WO2012127012A1 - Biphenyl tricyclic quinazoline compounds - Google Patents

Abstract

The invention is directed to certain novel compounds. Specifically, the invention is directed to compounds of formula (I): and salts thereof. The compounds of the invention are multiple inhibitors of tyrosine kinase activity.

Description

BIPHENYL TRICYCLIC QUINAZOLINE COMPOUNDS

FIELD OF THE INVENTION

The present invention relates to novel biphenyl tricyclic quinazoline compounds which are multiple inhibitors of the activity or function of tyrosine kinases, processes for their preparation, pharmaceutical compositions comprising the compounds, and the use of the compounds or the compositions in the treatment of various disorders. Specifically, the compounds of the invention are inhibitors of EGFR (Epidermal Growth Factor Receptor), VEGFR-2 (Vascular Endothelial Growth Factor Receptor 2), FGFR-1 (Fibroblast Growth Factor Receptor 1), PDGFRp (Platelet-Derived Growth Factor Receptor β), abl kinase and src kinase.

Compounds which are multiple-inhibitors of the activity or function of tyrosine kinases may be useful in the treatment of disorders such as cancer diseases, inflammatory diseases, autoimmune diseases, arthritis and hypervascularization diseases.

BACKGROUND OF THE INVENTION

Alterations of the cell cycle regulatory systems can lead to the onset of hyperproliferative diseases, such as for example cancer [Evan G.I., Vousden K.H., Nature, 2001, 411, 342-348]. Among the various proteins involved in cell signal transduction and amplification, the tyrosine kinases (TKs) play a pivotal role. These protein enzymes catalyze the transfer of a γ-phosphate from adenosine triphosphate (ATP) or from guanosine triphosphate (GTP) to the hydroxyl groups of a tyrosine residue in the protein substrate. The phosphorylated substrate is in turn able to promote the proliferative cascade through DNA transcription activation and apoptosis blockade. The TKs are divided in two groups, the transmembrane kinases (Receptor TKs, RTKs) and the soluble enzymes (Cytoplasmic TKs, CTKs). Both classes of TKs have been widely investigated as target for cancer treatment leading to a number of drug candidates. RTKs activate several transduction pathways as the Ras/MAPKs pathway and the PI3K/Akt pathway, thus leading to cell migration, cell survival and cell differentiation. Most of the RTKs are of fundamental importance only for embryonic development. This loss of importance for normal adult tissues, together with the TKs overexpression in the majority of tumor tissues, makes these enzymes a suitable target for cancer therapy. Among the RTKs, the Epidermal Growth Factor Receptor (EGFR), the Fibroblast Growth Factor Receptor-1 (FGFR-1), the Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and the Platelet-Derived Growth Factor Receptor-beta (PDGFRP) have attracted much attention. Among the CTKs, BCR-Abl and the oncogenic kinase Src play a key role in cancer growth and progression.

The RTKs are transmembrane receptors activated through the binding with specific proteins named growth factors (GFs) [Hubbard S.R., Miller W.T, Curr. Opin. Cell Biol. 2007, 19, 117-123]. Upon the binding with the GFs, the RTKs undergo to a dimerization/activation process that leads to their autophosphorylation. The RTKs activity regulation is strictly controlled in normal cells, whereas in almost all the tumors (both solid and liquid tumors) there is an over-expression or an over- activation of various TKs [Baselga J., Arribas J., Nat. Med., 2004, 10, 786-787].

Thus, the inhibition of the TKs is useful for the treatment of those pathologies in which the TKs are overexpressed as cancer, metastatic phenomena, arthritis and the hypervascularization diseases.

The inhibition of the TKs can be accomplished mainly through three different pharmacological strategies [Mendelsohn J., J. Clin. Oncol., 2002, 20, ls-13s] : 1) use of antisense oligonucleotides or ribozimes, that inhibit the synthesis of new TKs; 2) use of monoclonal antibodies, that inhibit the binding of the RTKs with the growth factors; 3) use of ATP-mimic compounds (TKs inhibitors, TKIs), that are small organic compounds able to inhibit the catalytic activity of the TKs.

Since the cancer cells activates a number of oncogenic signalling pathways and the selective inhibitors (as for example monoclonal antibodies and highly selective TKIs) block only a single pathway, these chemotherapeutic agents lead to drug resistance onset and to selection of mutated cell lines [Petrelli A., Giordano S., Curr. Med. Chem., 2008, 15, 422-432]. The drug resistance phenomena dramatically reduce the life expectancy of the patient and compromise the therapeutic effectiveness of the treatment. Moreover, the selection of mutated cell lines contributes to increase tumor aggressiveness and metastatic phenomena onset. To improve the efficacy of the cancer treatment protocol, it is useful to 1) use pharmacological associations of drugs (multi-agents therapy) with different mechanism of action (i.e. different targets); or 2) use multi-target agents (i.e. compounds able to interact with different proteins involved in the proliferative pathways). The multi-target therapies show a lower incidence of side effects when compared with the multi-agents therapies. Due to their intrinsic mechanism of action, the monoclonal antibodies act as selective inhibitors, so that they can only be used in association therapies. Moreover, the monoclonal antibodies can inhibit the RTKs but not the CTKs, that do not bear a growth factor binding domain. Finally, since the monoclonal antibodies are protein drugs, they can not be orally administered. Thus, the monoclonal antibodies are currently administered by intravenous or subcutaneous injection, impairing the patient compliance.

On the contrary, multi-target TKIs (MTTKIs) can be of use in multi-target therapy for oral and parenteral administration in the treatment of cancer diseases.

Thus, there is a need to find compounds which are multiple inhibitors of the activity or function of tyrosine kinases for the treatment of cancer diseases. US 5747498 describes 4-anilino quinazoline derivatives as selective Epidermal Growth Factor Receptor (EGFR) inhibitors.

Adriana Chilin et al. [J. Med. Chem., 2010, 53, 1862-1866] describes some dioxane, dioxalane and dioxepine quinazoline derivatives as Epidermal Growth Factor Receptor (EGFR) inhibitors.

Stephen W. Wright et al. [J. Med. Chem., 2002, 45, 3865-3877] describes certain anilinoquinazoline inhibitors of fructose-l,6-bisphosphatase.

The present invention address a need in the art by providing new quinazoline compounds which are multiple inhibitors of the activity or function of tyrosine kinases and as a such they are multi-target TKIs (MTTKIs). SUMMARY OF THE INVENTION

The invention is directed to certain novel biphenyl tricyclic quinazoline compounds. Specifically, the invention is directed to compounds of formula (I).

wherein X, Y, R, Ri, n and m are as defined below, or salts thereof.

The compounds are multiple inhibitors of tyrosine kinase activity. Particularly they are inhibitors of EGFR (Epidermal Growth Factor Receptor), VEGFR-2 (Vascular Endothelial Growth Factor Receptor 2), FGFR-1 (Fibroblast Growth Factor Receptor 1), PDGFRp (Platelet-Derived Growth Factor Receptor β), abl kinase and src kinase. More particularly they are inhibitors of at least 4 among the following tyrosine kinases EGFR, VEGFR-2, FGFR-1, PDGFRp, abl kinase and src kinase and as such they are multi-target TKIs (MTTKIs).

Compounds of the invention are useful in the treatment of conditions mediated by said tyrosine kinases. Compounds of formula (I) or pharmaceutically acceptable salts thereof may be of use in the treatment of a variety of cancers.

Accordingly, the invention is further directed to pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. The invention is still further directed to methods of inhibiting tyrosine kinase activity and treatment of disorders associated therewith using a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. The invention is yet further directed towards processes for the preparation for the compounds of the invention. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in a first aspect, a compound of formula (I) or a salt thereof,

(I)

wherein

R is hydrogen or C^.4 alkyl;

n is an integer from 1 to 5;

X and Y are each independently an oxygen or a sulphur atom;

is trifluoromethyl, a alkyl, a alkoxy, a trifluoromethoxy or a halogen group;

m is zero or an integer from 1 to 3; or salts thereof, (hereinafter "compounds of the invention").

