, tabletting, sugar-coating, or film-coating processes. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. The tablets and granules may be coated with a sugar or gelatin coating or if necessary with another suitable coating. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an alginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages. Capsules are made by preparing a powder mixture and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, macrogol 4000, purified soybean lecithin, polyoxyethylene (160), polyoxypropylene (30) glycol can also be added to improve the availability of the medicament when the capsule is ingested. The solid oral formulations may be also in the form of sustained release formulations that can be prepared in conventional manner, for instance, by applying an enteric coating to tablets and granules. Liquid oral preparations can be formulated e.g. as aqueous or oily suspensions or solutions, emulsions, syrups or elixir, or can be presented as freeze dried product to be regenerated by addition of water or a suitable vehicle before use. Said liquid preparations can contain conventional additives such as suspending agents, e.g. sorbitol, syrup, methylcellulose, gelatine, hydroxyethylcellulose, carboxymethylcellulose, alluminium stearate gel or hydrogenated edible fats, emulsifying agents, e.g. lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), e.g. almond oil, fractionated coconut oil, oily esters such as glycerin esters, propylene glycol, or ethyl alcohol; preservatives, e.g. methyl or propyl p-hydroxybenzoate or sorbic acid and, if desired, conventional flavours and dyes. The liquid dispersion for oral administration may be, e.g., solutions, syrups, emulsions and suspensions in aqueous or non-aqueous liquids, edible foams or whips. Oral fluids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. The syrups may contain as carrier, for example, saccharose, glycerine, mannitol and/or sorbitol.

The suspensions and emulsions may contain as carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol, viscosity increasing substances, e.g. sorbitol, dextran, and sodium carboxymethylcellulose. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be added.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like. Pharmaceutical compositions for parenteral injection comprise pharmaceutically- acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, in the form of sterile, aqueous, isotonic, saline solutions, isotonizing agents, a pH adjuster, anti- oxidants, buffers (e.g. phosphoric acid, phosphates, citric acid, acetic acid, aminocaproic acid, trometamol.), solubilizers (e.g. polyoxyethylene hardened castor oil, nicotinic acid amide, macrogol, castor oil fatty ethyl ester), stabilizers (e.g. sodium sulfite, sodium metasulfite, ether), preservatives (for example, methyl p-oxybenzoate, ethyl p-oxybenzoate, sorbic acid, phenol, cresol, chlorocresol), ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, fatty oils (e.g. sesame oil, olive oil), synthetic fatty acid esters, triglycerides and polyethylene glycol, and injectable organic esters such as ethyl oleate, glycols, e.g. propylene glycol, suspending agents (e.g. methyl cellulose, polysorbate 80, hydroxyethyl cellulose, arabic gum, tragacanth powder, carboxymethyl cellulose sodium, polyoxyethylene sorbitan monolaurate), thickening agents (e.g. cellulosic polymers such as hydroxypropylmethylcellulose and hydroxypropylcellulose, polyvinyl alcohol and polyvinylpyrrolidone) and, if desired, a suitable amount of lidocaine hydrochloride. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, glycerin, propylene glycol, sodium chloride, potassium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. For parenteral administration, it is possible to prepare fluid dosage units, containing the compound and a sterile vehicle. The compound, depending on the chosen vehicle and concentration, can be suspended or dissolved. Parenteral solutions are normally prepared by dissolving the compound in a vehicle, sterilising by filtration, filling suitable vials and sealing. Advantageously it is also possible to dissolve in the vehicle suitable adjuvants such as local anaesthetic, preservatives and buffering agents. In order to increase stability, the composition can be frozen after filling the vial and removing water under vacuum. Parenteral suspensions are prepared substantially in the same way, with the difference that the compound can be suspended rather than dissolved in the vehicle, and they can be sterilised by treatment with ethylene oxide before being suspended in the sterile vehicle. Advantageously, it is possible to include a surfactant or a wetting agent in the composition with the aim of easing the uniform distribution of the compound of the invention. The injectable formulations may be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Pharmaceutical preparations which are suitable for rectal or vaginal administration, e.g. suppositories, enemas, may be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as natural or synthetic triglycerides, cocoa butter, polyethylene glycol, paraffin hydrocarbons, polyoxyethylene, sorbitan fatty acid esters surfactants, lecithin, or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound and the like. Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations. The compounds of the invention may also be administered via transdermal release. Typical transdermal formulations include conventional aqueous and non-aqueous vectors, such as creams, oils, lotions or pastes or can be provided as membranes or medicated plasters. In an embodiment, a compound of the invention is dispersed in a pressure-sensible plaster adhering to the skin. This formulation allows the compound to be spread from the plaster to the patient through the skin. In order to obtain a sustained drug release through the cutis, natural rubber and silicon can be used as pressure-sensitive adhesives. The active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3 (6), 318 (1986).

Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye. In the forms for topical application, e.g. ointments, creams, gels, powders, lotions, solutions, suspensions, pastes, sprays, aerosols, oils or the like, for use in dermatological treatment may be prepared so as to contain liposomes, micelles, and/or microspheres. The active ingredient may be mixed with conventional oleoginous or emulsifying excipients. Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Ointments, as it is well known in the art of pharmaceutical formulation, are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Examples of ointments include oleaginous ointment bases, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum, emulsifiable ointment bases, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum, emulsion ointment bases, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid and water-soluble ointment bases prepared from polyethylene glycols of varying molecular weight. Creams, as also well known to those skilled in the art, are viscous liquids or semisolid emulsions, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually contains a humectant. The emulsifier in a cream formulation is chosen among non-ionic, anionic, cationic or amphoteric surfactants. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil. Preferred gelling agents are crosslinked acrylic acid polymers (such as "carbomer" polymers, e. g., carboxypolyalkylenes that may be obtained commercially under the Carbopol . Also preferred are hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. For the preparation of uniform gels, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring. Liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles, can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Compositions for topical administration, including those for inhalation, may be prepared as a dry powder which may be pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredient in finely divided form may be used in admixture with a larger-sized pharmaceutically-acceptable inert carrier comprising particles having a size, for example, of up to 100 micrometers in diameter. Suitable inert carriers include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers. Alternatively, the composition may be pressurized and contain a compressed gas, such as nitrogen or a liquified gas propellant. The liquified propellant medium and indeed the total composition is preferably such that the active ingredient does not dissolve therein to any substantial extent. The pressurized composition may also contain a surface active agent, such as a liquid or solid non-ionic surface active agent or may be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.

A further form of topical administration is to the eye. A compound of the invention is delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye, as for example the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera. The pharmaceutically- acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil or an encapsulating material. Alternatively, the compounds of the invention may be injected directly into the vitreous and aqueous humour.

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i. e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered, dose pressurised aerosols, nebulizers or insufflators.

The compounds of formula (I) may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

Compounds of the present invention may also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically- acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers (e.g. edetic acid, disodium edentate), preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, see, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.

The above described components for pharmaceutical composition administered are merely representative. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents. In general, the above compositions may be prepared in a conventional manner using conventional excipients. Further materials as well as processing techniques and the like are set out in Part 5 of Remington 's Pharmaceutical Sciences, 20 ^th Edition, 2000, Merck Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference. Compounds of the present invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in the incorporated materials in Remington 's Pharmaceutical Sciences.

The compounds of the present invention may be employed alone as a sole therapy or in combination with other therapeutic agents for the treatment of the above-mentioned conditions. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. When the compounds of this invention are in combination with others active ingredients, the active ingredients may be separately formulated into single-ingredient preparations of one of the above-described forms and then provided as combined preparations, or may be formulated together into a two- or more- ingredient preparation.

Active agents, that can be formulated with a compound of the invention or alternatively, can be administered in a combined method of treatment depend on the disease state to be cured and are, for instance, gamma globulin, immune globulin and monoclonal antibody products, antibiotics and antimicrobial products; antitumor agents (chemo therapeutics, for instance, Paclitaxel and Carboplatin); and the like. The term "antitumor agent" is meant to comprise both a single antitumor drug and "cocktails", i.e. a mixture of such drugs, according to clinical practice. In the field of medical oncology it is normal practice to use a combination of different forms of treatment to treat each patient with cancer. In particular, in cancer therapy the compounds of the invention can be administered alone or in association with an antitumor agent as herebelow defined, as well as combination with chemotherapy, surgical therapy and radiotherapy. Combination therapies according to the present invention thus comprise the administration of at least one compound of formula (I) and the use of at least one other cancer treatment method. Preferably, combination therapies according to the present invention comprise the administration of at least one compound of formula (I) and at least one other pharmaceutical active agent, preferably an anti-neoplastic agent. The compound (s) of formula (I) and the other pharmaceutical active agent (s) may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order. The amounts of the compound (s) of formula (I) and the other pharmaceutical active agent (s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. The compounds of formula (I) and at least one additional cancer treatment therapy may be employed in combination concomitantly or sequentially in any therapeutically appropriate combination with such other anti-cancer therapies. In one embodiment, the other anti- cancer therapy is at least one additional chemotherapeutic therapy including administration of at least one anti-neoplastic agent. The administration in combination of a compound of formula (I) thereof with other anti-neoplastic agents may be in combination in accordance with the invention by administration concomitantly in a unitary pharmaceutical composition including both compounds or separate pharmaceutical compositions each including one of the compounds. Alternatively, the combination may be administered separately in a sequential manner wherein one anti- neoplastic agent is administered first and the other second or vice versa. Such sequential administration may be close in time or remote in time. Anti-neoplastic agents may induce anti-neoplastic effects in a cell-cycle specific manner, i.e., are phase specific and act at a specific phase of the cell cycle, or bind DNA and act in a non cell-cycle specific manner, i.e., are non-cell cycle specific and operate by other mechanisms. Anti-neoplastic agents useful in combination with the compounds of formula (I) include the following:

(1) other antiangiogenic agents (for example linomide, inhibitors of integrin function, angiostatin, razoxin, thalidomide);

(2) cell cycle specific anti-neoplastic agents including, but not limited to, diterpenoids (such as paclitaxel and its analog docetaxel); antimitotic agents (for example vinca alkaloids like vinblastine, vincrisitine, vindesine, vinorelbine); taxoids (like taxol, taxotere); topoisomerase inhibitors (for example epipodophyllotoxins like etoposide, teniposide, amsacrine; gemcitabine); fluoropyrimidines (such as 5-fluorouracil and fluorodeoxyuridine); antimetabolites (such as allopurinol, fludurabine, tegafur, hydroxyurea, antifolates, like methotrexate, cladrabine, cytarabine, mercaptopurine and thioguanine, purine and adenosine analogues, cytosine arabinoside); camptothecins (such as 9-amino camptothecin, irinotecan, topotecan, CPT-11 and the various optical forms of 7- (4-methylpiperazino-methylene)-10, l l-ethylenedioxy-20-camptothecin); enzymes (for example asparaginase); thymidylate synthase inhibitors (for example raltitrexed);

(3) cytotoxic chemotherapeutic agents including, but not limited to, alkylating agents (such as nitrogen mustard, melphalan, chlorambucil, cyclophosphamide, mechlorethamine, hexamethylmelamine, busulfan, carmustine, lomustine, dacarbazine, ifosfamide, nitrosoureas, thiotepa); anti-tumour antibiotics (such anthracyclines like adriamycm, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum coordination complexes (such as cisplatin, carboplatin, and oxaliplatin);

(4) other chemotherapeutic agents including, but not limited to, antiestrogens (such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene); pro gestro gens (such as megestrol acetate); aromatase inhibitors (such as anastrozole, letrazole, vorazole, exemestane); antiprogestogens; antiandrogens (such as flutamide, nilutamide, bicalutamide, cyproterone acetate); LHRH agonists or antagonists (such as goserelin acetate, !euprorelin, buserelin); testosterone 5a-dihydroreductase inhibitors (such as finasteride); anti-invasion agents (for example metaHoproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function); inhibitors of growth factor function (for example growth factor antibodies, growth factor receptor antibodies, inhibitors of the functions of hepatocyte growth factor, erb- B2, erb-B4); inhibitors of the epidermal growth factor family (for example the EGFR tyrosine kinase inhibitors ZD1839, CP 358774CI 1033); platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR, and TIE-2); tyrosine kinase inhibitors such as inhibitors of CDK2 and CDK4 inhibitors; serine/threonine kinase inhibitors; fames yl transferase inhibitors;

(5) 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. due to enhanced receptor degradation or lowered expression levels);

(6) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense;

(7) 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;

(8) 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 dendritic cells), approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.

The mammal requiring treatment with a compound of the present invention is typically a human being.

According to another aspect of the invention, there is provided the use of a compound of the formula (I), as defined hereinbefore, as well as its stereoisomers, tautomers, polymorphs, solvates, mixtures and metabolites thereof, for the manufacture of a medicament for the prevention and/or treatment of cancers, autoimmune diseases, ocular diseases, vascular diseases, reproductive system diseases.

According to another aspect of the invention, there is provided the use of a compound of the formula (I), as defined hereinbefore, as well as its stereoisomers, tautomers, polymorphs, solvates, mixtures and metabolites thereof, for the manufacture of a medicament for the prevention and/or treatment of cancers, preferably colorectal cancer, metastatic breast cancer, lung cancer, gliblastoma and metastatic kidney cancer.

According to another aspect of the invention, there is provided the use of a compound of the formula I, as well as its stereoisomers, tautomers, polymorphs, solvates, mixtures and metabolites thereof, in the manufacture of a medicament for use in the treatment of both primary and metastatic solid tumors, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gall, bladder and bile ducts, small intestine, urinary tract, female genital tract, male genital tract, endocrine glands, skin, as well as haemangiomas, melanomas, sarcomas, tumors of the brain, nerves, eyes, and meninges, leukemias, lymphomas, multiple myeloma.