In one embodiment, compounds of formula (I) are in the form of pharmaceutically acceptable salts; In one embodiment, X and Y are both an oxygen atom;

In one embodiment, X and Y are both a sulphur atom;

In one embodiment, X is an oxygen atom and Y is a sulphur atom;

In one embodiment, Y is an oxygen atom and X is a sulphur atom; In one embodiment, R is hydrogen; In one embodiment, Ri is C_j^alkyl;

In one embodiment, R is methyl; In one embodiment, Ri is trifluoromethyl; In one embodiment, Ri is halogen;

In one embodiment, Ri is alkoxy;

In one embodiment, Ri is trifluoromethoxy;

In one embodiment, m is 0; In one embodiment, m is 1 or 2; In one embodiment, m is 3;

In one embodiment, n is an integer from 1 to 3; In one embodiment, n is 4 or 5;

It is to be understood that the present invention covers all combinations of substituent groups described hereinabove.

In one embodiment, the invention is directed to compounds according to formula(IA),

wherein n is an integer from 1 to 3 or pharmaceutical acceptable salts thereof.

In one embodiment, the invention is directed to the pharmaceutically acceptable salts of compounds of formula (IA).

In one embodiment, the invention is directed to compound of formula (IA) in the form of hydrochloride salts. Compounds of the invention include the compounds of Examples 1 to 3 :

([l,3]Dioxolo[4,5-g]quinazolin-8-yl)-(biphenyl-3'-yl)amine hydrochloride;

(7,8-Dihydro[l,4]dioxino[2,3-,g]quinazolin-4-yl)-(biphenyl-3'-yl)amine

hydrochloride;

(8,9-Dihydro-7H-[l,4]dioxepino[2,3-g]quinazolin-4-yl)-(biphenyl-3'-yl)amine hydrochloride.

Terms and Definition The term 'halogen' as used herein refers to a fluorine, chlorine, bromine or iodine atom.

The term 'C1- alkyl' as used herein refers to a group or a part of the group refers to a linear or branched saturated hydrocarbon group containing from 1 to 4 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert butyl, and the like. The term 'C1- alkoxy' as used herein refers to a group wherein an oxygen atom is bound to the rest of the molecule and to the above mentioned Ci-4alkyl group. Suitable examples include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy.

"Pharmaceutically acceptable" refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein the symbols and conventions used in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Medicinal Chemistry or the Journal of Biological Chemistry. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification.

Included within the scope of the "compounds of the invention" are all solvates (including hydrates), complexes, polymorphs, prodrugs, radiolabeled derivatives, stereoisomers and optical isomers of the compounds of formula (I) and salts thereof.

The compounds of the invention may exist in solid or liquid form. In the solid state, the compounds of the invention may exist in crystalline or noncrystalline form, or as a mixture thereof. For compounds of the invention that are in crystalline form, the skilled artisan will appreciate that pharmaceutically acceptable solvates may be formed wherein solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve nonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and EtOAc, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent that is incorporated into the crystalline lattice are typically referred to as "hydrates." Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The invention includes all such solvates.

The skilled artisan will further appreciate that certain compounds of the invention that exist in crystalline form, including the various solvates thereof, may exhibit polymorphism (i.e. the capacity to occur in different crystalline structures). These different crystalline forms are typically known as "polymorphs". The invention includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X- ray powder diffraction patterns, which may be used for identification. The skilled artisan will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents, used in making or recrystallising the compound. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, one polymorph may spontaneously convert to another polymorph under certain conditions.

The invention also includes isotopically-labelled compounds, which are identical to the compounds of formula (I) and salts thereof, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷0, ¹⁸0, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶C1, respectively. Certain isotopic variations of the invention, for example, those in which a radioactive isotope such as ³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the compounds of the invention can generally be prepared by conventional procedures such as by the illustrative methods or by the preparations described in the Examples hereafter using appropriate isotopic variations of suitable reagents. It is to be understood that the references herein to compounds of formula (I) and salts thereof covers the compounds of formula (I) as free bases, or as salts thereof, for example as pharmaceutically acceptable salts thereof. Thus, in one embodiment, the invention is directed to compounds of formula (I) as free base. In another embodiment, the invention is directed to compounds of formula (I) and salts thereof. In a further embodiment, the invention is directed to compounds of formula (I) and pharmaceutically acceptable salts thereof.

The skilled artisan will appreciate that pharmaceutically acceptable salts of the compounds according to formula (I) may be prepared. Indeed, in certain embodiments of the invention, pharmaceutically acceptable salts of the compounds according to formula (I) may be preferred over the respective free base because such salts impart greater stability or solubility to the molecule thereby facilitating formulation into a dosage form. Accordingly, the invention is further directed to compounds of formula (I) and pharmaceutically acceptable salts thereof.

As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects. These pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in free base form with a suitable acid. Salts and solvates having non-pharmaceutically acceptable counter-ions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of formula (I) and their pharmaceutically acceptable salts. Thus one embodiment of the invention embraces compounds of formula (I) and salts thereof.

In certain embodiments, compounds according to formula (I) may contain a basic functional group and are therefore capable of forming pharmaceutically acceptable acid addition salts by treatment with a suitable acid. Suitable acids include pharmaceutically acceptable inorganic acids and pharmaceutically acceptable organic acids. Representative pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, sulfamate, phosphate, acetate, hydroxyacetate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicyclate, glycollate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, naphthoate, hydroxynaphthoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate), and napthalene-2 -sulfonate.

As used herein, "treat" in reference to a disorder means: (1) to ameliorate or prevent the disorder or one or more of the biological manifestations of the disorder, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the disorder or (b) one or more of the biological manifestations of the disorder, (3) to alleviate one or more of the symptoms or effects associated with the disorder, or (4) to slow the progression of the disorder or one or more of the biological manifestations of the disorder. As indicated above, "treatment" of a disorder includes prevention of the disorder. The skilled artisan will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a disorder or biological manifestation thereof, or to delay the onset of such disorder or biological manifestation thereof.

As used herein, "safe and effective amount" in reference to a compound of formula (I) or a pharmaceutically acceptable salt thereof or other pharmaceutically-active agent means an amount of the compound sufficient to treat the patient's condition but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment. A safe and effective amount of a compound will vary with the particular compound chosen (e.g. consider the potency, efficacy, and half-life of the compound); the route of administration chosen; the disorder being treated; the severity of the disorder being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be routinely determined by the skilled artisan.

As used herein, "patient" refers to a human (including adults and children) or other animal. In one embodiment, "patient" refers to a human.

Compound Preparation

The compounds of the invention may be made by a variety of methods, including standard chemistry. Any previously defined variable will continue to have the previously defined meaning unless otherwise indicated. Illustrative general synthetic methods are set out below and then specific compounds of the invention are prepared in the Examples section. The present invention also provides a process for the preparation of the compound of formula (I) or a salt thereof, which process comprises:

(II) (III) reacting a compound of formula (II), wherein W is a suitable leaving group such as chlorine [for examples see J. March, Advanced Organic Chemistry: reactions, mechanisms, and structure, John Wiley & Sons (1992), 4^th Ed., p352)], X, Y and n are as defined in formula(I), with a phenyl aniline derivative of formula (III), wherein R, Rl and n have the meanings defined in formula(I), in a suitable solvent such as alcohol at a temperature ranging from 20-100°C, to obtain compounds of formula(I). Compounds of formula (II) wherein X, Y and n are as defined in formula(I), may be prepared by reaction of a quinazolinone derivative (IV) with a halogenating agent such as for example POCI3 optionally in the presence of

a suitable organic base such as for example triethylamine at a temperature ranging from 20°C to reflux temperature.