According to another aspect of the invention, there is provided the use of a compound of the formula (I), as defined hereinbefore, as well as its stereoisomers, tautomers, polymorphs, solvates, mixtures and metabolites thereof, for the manufacture of a medicament for the prevention and/or treatment of autoimmune diseases; various ocular diseases, such as diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, age-related macular degeneration, uveitis, ocular diseases with retinal vessel proliferation, proliferating retinitis, diabetic retinitis, hypoxia, angiogenesis in the eye associated with infection or surgical intervention, eye diseases associated with choroidal neovascularization and iris neovascularization; excessive scar formation and adhesions; blood vessel, vascular diseases such as capillary proliferation within atherosclerotic plaques, atheroma; Osier-Webber Syndrome; myocardial angiogenesis, myocardial infarction; obstructive arteriosclerosis, plaque neovascularization, diabetic angiopathy, vascular malformation, hypertonia, angina pectoris, cerebral infarction; telangiectasia; hemophiliac joints; angiofibroma, arterial restenosis; and wound granulation; reproductive system disease such as uterus dysfunction, endometriosis, dysfunctional uterine bleeding, placental dysfunction, ovarian hyperergasia or follicle cyst; transplanted organ rejection; central nervous system disease such as retinosis, cerebral apoplexy, vascular dementia or Alzheimer disease; inhibition of ovulation and establishment of the placenta; cat scratch disease (Rochele minalia quintosa) and ulcers.

According to another aspect of the invention, there is provided a method for the prevention and/or treatment of diseases states associated with angiogenesis and/or increased vascular permeability comprising cancers, autoimmune diseases; various ocular diseases, such as diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, age-related macular degeneration, uveitis, ocular diseases with retinal vessel proliferation, proliferating retinitis, diabetic retinitis, hypoxia, angiogenesis in the eye associated with infection or surgical intervention, eye diseases associated with choroidal neovascularization and iris neovascularization; blood vessel, vascular diseases such as capillary proliferation within atherosclerotic plaques, atheroma; Osier-Webber Syndrome; myocardial angiogenesis, myocardial infarction; obstructive arteriosclerosis, plaque neovascularization, diabetic angiopathy, vascular malformation, hypertonia, angina pectoris, cerebral infarction; telangiectasia; hemophiliac joints; angiofibroma, arterial restenosis; and wound granulation; reproductive system disease such as uterus dysfunction, endometriosis, dysfunctional uterine bleeding, placental dysfunction, ovarian hyperergasia or follicle cyst; transplanted organ rejection; central nervous system disease such as retinosis, cerebral apoplexy, vascular dementia or Alzheimer disease; inhibition of ovulation and establishment of the placenta; cat scratch disease (Rochele minalia quintosa) and ulcers, comprising administering a composition of the invention.

According to another aspect of the invention, there is provided a method for the prevention and/or treatment of a cancer of both primary and metastatic solid tumors, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gall, bladder and bile ducts, small intestine, urinary tract, female genital tract, male genital tract, endocrine glands, skin, as well as haemangiomas, melanomas, sarcomas, tumors of the brain, nerves, eyes, and meninges, leukemias, lymphomas, multiple myeloma, comprising administering a composition of the invention.

Further object of the present invention is a method for inhibiting angiogenesis, comprising administering an effective amount of a compound of the present invention in a pharmaceutically acceptable carrier to a site where inhibition of blood vessel ingrowth is desired to inhibit angiogenesis, in the absence of cytotoxicity to normal cells at the site, e.g. tumor.

Further object of the present invention is a method for inhibiting angiogenesis wherein the effective dose is a dose effective in diminishing the number of blood vessels growing into a tumor, autoimmune diseases, ocular diseases, vascular diseases, reproductive system diseases, diseases characterized by abnormal neovascularization.

Further object of the present invention is a method for improving the conditions of a patient having cancer of both primary and metastatic solid tumors, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gall, bladder and bile ducts, small intestine, urinary tract, female genital tract, male genital tract, endocrine glands, skin, as well as haemangiomas, melanomas, sarcomas, tumors of the brain, nerves, eyes, and meninges, leukemias, lymphomas, multiple myeloma, said method comprising administering to said patient an effective amount of a compound of formula (I), as defined above alone or in association with an antitumor agent.

A further object of the present invention is a combined method of treatment of the above mentioned pathological conditions in mammals, including humans, in need of such treatment, said method comprising administering:

1) an angiogenesis inhibitor according to the invention and

2) a different pharmaceutically active agent, typically an antitumor agent, in amounts and close enough together in time sufficient to produce a therapeutically useful effect.

The present invention also provides products containing an angiogenesis inhibitor of the invention and an antitumor agent as a combined preparation for simultaneous, separate or sequential use in anti-cancer therapy.

The angiogenesis inhibitors of the invention can therefore be used in a treatment to ameliorate a cancer. They may be administered to a patient suffering from a cancer treatable with an antitumor agent, for example an anthracycline glycoside such as doxorubicin, daunomycin, epirubicin or idarubicin as mentioned above, together with the antitumor agent.