Compounds of formula (IV) wherein X, Y and n are as defined in formula(I), may be prepared by oxidation of a derivative (V)

with Cerium (IV) ammonium nitrate or Cromium (VI) oxide in the presence of an organic acid such as acetic acid and in suitable solvent such as water at room temperature. Compound of formula (V) may be prepared by treatment of a carbamate derivative of formula(VI)

with hexamethylenetetramine (urotropine) in a suitable organic acid such as TFA at temperature ranging from 0 to 120°C followed by treatment with potassium hexacyanoferrate (III) in aqueous ethanolic alkali such as potassium hydroxide at a temperature ranging from 20°C to reflux.

Compound of formula (VI) may be prepared by an amine derivative of formula (VII)

with ethyl haloformiate in a suitable solvent such as tetrahydrofuran and in the presence of a suitable organic base such as for example triethylamine.

The compounds (III) and (VII) are known compounds or may be prepared with analogous methods of those of known compounds.

When it is desired to isolate a compound of formula (I) as a salt, for example a pharmaceutically acceptable salt, this may be achieved by reacting the compound of formula (I) in the form of the free base with an appropriate amount of a suitable acid and in a suitable solvent such as an alcohol (e.g. ethanol or methanol), an ester (e.g. ethyl acetate) or an ether (e.g. diethyl ether or tetrahydrofuran). Pharmaceutically acceptable salts may also be prepared from other salts, including other pharmaceutically acceptable salts, of the compounds of formula (I) using conventional methods. The compounds of formula (I) may readily be isolated in association with solvent molecules by crystallisation from or evaporation of an appropriate solvent to give the corresponding solvates.

Methods of Use

Compounds of the invention and salts thereof are multiple inhibitors of the activity or function of tyrosine kinases. Specifically, the compounds of the invention are inhibitors of EGFR , VEGFR-2 , FGFR-1, PDGFRp, abl kinase and src kinase. Particularly they have affinity for at least four kinases among EGFR, VEGFR-2, FGFR- 1, PDGFRp, abl e src.

Compounds of the invention are useful in the treatment of conditions mediated by said tyrosine kinases.

Compounds of the invention may be of use in the treatment of a variety of cancers, including the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; and other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma. Due to the key role of kinases in the regulation of cellular proliferation in general, inhibitors could act as reversible cytostatic agents which may be useful in the treatment of any disease process which features abnormal cellular proliferation, e.g., benign prostate hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, endotoxic shock, and fungal infections. Compounds of the invention may also be of use in the treatment of a range of disorders such as transplant (such as organ transplant, acute transplant), protection from ischemic or reperfusion injury such as ischemic or reperfusion injury incurred during organ transplantation, myocardial infarction, stroke or other causes, transplantation tolerance induction; diabetes, psoriasis, arthritis (such as rheumatoid arthritis, psoriatic arthritis or osteoarthritis) Kaposi's sarcoma, haemangioma, obesity, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases including Autoimmune Hyperthyroidism, such as Graves' Disease; Addison's disease, Autoimmune polyglandular diseases, autoimmune alopecia, pernicious anemia, vitiligo, autoimmune hypopituatarism; Guillain-Barre syndrome, psoriasis, multiple sclerosis, inflammatory bowel diseases including ulcerative colitis and Crohn's disease, lupus (systemic lupus erythematosis).

Compounds of the invention may be also active in a number of diseases associated with deregulated angiogenesis, such as diseases caused by ocular neovascularisation, retinopathies associated with retinal vessel proliferation such as diabetic retinopathy or age related macula degeneration.

Compounds of the invention are particularly useful in the treatment or prevention of head and neck, breast, lung, colon, ovary, pancreatic, and prostatic cancer. The invention therefore provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in therapy.

In another aspect, the invention provides a compound of Formula (I) or pharmaceutically acceptable salt thereof for use in the treatment of conditions mediated by tyrosine kinases.

The invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use as a therapeutic substance in the treatment or prophylaxis of cancer diseases.

The invention further provides a method of treatment or prophylaxis of conditions mediated by tyrosine kinases in mammals including humans, which comprises administering to the sufferer a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In a further aspect, the invention further provides a method of treatment or prophylaxis of cancer diseases in mammals including humans, which comprises administering to the sufferer a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of conditions mediated by tyrosine kinases.

In another aspect, the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment or prophylaxis of the cancer diseases. Compositions

The compounds of formula (I) and pharmaceutically acceptable salts thereof will normally, but not necessarily, be formulated into pharmaceutical compositions prior to administration to a patient. Accordingly, in another aspect the invention is directed to pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically- acceptable excipients.

The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein a safe and effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof can be extracted and then given to the patient such as with powders or syrups. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a compound of formula (I) or a pharmaceutically acceptable salt thereof. When prepared in unit dosage form, the pharmaceutical compositions of the invention typically may contain, for example, from 0.5mg to lg, or from lmg to 700mg, or from 5mg to lOOmg of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The pharmaceutical compositions of the invention typically contain one compound of formula (I) or a pharmaceutically acceptable salt thereof.

As used herein, "pharmaceutically-acceptable excipient" means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the compound of formula (I) or a pharmaceutically acceptable salt thereof when administered to a patient and interactions which would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided. In addition, each excipient must of course be pharmaceutically-acceptable e.g. of sufficiently high purity. The compound of formula (I) or a pharmaceutically acceptable salt thereof and the pharmaceutically-acceptable excipient or excipients will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration. For example, dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixers, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols, solutions, and dry powders; and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting of the compound or compounds of formula (I) or pharmaceutically acceptable salts thereof once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.

Suitable pharmaceutically-acceptable excipients include the following types of excipients: Diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweetners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically-acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other excipients are present in the formulation.

Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically-acceptable excipients in appropriate amounts for use in the invention. In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically-acceptable excipients and may be useful in selecting suitable pharmaceutically-acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

Accordingly, in another aspect the invention is directed to process for the preparation of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically- acceptable excipients which comprises mixing the ingredients. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof may be prepared by, for example, admixture at ambient temperature and atmospheric pressure.

In one embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof will be formulated for oral administration. In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof will be formulated for parenteral administration.

In one aspect, the invention is directed to a solid oral dosage form such as a tablet or capsule comprising a safe and effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g. corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives (e.g. microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch (e.g. corn starch, potato starch, and pre-gelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesuim stearate, calcium stearate, and talc.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The composition 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.

The compounds of formula (I) or pharmaceutically acceptable salts thereof may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamide-phenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of formula (I) or pharmaceutically acceptable salts thereof 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.

In another aspect, the invention is directed to a liquid oral dosage form. Oral liquids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Syrups can be prepared by dissolving the compound of formula (I) or a pharmaceutically acceptable salt thereof in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound of formula (I) or a pharmaceutically acceptable salt thereof 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.

In another aspect, the invention is directed to a dosage form adapted for administration to a patient by inhalation, for example, as a dry powder, an aerosol, a suspension, or a solution composition. Preferably, the invention is directed to a dry powder composition adapted for inhalation comprising compound of formula (I) or a pharmaceutically acceptable salt thereof.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the patient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986).

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Ointments, creams and gels, may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agent and/or solvents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil, or a solvent such as polyethylene glycol. Thickening agents and gelling agents which may be used according to the nature of the base include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethylene and cellulose derivatives, and/or glyceryl monostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents, suspending agents or preservatives.

Topical preparations may be administered by one or more applications per day to the affected area; over skin areas occlusive dressings may advantageously be used. Continuous or prolonged delivery may be achieved by an adhesive reservoir system. For treatments of the eye or other external tissues, for example mouth and skin, the compositions may be applied as a topical ointment or cream. When formulated in an ointment, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound of formula (I) or pharmaceutically acceptable salt thereof may be formulated in a cream with an oil-in-water cream base or a water-in- oil base. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (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.

The compound and pharmaceutical formulations according to the invention may be used in combination with or include one or more other therapeutic agents.

The compounds of this invention may also be useful in combination with known anti-cancer and cytotoxic agents and treatments, including radiation. If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described below and the other pharmaceutically active agent within its approved dosage range. Compounds of formula I may be used sequentially with known anticancer or cytotoxic agents and treatment, including radiation when a combination formulation is inappropriate.