Further object of the present invention is a kit for the treatment of cancer, comprising: (a) an effective amount of an angiogenesis inhibitor of the formula (I) and

(b) as a separate composition, an effective amount of an antitumor agent.

With the aim to better illustrate the present invention, without posing any limitation to it, the following examples are now given.

Examples

Materials

Fluorescence microscopy was used (Zeiss Axiophot, Thornwood, USA);

Avastin ^® (Bevacizumab) is a product of Genentech (San Francisco, USA);

DMSO (dimethyl sulfoxide) was purchased at Sigma- Aldrich, Buchs, Switzerland;

The eggs were closed after all treatments by Laboratory Wrapping Film, Parafilm (Pechiney, Menasha, USA);

Embryos were from Animalco AG (Staufen, Switzerland);

Fluorescein-dextran is a product of Sigma, St.Louis, USA.

FITC-dextran (Fluorescein isothiocyanate dextran, 20 kDa, 25 mg/ml) was purchased at Sigma-Aldrich (Buchs, Switzerland);

India ink was purchased at Pebeo S.A. (Romanel-sur-Morges, Switzerland) and filtered prior to use with a sterile cellulose acetate membrane (0.2 μπι pores, Renner GmbH, Darmstadt, Germany);

MicroliterTM syringes are equipped with 33-gauge metal Hub (N) needles, both from the Hamilton (Reno, USA);

0.9% NaCl solution, which was used as a solvent for Avastin ^® or by itself as a control, is a product of Bichsel AG (Interlaken, Switzerland);

Pasteur pipettes were purchased from Copan (Brescia, Italy)

The following abbreviations have been used:

BD Matrigel (Basement Membrane Matrix growth factor-reduced); CAM (Chorioallantoic Membrane); DMSO (dimethyl sulfoxide); ECGS (endothelial cell grow supplement); EDD (embryo development days); EGF (Epidermal Growth Factor); FBS (fetal bovine serum); FITC (Fluorescein isothiocyanate); GNX-686 (l-(3-chlorophenyl)-3-phenyl-pyrrole-2,5- dione); HMEC-1 cells (human microvascular endothelial cells-1); HUVEC-2 (human umbilical vein endothelial cells) from BD Biosciences; MCDB (Molecular, Cellular, and Developmental Biology); P7 (postnatal day 7); P18 (postnatal day 18); PBS (phosphate buffered saline); ROP (Retinopathy of Prematurity)

The 10 mM stock solution of the GNX-686 was prepared in 100% DMSO. Then, 1 μΐ of this solution was dropped into 1 ml of 0.9% NaCl while mixing (Vortex- Genie2; Scientific Industries, New York, USA), giving a 10 uM GNX-686 solution in 0.1% DMSO/NaCl. Cell culture

All cells were cultured in 10 cm culture dishes and maintained at 37 °C in a humidified atmosphere containing 95% air and 5% C0 ₂ . HMEC-1 cells were maintained in MCDB- 131medium supplemented with 10% [v/v] heat-inactivated fetal bovine serum (NA FBS), 1% glutamine, 1% penicillin and streptomycin, 1 mg/ml hydrocortisone and 10 ng/ml EGF. HUVEC-2 were maintained on gelatin coated plates in MCDB-131 medium supplemented with 20% [v/v] heat-inactivated fetal bovine serum (NA FBS), 1% glutamine, 1% penicillin and streptomycin, ImM Na pyruvate, 50 mg/ml ECGS and 100 mg/ml heparin.

Cell proliferation

Cell proliferation was assessed using the CellTiter-Glo ^® Luminescent Cell Viability Assay (Promega). Cells (3xl0 ^J cells/well) were seeded on 96-well white tissue culture plates (Costar#3610) (gelatin coated for HUVEC) in the appropriate growth medium and allowed to attach overnight. Media was removed and replaced with 150 μΐ of tissue culture medium containing GNX-686 (0.5 - 32 μΜ) and grown for a further 24, 48 and 72 h. At each time point, cell proliferation was evaluated by addition of 150 μΐ of CellTiter-Glo ^® reagent to each well and luminescence was read on a Tecan infinity F200 plate reader.

In vitro tube formation assay

HUVEC-2 cells were plated on 48-well plates (2.5x10 ⁴ cells per well), previously coated with 140 μΕ of BD Matrigel, in the presence of GNX-686 (4 μΜ) or control (0.05% DMSO) in MCDB 10% FBS and ECGS 50 mg/ml. Tube formation was assessed by counting the number of capillary-like structures that had formed after a further 15 h incubation at 37°C.