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 medical oncology the other component(s) of such conjoint treatment in addition to the antiproliferative treatment defined herein before may be: surgery, radiotherapy or chemotherapy. Such chemotherapy may cover three main categories of therapeutic agent: (i) antiangiogenic agents that work by different mechanisms from those defined hereinbefore (for example, linomide, inhibitors of integrin ανβ3 function, angiostatin, razoxane); (if) cytostatic agents such as antiestrogens (for example, tamoxifen, toremifene, raloxifene, droloxifene, iodoxifene), progestogens (for example, megestrol acetate), aromatase inhibitors (for example, anastrozole, letrozole, borazole, exemestane), antihormones, antiprogestogens, antiandrogens (for example, flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (for example, gosereline acetate, leuprolide), inhibitors of testosterone 5a-dihydroreductase (for example, finasteride), farnesyltransferase inhibitors, anti-invasion agents (for example, metalloproteinase inhibitors such as marimastat and inhibitors of urokinase plasminogen activator receptor function) and inhibitors of growth factor function, (such growth factors include for example, EGF, FGF, platelet derived growth factor and hepatocyte growth factor, such inhibitors include growth factor antibodies, growth factor receptor antibodies such as Avastin® (bevacizumab) and Erbitux® (cetuximab); tyrosine kinase inhibitors, serine/threonine kinase inhibitors and inhibitors of insulin growth receptor); and (iii) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as antimetabolites (for example, antifolates such as methotrexate, fluoropyrimidines such as 5-fluorouracil, purine and adenosine analogues, cytosine arabinoside); Intercalating antitumour antibiotics (for example, anthracyclines such as doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (for example, cisplatin, carboplatin); alkylating agents (for example, nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide nitrosoureas, thiotepa; antimitotic agents (for example, vinca alkaloids like vincristine, vinorelbine, vinblastine and vinflunine, and taxoids such as Taxol® (paclitaxel), Taxotere® (docetaxel) and newer microbtubule agents such as epothilone analogs, discodermolide analogs, and eleutherobin analogs); topoisomerase inhibitors (for example, epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan, irinotecan); cell cycle inhibitors (for example, flavopyridols); biological response modifiers and proteasome inhibitors such as Velcade® (bortezomib). Compounds of the invention may be also usefully associated with COX-2 inhibitors (such as for example apricoxib) for the treatment of the aforementioned pathologies. The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical composition and thus pharmaceutical compositions comprising a combination as defined above together with a pharmaceutically acceptable diluent or carrier represent a further aspect of the invention.

The individual compounds of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. In one embodiment, the individual compounds will be administered simultaneously in a combined pharmaceutical formulation. Appropriate doses of known therapeutic agents will readily be appreciated by those skilled in the art.

The invention thus provides, in a further aspect, a pharmaceutical composition comprising a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof together with another therapeutically active agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The figure shows the pro-apoptotic effect of Examples 1, 2 and 3 on A431 cells. After a 24 h incubation period with 10 μΜ compounds, cells were lysated and the amount of mono- and oligonucleosomes in supernantants was determined using a sandwich-enzyme immunoassay. Results, expressed as percent change from control non treated cultures (taken as 0), are the mean ± SD of three independent experiments *= p<0.05 vs control cultures, Student t test.

Figure 2. The figure shows the effect of Examples 1, 2 and 3 on FGF-2 release determined by means of an immuno-enzymatic assay. After a 72 h incubation period of HUVECs with 0.1 μΜ compounds, culture media were collected and cytokine concentration was measured. Results, means of three experiments, were expressed as pg/mL of FGF-2. *= p< 0.05 vs control cultures, Student t test. Figure 3. The figure shows the morphogenesis of HUVECs seeded on Matrigel and treated for 18 h with compounds at the highest concentration not inducing decrease in cell viability (0.1 μΜ). Quantitative analysis of the effects of compounds on the dimensional (percent area covered by HUVECs and total length per field), and topological parameters (number of branching points per field, number of mesh per field and percent area inside meshes) of the HUVEC meshwork are reported. Bars: black, Example 1; dark grey, Example 2; light grey, Example 3. Results, expressed as percent change from control non treated cultures (taken as 0), are the mean ± SD of at least three independent experiments. * = p<0.05 vs control cultures, Student t test.

Figure 4. The figure shows the morphogenesis of HUVECs seeded on Matrigel and treated for 18 h with 50 ng/mL of FGF-2 (A) or Ο.ΙμΜ Examples 1, 2 and 3 plus 50 ng/mL FGF-2 (B) Bars: black, Example 1; dark grey, Example 2; light grey, Example 3. Results, obtained from three independent experiments, are expressed as mean ± SD. * = p<0.05 vs non treated cultures (A) or FGF-2 treated cultures, Student t test.

Figure 5. The figure shows the morphogenesis of HUVECs seeded on Matrigel and treated for 18 h with 20 ng/mL of VEGF (A) or Ο.ΙμΜ Examples 1, 2 and 3 plus 20 ng/mL VEGF (B) Bars: black, Example 1; dark grey, Example 2; light grey, Example 3. Results, obtained from three independent experiments, are expressed as mean ± SD. * = p<0.05 vs non treated cultures (A) or VEGF treated cultures, Student t test.

Figure 6. The figure shows the haemoglobin (Hb) content in the Matrigel plugs containing 200 ng/mL FGF-2 with or without 1 μΜ Examples 1, 2 and 3. At 7 day, the plugs were removed and haemoglobin was determined by means of a colorimetric assay using the Drabkin's Reagent. Results, obtained from three experiments and expressed as mg/mL haemoglobin, are reported as mean ± SD. *= p< 0.05 vs FGF-2; Student t test. Figure 7. The figure shows sections of plugs stained with hematoxylin and eosin (magnification x200). (A) control; (B) 200 ng/mL FGF-2; (C) 200 ng/mL FGF-2 plus

1 μΜ Example 1; (D) 200 ng/mL FGF-2 plus 1.0 μΜ Example 2; (E) 200 ng/mL FGF-

2 plus 1.0 μΜ Example 3.

EXPERIMENTAL

The following Intermediates and Examples illustrate the preparation of compounds of the invention.

In the procedures that follow, after each starting material, reference to a description is typically provided. This is provided merely for assistance to the skilled chemist. The starting material may not necessarily have been prepared from the batch referred to.

The yields were calculated assuming that products were 100% pure if not stated otherwise.

Compounds are named according to IUPAC rules.

All commercial chemicals and solvents used were analytical grade and were used without further purification.

Analytical thin layer chromatography (tic) was performed on pre-coated silica gel plates (Merck 60-F-254, 0.25 mm) eluting with CHCl₃/MeOH mixture (1: 1, v/v). Melting points were determined on a Gallenkamp MFB-595-010M melting point apparatus and are uncorrected.