The in ovo CAM model

Fertilized chicken eggs were labelled and transferred into a hatching incubator with a relative air humidity of 65% and a temperature of 37 °C. This incubator was equipped with an automatic rotator (Savimat, Chauffry, France). On EDD 3, a hole of approximately 3 mm in diameter was opened in the eggshell and covered with Parafilm ^® to prevent dehydratation and possible infections. The eggs were returned to the incubator in a static position until use. On EDD 7, the shell above the air pouch of the egg is extended to a diameter of approximately 3 cm in order to provide access to the chorioallantoic membrane. In order to quantify the vascular effects, to characterize and quantify the growth of vessels for the period ranging between EDD 7 and EDD 9, three descriptors were selected:

(i) the number of branching points/mm (all equivalent and non-equivalent bifurcations, > 3 neighbours);

(ii) the mean area of the vessel network meshes defined as the closed area surrounded by capillaries and/or vessels, expressed in the units of 10 ² μπι ² ;

(iii) the mean of the 3 rd quartile of the mesh area histogram, also expressed in units of 10 ² μπι ² .

These parameters were then, in the end, used to quantify the "immaturity" of the vascularization, or the decreased vascularization due to the administration of antiangiogenic agents.

For the topical application of the tested agents and their controls on EDD 7, polyethylene rings were cut in sterile conditions and deposited on the CAM. These polyethylene rings (diameter 5 mm; wall thickness 0.5 mm, 1 mm height) were used to confine the topical drug to only one small and well-defined part of the CAM surface. They were sliced from Pasteur pipettes. Subsequently, 20 μΐ of freshly prepared GNX 686 (0.001- 0.01 μπιοΐ/ml in DMSO/0.9%NaCl) or Avastin ^® (0.001-0.01 μπιοΐ/ml in 0.9% NaCl) or their controls (DMSO/0.9%NaCl or 0.9% NaCl, respectively) were deposited topically (PipetmanNeo, Gilson, Middleton, USA) onto the CAM surface within the ring. The embryos were then labelled and returned to the incubator in a static position for 24 h. The second topical administration of a second dose of 20 μΐ of GNX-686 and Avastin solutions, or of their control solutions, took place in the same way on EDD 8. Again, the embryos were returned to the incubator in a static position for 24 h for later analysis on EDD 9.

Microscopy on the CAM and image acquisition

Fluorescence images were obtained with an epi-fluorescence microscope (Nikon Eclipse E600 FN, Japan) equipped with a Nikon objective (CFI achromat; magnification x 10; N.A.: 0.30, Working distance: 30 mm) coupled to an F-view II 12-bit monochrome Peltier- cooled digital CCD camera (Soft Imaging System GmbH, Munster, Germany) driven with the "analySIS DOCU" software (Soft Imaging System GmbH, Munster, Germany). A 10 x objective is minimally necessary for a proper quantitative visualization of the capillaries.

Fluorescence images (1280 x 1024 pixels with 4095 grey level, i.e. 12 bits) of the CAM superficial vessels were recorded by exciting the fluorescence with a filtrated (Agitation = 450 - 490 nm) Hg-arc lamp (Osram, GmbH, Augsburg, Germany) together with a long pass emission filter (λ > 520 nm) for observation of the FITC-dextran fluorescence. The images were stored in a 16-bit TIF file. These images were then classified in terms of the quantification of the capillary features (size, and quantity) in the treated area and then used to study the GNX-686 or Avastin ^® -induced changes in comparison with a similar area treated with the control solution. The latter was in general not performed on the same embryo.

To obtain the fluorescence images, a volume of 20 μΐ of FITC-dextran (20 kDa) was injected into one of the principle vessels of the CAM (-200 μπι in diameter), directly under the microscope. In order to increase the quality (contrast) of the recorded angiograms 30 μΐ of well known light absorber, India ink, was injected shortly after into the extra-embryonic cavity, just under the deposited polyethylene ring, in order to decrease the embryo's interfering fluorescence from deeper located vessels. This interfering luminescence can change rapidly with time due to the embryo's movement. Prior to injection, the India ink was filtered using a sterile cellulose acetate membrane.

The potency of antiangiogenic agents is illustrated by reporting graphically the differences obtained, for the three descriptors mentioned above, between solutions containing these agents and their controls. For each GNX-686 concentration, the percentage of DMSO was changing respectively. Each point on the reported plots represents the average of measurements performed on 9-10 embryos. At least 5 images taken at different areas of (1.4 x 1.12 mm ) were recorded and analyzed with the objective 10 X on each individual CAM. These images were located within the polyethylene ring that delineated the agent- treated zone.