The ^!H-NMR (Nuclear Magnetic Resonance) spectra were recorded on a Bruker 300- AMX spectrometer with TMS as an internal standard. Chemical shifts are reported in ppm (δ). Coupling constants are given in Hz and the relative peaks area were in agreement with all assignment. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet. Elemental analysis were performed on a Perkin-Elmer 2400 analyzer. Mass spectra were performed on an Applied Biosystem Mariner System 5220 with direct injection of the sample. Microwave assisted reactions were performed on a CEM Discover® monomode reactor with the temperature monitored by a built-in infrared sensor and the automatic control of the power; all the reactions were performed in closed devices with pressure control. The following table lists the abbreviations used:

AcOH Acetic acid

ATP Adenosine triphosphate

BSA Bovine Serum Albumin

CAN Cerium (IV) Ammonium Nitrate

DMSO Dimethyl sulfoxide

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

EGTA Ethylene glycol tetraacetic acid

ESI-TOF Electrospray Ionization - Time of Flight

EtOAc Ethyl acetate

EtOH Ethanol

FCS Fetal Calf serum

h hour

HMTA Hexamethylenetetramine

HRMS High Resolution Mass Spectrometry

HUVEC Human Umbelical Vein Endothelial Cell

i-PrOH Isopropanol

MeOH Methanol

min minute MOPS 3-(N-morpholino)propansulphonic acid

mp Melting point

MTT 3 - (4, 5 -dimethylthiazol-2 -yl) -2, 5 -diphenyltetrazole bromide

NMR Nuclear Magnetic Resonance

PBS Phosphate buffer solution

SDS-PAGE Sodium dodecyl sulphate Polyacrilamide Gel Electrophoresis

TEA Triethylamine

TFA Trifluoroacetic acid

THF Tetrahydrofuran

tic Thin layer chromatography

TMS Tetramethylsilane

Intermediate 1

Ethyl (l,3-benzodioxolan-5-yl)carbamate

A mixture of 5-aminobenzo [l,3] dioxolane (5.0 g, 36.5 mmol), ethyl chloroformate (6.9 mL, 72.9 mmol) and TEA (10.2 mL, 72.9 mmol) in anhydrous THF (440 mL) was stirred at room temperature for 30 min. The solid was filtered off and the solvent was evaporated under reduced pressure to give the title compound. Yield 98%; mp < 45 °C; NMR (300 MHz, DMSO-c ₆) : δ 9.47 (broad s, 1H, NH], 7.11 (s, 1H, 4-H), 6.87 - 6.78 (m, 2 H, 6-H and 7-H), 5.95 (s, 2H, OCH₂0), 4.09 (q, / = 7.1 Hz, 2H, COOC//2CH3), 1.22 (t, / = 7.1 Hz, 3 H, COOCH2C//3) . Anal, calcd. for C10H11NO4: C, 57.41; H, 5.30; N, 6.70; found: C, 57.45; H, 5.25; N, 6.72. HRMS (ESI-TOF) for C10H12NO4 [M + H]⁺: calcd.: 210.0761, found: 210.1658.

Intermediate 2

[l,3]Dioxolo[4,5-flf]quinazoline

A mixture of ethyl (l,3 -benzodioxolan-5-yl)carbamate (intermediate 1) (7.2 g, 34.0 mmol) and HMTA (4.2 g, 34.0 mmol) in TFA (90 mL) was microwave irradiated at 110 °C (power set point 80 W; ramp time 1 min; hold time 10 min). After cooling the mixture was diluted with aqueous ethanolic (water/EtOH: 1/1) 10% KOH (1200 mL), and KsFe(CN)6 (89.5 g, 272.0 mmol) was added to the solution, which was refluxed under stirring for 4 hours. After cooling the mixture was diluted with water (1200 mL) and extracted with CHCI3 (3 x 500 mL). The organic phase was evaporated under reduced pressure to give the title compound (5.0 g, yield 83%); mp: 158 °C; Ή NMR (300 MHz, CDCh-d): δ 9.12 (s, 1H, 8-H), 9.10 (s, 1H, 6-H), 7.29 (s, 1H, 4-H or 9-H), 7.12 (s, 1H, 4-H or 9-H), 6.17 (s, 2H, OCH₂0). Anal, calcd. for C9H6N2O2: C, 62.07; H, 3.47; N, 16.09; foud: C, 62.15; H, 3.50; N, 16.02. HRMS (ESI- TOF) for C9H7N2O2 [M + H]⁺: calcd.: 175.0502, found: 175.0498.

Intermediate 3

[l,3]Dioxolo[4,5-,gf]quinazolin-8(7H)-one

To a solution of [l,3]dioxolo[4,5-,g]quinazoline (intermediate 2) (0.5 g, 2.9 mmol) in AcOH (1.0 mL) a solution of CAN (6.4 g, 11.6 mmol) in water (13.0 mL) was added dropwise. The white precipitate formed was collected to give the title compound (0.4 g, yield 68%); mp > 300 °C; Ή NMR (300 MHz, DMSO-c ₆): δ 7.97 (s, 1H, 6-H), 7.41 (s, 1H, 4-H or 9-H), 7.12 (s, 1H, 4-H or 9-H), 6.20 (s, 2H, OCH₂0). Anal, calcd. for C9H6N2O3: C, 56.85; H, 3.18; N, 14.73; C, 56.79; H, 3.22; N, 14.74. HRMS (ESI-TOF) for C9H7N2O3 [M + H]⁺: calcd.: 191.0451, found: 191.0369.

Intermediate 4

8-Chloro-[l,3]dioxolo[4,5-flf]quinazoline

A suspension of [l,3]dioxolo[4,5-g]quinazolin-8(7//)-one (intermediate 3) (0.8 g, 4.3 mmol) in P0C1₃ (4.0 mL, 43.0 mmol) and TEA (3.0 mL) was refluxed for 3 h. After cooling the mixture was concentrated under reduced pressure and the solid residue was dissolved in EtOAc (50 mL) and washed with a saturated aqueous NaHCC solution (2 x 25 mL). The organic phase was evaporated under reduced pressure to give the title compound (0.6 g, yield 72%); mp: 168 °C; Ή NMR (300 MHz, CDCl₃-cf): δ 8.85 (s, 1H, 6-H), 7.49 (s, 1H, 4-H or 9-H), 7.32 (s, 1H, 4-H or 9-H), 6.21 (s, 2H, OC//2O). Anal, calcd. for C₉H₅C1N₂02: C, 51.82; H, 2.42; CI, 17.00; N, 13.43; found: C, 51.95; H, 2.39; CI, 17.05; N, 13.39. HRMS (ESI-TOF) for C9H6CIN2O2 [M + H]⁺: calcd.: 209.0112, found: 209.0123. Intermediate 5

Ethyl (l,4-benzodioxan-6-yl)carbamate

A mixture of 6-aminobenzo [l,4] dioxane (1.9 g, 12.5 mmol), ethyl chloroformate (2.4 mL, 25.0 mmol) and TEA (3.5 mL, 25.0 mmol) in anhydrous THF (150 mL) was stirred at room temperature for 30 min. The solid was filtered off and the solvent was evaporated under reduced pressure to give the title compound (2.2 g, yield 77%); mp: 117 °C; Ή NMR (300 MHz, DMSO-c ₆) : δ 9.38 (broad s, 1H, NH], 7.03 (d, / = 2.1 Hz, 1H, 5-H), 6.85 (dd, / = 8.7 Hz, 2.1 Hz, 1H, 7-H), 6.74 (d, / = 8.7 Hz, 1H, 8-H), 4.22 - 4.14 (m, 4H, OC//₂C//₂0), 4.08 (q, / = 7.1 Hz, 2 H, COOCtf₂CH₃), 1.21 (t, / = 7.1 Hz, 3 H, COOCH₂Ctf₃). Anal, calcd. for C11H13NO4: C, 59.19; H, 5.87; N, 6.27; found: C, 59.25; H, 5.76; N, 6.23. HRMS (ESI-TOF) for C11H14NO4 [M + H]⁺: calcd.: 224.0917, found: 224.1001.

Intermediate 6

7,8-Dihydro[l,4]dioxino[2,3-flf]quinazoline

A mixture of ethyl (l,4-benzodioxan-6-yl)carbamate (intermediate 5) (5.0 g, 22.4 mmol) and HMTA (3.1 g, 22.4 mmol) in TFA (70 mL) was microwave irradiated at 110 °C (power set point 80 W; ramp time 1 min; hold time 10 min). After cooling the mixture was diluted with aqueous ethanolic (water/EtOH: 1/1) 10% KOH (800 mL), and K₃Fe(CN)6 (59.0 g, 179.2 mmol) was added to the solution, which was refluxed under stirring for 4 hours. After cooling the mixture was diluted with water (800 mL) and extracted with CHC1₃ (3 x 500 mL). The organic phase was evaporated under reduced pressure to give the title compound (3.6 g, yield 86%); mp: 109 °C;

NMR (300 MHz, DMSO-c ₆) : δ 9.30 (s, 1H, 4-H), 9.03 (s, 1H, 2 -H), 7.55 (s, 1H, 5-H or 10-H), 7.36 (s, 1H, 5-H or 10-H), 4.47 - 4.38 (m, 4H, OCtf₂Ctf₂0). Anal, calcd. for CioH₈N₂0₂: C, 63.82; H, 4.28; N, 14.89; found: C, 63.95; H, 4.23; N, 14.88. HRMS (ESI- TOF) for CioH₉N₂0₂ [M + H]⁺: calcd.: 189.0659, found: 189.0599.