Quantitative image analysis

The quantitative image analysis method was used for in vivo fluorescence measurements taken during the CAM's growth. The CAM capillary growth with and without the topical administration of GNX 686 or Avastin ^® were compared. The method was described in detail by Nowak-Sliwinska P, Ballini JP, Wagnieres G, van den Bergh H. Microvasc Res. 2010 Jan, 79(1), 21-8.

Analysis of the fluorescence images was performed following the sequence indicated in Figure 2, using a macro written for ImageJ (version 1.40 a; National Institutes of Health; Bethesda, USA; Abramoff MD, Magelhaes PJ, Ram SJ. J. Biophotonics International. 2004, l l(7):36-42; Rasband WS. http://rsb.info.nih.gov/ij/. 2007) together with a set of already existing plugins. The latter include an "a posteriori" shading correction (#514, version 3, Bonnet, 2005), the use of a rolling ball background filter (Castle M, Keller J. Mental Health Res Inst, Univ Michigan. 2008), a featureJ Laplacian (FJ) version 1.4.0 (Meijering E. Biomedical Imaging Group, Univ Med Center Rotterdam, Netherlands, http://imagescience.org/meijering/software/featurej/ 2007), the use of a region removal plugin (Holen R, Hagen M. www.pvv.org/~perchrh/papers/mastersthesisHendenBacheWiig.pdf 2005), and finally the application of a morphology binary connectivity (Landini G. http://www.dentistry.bham.ac.uk/landinig/software/software.h tml 2006).

The applications of the compounds were performed inside polyethylene rings (one ring per egg) that were applied on the CAM. The application of this ring on the CAM did not cause, by itself, any visible morphological change of the vascular network. GNX-686 and Avastin® treatments started at EDD 7 (first topical application of a 20 μΐ solution within the ring) and was repeated at EDD 8 (second topical application of a 20 μΐ solution within the ring) with concentrations of actives ranging between 0.001 and 0.01 μπιοΐ/ml (see Table 1). The smallest concentration (0.001 μπιοΐ/ml) corresponds to concentration of Avastin® used in the clinics for injections in the human vitreous.

Table 1 : Concentrations of the actives in the 20 μΐ solutions administered topically on the

CAM

The curves presented in Figure 2 illustrate the efficacy of the tested agents for different concentrations. The values of the descriptors obtained with the solutions containing the actives have been divided by those obtained with the solutions only (control). Concentrations of GNX-686 > 0.005 μπιοΐ/ml effectively influenced the capillary proliferation in the CAM.

At concentrations ranging between 0.0037 and 0.01 μπιοΐ/ml the inhibitory efficiency of GNX-686 was comparable to that of Avastin®. For the lowest tested concentration (0.001 μπιοΐ/ml), all three descriptors indicate a smaller effect for GNX-686 than for Avastin®. The effects induced by 0.9% NaCl, GNX-686 (0.01 μιηοι/ml) and Avastin® (0.01 μπιοΐ/ml) are given in Figure 3. Black surfaces correspond to non-vascularized regions. Mouse ROP model

Neonatal mice (C57BL/6J) were obtained from breeding colonies maintained within the Gemelli Hospital (Rome, Italy). Litters of P7 pups were placed in either hyperoxia (75% (¾) or normoxia (20% 0 ₂ ) for 5 days. Neonatal P7 mice were placed in an oxygen chamber together with their mother (oxygen content was regulated by an automated 0 ₂ /N ₂ mixer to 75% ± 2% 0 ₂ ) with sufficient food and water to sustain them for 5 days. The cage temperature was maintained at 23°C ± 2°C. At postnatal day 12 mice were returned to normoxia and maintained until postnatal day 18. At postnatal day 12 "hyperoxic" animals were treated with GNX-686 (0.4 μΜ final concentration in the vitreous) in the right eye and 1 % DMSO in the left eye (control). GNX-686 (1 μΐ of 4 mM solution in 10% DMSO) or DMSO was injected directly into the ocular vitreous using a sterile Hamilton syringe. At postnatal day 18 success of the model was assessed in a satellite group of animals (normoxic vs hyperoxic) by fluorescein-dextran angiography. Quantitation of neoangiogenesis in the presence or absence of compound was assessed by immunohistochemistry.

Image Aquisition of the ROP

Mice were anesthetized intraperitoneally with tribromoethanol (0.2 ml/10 g body weight) and then perfused through the left ventricle with 1 ml of PBS containing 50 mg of 2 X 10 ⁶ molecular weight fluorescein-dextran. Before use, the fluorescein-dextran solution was clarified by centrifugation for 5 minutes at 10,000 rpm (Fisher Scientific, Springfield, USA, model 235C). The eyes were marked for orientation, enucleated, and placed in 4% paraformaldehyde for 3 to 24 hours. Lenses were removed and peripheral retinas were cut to allow flat-mounting with glycerol-gelatin. The flat-mounted retinas were viewed by fluorescence microscopy and photographed.