Intermediate 7

7,8-Dihydro[l,4]dioxino[2,3-.gf]quinazolin-4(3H)-one To a solution of 7,8-dihydro[l,4] dioxino[2,3 -g]quinazoline (intermediate 6) (1.7 g, 9.1 mmol) in AcOH (4.0 mL) a solution of CAN (20.0 g, 36.4 mmol) in water (48.0 mL) was added dropwise. The white precipitate formed was collected to give the title compound (1.1 g, yield 65%); mp: 282 °C; Ή NMR (300 MHz, DMSO-c ₆) : δ 12.01 (broad s, 1H, NH], 7.92 (s, 1H, 2-H), 7.45 (s, 1H, 5-H or 10-H), 7.08 (s, 1H, 5-H or 10- H), 4.39 - 4.30 (m, 4H, OC//₂C//₂0). Anal, calcd. for Ci₀H₈N₂O₃: C, 58.82; H, 3.95; N, 13.72; found: C, 58.89; H, 4.00; N, 13.65. HRMS (ESI-TOF) for Ci₀H₉N₂O₃ [M + H]⁺: calcd.: 205.0608, found: 205.0599. Intermediate s

4-Chloro-7,8-dihydro[l,4]dioxino[2,3-#]quinazoline

A suspension of 7,8-dihydro[l,4] dioxino[2,3 -g]quinazolin-4(3//)-one (intermediate 7) (0.2 g, 1.0 mmol) in P0C1₃ (1.0 mL, 10.7 mmol) and TEA (1.0 mL) was refluxed for 3 h. After cooling the mixture was concentrated under reduced pressure and the solid residue was dissolved in EtOAc (50 mL) and washed with a saturated aqueous NaHC03 solution (2 x 25 mL). The organic phase was evaporated under reduced pressure to give the title compound (0.2 g, yield 70%); mp: 216 °C; *H NMR (300 MHz, CDCls-cT) : δ 8.86 (s, 1H, 2 -H), 7.66 (s, 1H, 5-H or 10-H), 7.50 (s, 1H, 5-H or 10- H), 4.47 - 4.39 (m, 4H, OCtf₂Ctf₂0). Anal, calcd. for: Ci₀H₇ClN₂O₂: C, 53.95; H, 3.17; CI, 15.92; N, 12.58; found: C, 54.01; H, 3.12; CI, 15.98; N, 12.52. HRMS (ESI-TOF) for CioH₈ClN₂0₂ [M + H]⁺: calcd.: 223.0269, found: 223.0305.

Intermediate 9

Ethyl (3,4-dihydro-2H-l,5-benzodioxepin-7-ylJcarbamate

A mixture of 7-amino-3,4-dihydro-2//-l,5-benzodioxepine (2.3 g, 13.9 mmol), ethyl chloroformate (2.7 mL, 27.8 mmol) and TEA (3.9 mL, 27.8 mmol) in anhydrous THF (150 mL) was stirred at room temperature for 30 min. The solid was filtered off and the solvent was evaporated under reduced pressure to give the title compound (3.3 g, yield 98%); mp < 45 °C; Ή NMR (300 MHz, DMSO-c ₆) : δ 9.48 (broad s, 1H, NH], 7.12 (d, / = 2.4 Hz, 1H, 6-H), 7.00 (dd, / = 8.7 Hz, 2.4 Hz, 1H, 8-H) , 6.86 (d, / = 8.7 Hz, 1H, 9-H), 4.13 - 4.01 (m, 6H, OCH₂CH₂CH₂0 and COOCH₂CH₃), 2.05 (quint, / = 5.3 Hz, 2H, OCH2C//2CH2O), 1.22 (tj = 7.1 Hz, 3H, COOCH₂C¾). Anal, calcd. for Ci₂Hi₅N0₄: C, 60.75; H, 6.37; N, 5.90; found: C, 60.63; H, 6.42; N, 5.92. HRMS (ESI-TOF) for C12H16NO4 [M + H]⁺: calcd.: 238.1074, found: 238.1098. Intermediate 10

8,9-Dihydro-7H-[l,4]dioxepino[2,3-g]quinazoline

A mixture of ethyl (3,4-dihydro-2//-l,5-benzodioxepin-7-yl)carbamate (intermediate 9) (3.3 g, 13.9 mmol) and HMTA (1.9 g, 13.9 mmol) in TFA (42 mL) was microwave irradiated at 110 °C (power set point 80 W; ramp time 1 min; hold time 10 min). After cooling the mixture was diluted with aqueous ethanolic (water/EtOH: 1/1) 10% KOH (420 mL), and K₃Fe(CN)₆ (41.2 g, 125.1 mmol) was added to the solution, which was refluxed under stirring for 4 hours. After cooling the mixture was diluted with water (500 mL) and extracted with CHCI3 (3 x 300 mL). The organic phase was evaporated under reduced pressure to give the title compound (2.5 g, yield 89%); mp: 87 °C; Ή NMR (300 MHz, DMS0-c ₆): δ 9.37 (s, 1H, 4-H), 9.11 (s, 1H, 2-H), 7.68 (s, 1H, 5-H or 11-H), 7.46 (s, 1H, 5-H or 11-H), 4.37 (tj = 5.7 Hz, 2H, OC//2CH2CH2O), 4.31 (t,/ = 5.7 Hz, 2H, OCH₂CH₂C//₂0); 2.23 (quint,/ = 5.7 Hz, 2H, OCH2C//2CH2O). Anal, calcd. for CiiHi₀N₂O₂: C, 65.34; H, 4.98; N, 13.85; found: C, 65.28; H, 5.01; N, 13.87. HRMS (ESI-TOF) for C11H11N2O2 [M + H]⁺: calcd.: 203.0818, found: 203.0836.

Intermediate 11

8,9-Dihydro-7H-[l,4]dioxepino[2,3-,gf]quinazolin-4(3H)-one

To a solution of 8,9-dihydro-7//-[l,4]dioxepino[2,3-g]quinazoline (intermediate 10) (2.5 g, 12.3 mmol) in AcOH (5.0 mL) a solution of CAN (27.0 g, 49.2 mmol) in water (60.0 mL) was added dropwise. The white precipitate formed was collected to give the title compound (2.0 g, yield 73%; mp: 180 °C; Ή NMR (300 MHz, DMSO-c/₆): δ 7.97 (s, 1H, 2-H), 7.56 (s, 1H, 5-H or 11-H), 7.16 (s, 1H, 5-H or 11-H), 4.28 (t, / = 5.6 Hz, 2H, OC//2CH2CH2O), 4.22 (t,/ = 5.6 Hz, 2H, OCH₂CH₂C//₂0), 2.17 (quint,/ = 5.6 Hz, 2H, OCH2C//2CH2O). Anal, calcd. for: CiiHi₀N₂O₃: C, 60.55; H, 4.62; N, 12.84; found: C, 60.60; H, 4.55; N, 12.87. HRMS (ESI-TOF) for C11H11N2O3 [M + H]⁺: calccL 219.0764, found: 219.0808.