Quantification of Neovascular Proliferative Retinopathy

At PI 8, mice were sacrificed with intraperitoneal tribromoethanol (0.1 ml/g body weight). The eyes were enucleated, immersed in 4% paraformaldehyde in PBS for at least 24 hours, and embedded in paraffin. Serial sections (6 mm) of whole eyes were cut through the cornea and parallel to the optic nerve and stained with eosin and hematoxylin. Nuclei from new vessels and vessel profiles could be distinguished from other structures in the retina and counted in cross-section with light microscopy (magnification x 400). Approximately 150 serial sections were cut from each eye. In the central half of the globe, 10 sections on each side of the optic nerve, 30 to 90 nm apart, were counted for neovascularization. Vascular cell nuclei, identified under light microscopy with hematoxylin staining, were considered to be associated with new vessels if they were found on the vitreal side of the internal limiting membrane.

ROP mouse model

The structure of the retinal blood vessel tree was evaluated in fluorescein-injected flat- mounted retinas (Figure 4A) at 5 days after transferring of mice from hyperoxic to normoxic conditions. The representative retinal images are shown in Figure 4 (B: DMSO- treated retinas; C: GNX-686-treated, 4 μΜ). The vascular network of GNX-686-treated mice in the model of ROP was significantly altered compared with the vascular network of control (DMSO-treated) mice. The control samples were highly tortuous and contained neovascular tufts, as indicated with arrows in Figure 4A. These alterations appeared as a severe loss of retinal microvasculature. Moreover, the tortuosity was less pronounced in GNX-686-treated eyes (see Figure 4B).

Vascular cell nuclei, identified under light microscopy with hematoxylin staining, associated with new vessels if they were found on the vitreal side of the internal limiting membrane (see Figure 5 A and B - indicated by arrows). These nuclei were not present in the GNX-686 treated eyes (figure 5C and D). A reduction in retinal neovascularization of > 50% was observed in animals treated with 4 uM GNX-686, see Figure 4E.

GNX-686 inhibits endothelial tube formation

In order to investigate the angiostatic activity of the GNX-686, we performed endothelial growth and sprouting assays (Figure 6). One of the hallmark of angiogenesis is endothelial cell migration. This parameter can be tested in the tube formation assay. When HUVECs were cultured on a 3-dimensional gel (Matrigel), cellular reorganization and tubular network formation of endothelial cells can be observed (Figure 6A). This sprouting was efficiently inhibited by exposure of cells to 4 μΜ of GNX-686 (Figure 6B). Quantification of these results is shown in Figure 6C.

Growth of endothelial cells was tested using the Cell Titer Glo assay, a colorimetric assay that quantifies metabolic activity by detection of APT concentration in the cell culture. GNX-686 in concentrations lower than 16 μΜ was found to have a minor effect on the growth of primary macrovascular human umbilical vein endothelial cells (HUVEC), in standard serum concentration (see Figure 6D, dark bars), as well as in reduced serum amount in the growing medium (see Figure 6D, dark bars). At the concentration of 16 μΜ, an inhibitory activity of approximately 30-50%, and was serum content dependent. All tested concentrations did not affect the growth of the microvascular endothelial cell line HMEC (Figure 6E). HeLa cells were used as a control and activity was neither seen in these cells.

Example 1

Assay For Determining angiogenesis in vivo

The representative assay for studying angiogenesis in vivo is the chicken's embryo chorioallantoic membrane ((CAM) model: Ausprunk DH, Knighton DR., Folkman J. Dev Biol. 1974 Jun, 38(2), 237-48; Ausprunk DH, Knighton DR., Folkman J. Am. J. Pathol. 1975 Jun, 79(3), 597-618; Ehrman RL, Knoth M. J. Nat. Cancer Inst. 1968, 41 : 1329-41. CAM is a vascular fetal membrane composed of the fused chorion and adjacent wall of the allantois. The CAM is formed on day 4 of incubation by fusion of the chorion and allantois. Rapid capillary proliferation continues until day 11 and the vascular system attains its final arrangement on day 18, just before hatching.

Blood vessel growth inhibition and changes in the architecture of the capillary plexus were induced after topical application of this drug on the CAM surface. Vascular effects were monitored in ovo, between day 7 and 9 of embryo development, using an epi-fluorescence microscope equipped with a sensitive camera following the intravenous injection of a fluorescent agent. The fluorescence angiograms of the CAM vasculature were recorded and analyzed by a multistep mathematical procedure to obtain a skeleton representation of the vessels and capillaries. GNX-686 decreased the capillary growth in a dose-dependent manner. Moreover, in the tested dose range, the inhibitory effect obtained by GNX-686 was comparable to that of Avastin ^® for identical concentration expressed in μπιοΐ/ml.