Intermediate 12

4-Chloro-8,9-dihydro-7H-[l,4]dioxepino[2,3-flf]quinazoline

A suspension of 8,9-dihydro-7//-[l,4]dioxepino[2,3 -g]quinazolin-4(3 H)-one (intermediate 11) (0.8 g, 3.7 mmol) in P0C1₃ (3.5 mL, 37.5 mmol) and TEA (3.0 mL) was refluxed for 3 h. After cooling the mixture was concentrated under reduced pressure and the solid residue was dissolved in EtOAc (50 mL) and washed with a saturated aqueous NaHC03 solution (2 x 25 mL) . The organic phase was evaporated under reduced pressure to give the title compound (0.2 g, yield 93%); mp: 194 °C;

NMR (300 MHz, DMSO-c ₆) : δ 8.92 (s, 1H, 2 -H), 7.68 (s, 1H, 5-H or l l.H), 7.56 (s,

IH, 5-H or 11-H); 4.42 (t, / = 5.8 Hz, 2H, OCtf₂CH₂CH₂0), 4.36 (t, / = 5.8 Hz, 2H, OCH₂CH₂Ctf₂0), 2.26 (quint, / = 5.8 Hz, 2H, OCH₂Ctf₂CH₂0). Anal, calcd. for CiiH₉ClN₂0₂: C, 55.83; H, 3.83; CI, 14.98; N, 11.84; found: C, 55.85; H, 3.79; CI, 15.02; N, 11.90. HRMS (ESI-TOF) for CiiHi₀ClN₂O₂ [M + H]⁺: calcd.: 237.0425, found: 237.0505.

Example 1

([l,3]Dioxolo[4,5-flf]quinazolin-8-yl)-(biphenyl-3'-yl)amine hydrochloride

A mixture of 8-chloro-[l,3]dioxolo [4,5-g]quinazoline (intermediate 4) (0.1 g, 0.5 mmol) and 3 -phenylaniline (84.6 mg, 0.5 mmol) in /-PrOH (2 mL) was microwave irradiated at 80 °C (power set point 60 W; ramp time 1 min; hold time 15 min) . After cooling, the obtained precipitate was collected by filtration to give the title compound (0.2 g, yield 85%); mp > 300 °C; Ή NMR (300 MHz, DMS0-c ₆) : δ 10.66 (broad s, 1H, NH], 8.77 (s, 1H, 6-H), 8.18 (s, 1H, 4-H or 9-H), 8.03 (s, 1H, H_ar), 7.78- 7.67 (m, 3 H, H_ar), 7.60-7.47 (m, 4H, H_ar), 7.45-7.37 (m, 1H, H_ar), 7.30 (s, 1H, 4-H or 9- H), 6.37 (s, 2 H, 0Ctf₂0). Anal, calcd. for C₂iHi₆ClN₃0₂: C, 66.76; H, 4.27; CI, 9.38; N,

I I.12; found: C, 66.78; H, 4.26; CI, 9.35; N, 11.10. HRMS (ESI-TOF) for C₂iHi₆N₃0₂ [M + H]⁺: calcd.: 342.1237, found: 342.1260. Example 2

(7,8-Dihydro[l,4]dioxino[2,3-flf]quinazolin-4-yl)-(biphenyl-3'-yl)amine hydrochloride

A mixture of 4-chloro-7,8-dihydro[l,4]dioxino[2,3 -g]quinazoline (intermediate 8) (0.1 g, 0.5 mmol) and 3 -phenylaniline (84.6 mg, 0.5 mmol) in /-PrOH (2 mL) was microwave irradiated at 80 °C (power set point 60 W; ramp time 1 min; hold time 15 min). After cooling, the obtained precipitate was collected by filtration to give the title compound (0.2 g, yield 88%); mp > 300 °C; Ή NMR (300 MHz, DMS0-c ₆) : δ 11.01 (broad s, IH, NH], 8.82 (s, IH, 2 -H), 8.34 (s, IH, 5-H or 10-H), 8.03 (s, IH, H_ar), 7.78-7.66 (m, 3 H, H_ar), 7.64-7.47 (m, 4H, H_ar), 7.44-7.37 (m, IH, H_ar), 7.31 (s, IH, 5-H or 10-H), 4.55 - 4.41 (m, 4H, OC//₂C//₂0). Anal, calcd. for C₂₂Hi₈ClN₃0₂: C, 67.43; H, 4.63; CI, 9.05; N, 10.72; found: C, 67.44; H, 4.66; CI, 9.08; N, 10.71. HRMS (ESI-TOF) for C₂₂Hi₈N₃0₂ [M + H]⁺: calcd.: 356.1394, found: 356.1385. Example 3

(8,9-Dihydro-7H-[l,4]dioxepino[2,3-flf]quinazolin-4-yl)-(biphenyl-3'-yl)amine hydrochloride

A mixture of 4-chloro-8,9-dihydro-7H-[l,4] dioxepino [2,3 -g] quinazoline (intermediate 12) (0.1 g, 0.5 mmol) and 3 -phenylaniline (84.6 mg, 0.5 mmol) in /- PrOH (2 mL) was microwave irradiated at 80 °C (power set point 60 W; ramp time 1 min; hold time 15 min). After cooling, the obtained precipitate was collected by filtration to give the title compound (0.2 g, yield 89%); mp > 300 °C; ^XH-NMR (300 MHz, DMS0-c/₆) : δ 10.73 (broad s, IH, NH], 8.78 (s, IH, 2 -H), 8.39 (s, IH, 5-H or 11- H), 8.06 (s, IH, H_ar), 7.82 -7.66 (m, 3H, H_ar), 7.59-7.46 (m, 4H, H_ar), 7.45-7.37 (m, IH, H_ar), 7.35 (s, IH, 5-H or 11-H), 4.42 (t, / = 5.9 Hz, 2 H, OCH₂CH₂CH₂0), 4.35 (t, / = 5.9 Hz, 2H, OCH₂CH₂CH₂0), 2.27 (quint, / = 5.4 Hz, 2H, OCH₂CH₂CH₂0). Anal, calcd. for C₂₃H₂₀ClN₃O₂: C, 68.06; H, 4.96; CI, 8.73; N, 10.35; found: C, 68.09; H, 4.95; CI, 8.73; N, 10.37. HRMS (ESI-TOF) for C₂₃H₂₀N₃O₂ [M + H]⁺: calcd.: 370.1550, found: 370.1521. Biological Data Compounds of the invention may be tested for in vitro and in vivo biological activity in accordance with the following assays:

1. In vitro evaluation of the inhibitory effects on isolated tyrosine kinases

Examples 1, 2 and 3 were tested in vitro at 0.1 μΜ to determine the inhibitory effect on selected tyrosine kinases (EGFR, FGFR-1, VEGFR-2, PDGFR-β, src and abl). Intracellular catalytic domains of tyrosine kinases were solubilized and then diluted in dilution buffer (5mM MOPS pH 7.2, 2.5 mM glycerol 2 -phosphate, 5 mM MgC12, 0.4 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT, 0.5 mM BSA). The final reactions were executed in pre-cooled microcentrifuge tubes. To the active kinases (final concentration 200 ng/ml) the Examples 1, 2 and 3 were added as well as 0.2 mg/ml of substrate solution (Myelin Basic Protein), 0.05 mM ATP and 0.25 μα of [γ- ³²P]ATP [PerkinElmer, Monza, MI) (final volume of the reaction mixture 25 μί,). Negative controls were prepared replacing the substrate solution with water whereas positive controls were set up replacing inhibitors tested with water. Reactions were carried out at 30°C for 20 min and stopped by addition of loading buffer containing 0.25 mM β-mercaptoethanol. Samples were then subjected to electrophoresis on 10% w/v SDS-PAGE gel. Gels were dried, and phosphorylated myelin basic protein was identified by autoradiography. The VersaDoc Quantity One software (BioRad) was used for densitometric analysis. Data were collected in Table 1.

Tumor growth and angiogenesis is supported by several growth factors, such as basic Fibroblast Growth Factor (bFGF), Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF) and Epithelial Growth Factor (EGF), that induce cell proliferation through the binding with transmembrane tyrosine kinases (or Receptor Tyrosine Kinases). Thus, the blockade of these receptors lead to an inhibition of cell growth. The data show that the Examples 1, 2 and 3 are able to inhibit a large panel of kinases demonstrating their ability to act as multi-kinase inhibitors. Inhibition of purified kinases

Compound

VEGFR- s EGFR FGFR-1 PDGFR-β src abl

2

1 M (+) (+) M (+) (+)

2 (-) M (-) (+) (+) (+)

3 M (+) (-) (+) M (+)

Table 1. Kinase inhibition profile of Example 1, 2 and 3 . (+) = IC50 equal or less than 1.0 μΜ; (-) = IC₅₀ more than 1.0 μΜ. 2. Evaluation of effects on cell proliferation

The 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT)- tetrazolium dye assay [Denizot F., Lang R., /. Immunol. Methods, 1986, 89, 271-277; Wemme H. et al., Immunobiology, 1992, 185, 78-89] was carried out to evaluate the antiproliferative potential of compounds. This assay determines the ability of the Examples to kill the cells.

The cells were seeded into a 96-well plates [Becton Dickinson Falcon) using complete medium (150 \iL for each well). After a 24 h incubation period at 37°C, media were replaced with ones containing various concentrations (ranging from 0.01 to 10 μΜ, 200 \iL for each well) of Examples 1, 2, and 3. Control cultures were represented by non treated cells. After a 68 h incubation period at 37°C, 20 \iL of 5 mg/mL MTT [Sigma) in PBS were added to each well and cultures were incubated for 4 h. The purple-coloured formazan crystals were dissolved in 75 \iL acid isopropanol [Carlo Erba) shaking the plate for 20 min in the dark. The optical densities were measured on Microplate autoreader EL 13 a 570 nm. The results were expressed as EC50 (the concentrations of the compounds which induced a 50% reduction of viability in comparison with non treated cultures) and shown in Table 2. The results indicate that Examples 1, 2, and 3 at very low concentrations (μ ) induce antiproliferative effects on tumor cell lines and human umbilical vein endothelial cells (HUVECs). Compoun Cell lines

d A431 NIH3T3 HUVEC MCF-7 HT-29 HeLa HepG2

0.81 +0.0 0.60±0.0 0.75±0.0 0.77±0.0 0.95±0.0 0.77±0.0

1 >10

6 2 1 2 6 2

0.08±0.0 0.60±0.2 0.08±0.0 0.74±0.0 0.68±0.3 0.62±0.0 2.10±0.3

2

1 0 1 6 6 1 9

0.66±0.0 0.92±0.0 0.91 +0.0 0.70±0.1 0.71±0.0 5.49±0.6

3 >10

8 6 6 1 1 2

Table 2. Cytotoxicity of Examples 1, 2 and 3 determined on various cell lines by MTT assay. The results are expressed as EC50 (μΜ) ± S.D.

3. Evaluation ofapoptosis rate on A431 cell line

The Cell Death Detection ELISA ^PLUS [Roche) was used to determine the apoptosis rate in cell cultures treated with 10 μΜ Examples 1, 2 and 3 . The assay was carried out according to the manufacturer's instructions. The results, shown in Figure 1 and mean of at least three separate experiments, were expressed as percent change from control non treated cultures. Their statistical comparison was performed by analysis of variance, followed by Student's t-test. After an incubation period of 24 h, all compounds significantly increases the apoptotic rate respect to control not treated cultures.

These results indicate that the cytotoxicity of the compounds is related to a proapototic activity. The Examples 1, 2 and 3 kill the cells by inducing a programmed death process (apoptosis) and not necrosis (that leads to inflammatory responses). 4. Assessment ofFGF-2 release from HUVECs

The kit Human FGF basic Immunoassay [Quantikine) was used to evaluate the inhibitory effects of 0.1 μΜ Examples 1, 2 and 3 on FGF-2 release. The assay was carried out according to the manufacturer's instructions. The results, shown in Figure 2 and mean of at least three separate experiments, were expressed as pg/mL FGF-2. Their statistical comparison was performed by analysis of variance, followed by Student's t-test. Angiogenesis leads to formation of new vessels from the preexisting ones and is tightly regulated in physiological conditions. An impairment of this process is involved in several pathological conditions. An excess of angiogenesis is characteristic of tumor and is responsible of tumor growth and metastasis, because blood vessels feed tumor cells that then enter the blood flow and colonize other tissues and organs. Furthermore, tumor cells together with endothelial cells produce large amount of proangiogenic factor, such as VEGF and FGF-2. All compounds significantly decrease FGF-2 release respect to that determined in control non treated cultures. These results indicate that the antiangiogenic effects (see below) of Examples 1, 2 and 3 may be due to their interaction with TKs and also to an inhibitory effect on the release of FGF-2. 5. Evaluation of effects on in vitro morphogenesis

Morphogenesis analysis was carried out seeding HUVECs on Matrigel [B&D Biosciences). Matrigel was thawed on ice overnight, spread evenly over each well (50 μί,) of a 24-well plate [Falcon), and allowed to polymerise for 1 h at 37°C. HUVECs (7,5xl0⁴/l mL for each well) were seeded on Matrigel and cultured in basal medium supplemented with 1% FCS [Promocell) and Examples 1, 2 and 3 at non cytotoxic concentration (0.1 μΜ), as determined by MTT assay (data not shown). After 18 h of incubation at 37°C and rinsing with PBS, cultures were fixed with 2% glutaraldehyde [Merck) in 0.1 M cacodylate buffer [Sigma), pH 7.2 [Albini A. et al, Int. J. Dev. Biol., 2004, 48, 563-571]. Samples were then photographed (5 fields for each well: the four quadrants and the center) at a x50 magnification. Phase contrast images were recorded using a digital camera and image analysis was carried out using the ImageJ-Matrigel Assay software. The following dimensional parameters (percent area covered by HUVECs, total length of HUVEC network per field and percent area inside the meshes) and topological parameters (number of meshes, and branching points per field) were estimated. The results, mean of at least three experiments, were expressed as percent change from control non treated cultures. The statistical analysis was performed by analysis of variance, followed by Student's t-test. The results are shown in Figure 3. Angiogenesis is a multi step process. In the first step the basement membrane is degradated by proteolytic enzymes, such as matrix metalloproteinases (MMPs) and plasminogen activators, to allow the migration of endothelial cells (ECs) into the perivascular stroma. The angiogenic stimuli activate ECs that proliferate and then organize into tubular structures leading to the development of a circulatory network. Finally, neo-vessels are stabilized by the perivascular apposition of smooth muscle cells and pericytes. The morphogenesis assay evaluate the capacity of compounds to affect the formation of tubular structures. The results indicate that the compounds exert antigiogenic activity by inhibiting this step of the angiogenic process. Indeed, Example 2 significantly decreased all dimensional and topological parameters, whereas Examples 1 and 3 affected only the number of meshes and percent area covered by meshes.

The assay was also carried out using reduced Matrigel and seeding the cells with 1 mL basal medium supplemented with 1% FCS, 50 ng/mL FGF-2 (Figure 4A), FGF-2 plus Examples 1, 2 and 3 (Figure 4B), 20 ng/ml VEGF (Figure 5A), and VEGF plus Examples 1, 2 and 3 (Figure 5b). The Examples were added at non cytotoxic concentration (0.1 μΜ). The results, mean of at least three experiments, were expressed as percent change from control non treated cultures. The statistical analysis was performed by analysis of variance, followed by Student's t-test. Only Example 2 was able to abolish the increase in cell proliferation induced by FGF-2 and VEGF. The increases in both dimensional and topological parameters induced by FGF-2 were reversed by Examples 1 and 2, whereas Example 3 affected the number of branching points and meshes, and percent area covered by meshes. The stimulatory effects induced by VEGF on in vitro morphogenesis were almost completely abolished by all Examples. These results indicate that the compounds are able to counteract the angiogenesis induced by FGF-2 and VEGF, well known proangiogenic factors contained in the tumor microenvironment. 6. In vivo evaluation of antiangiogenic effects

Three-month-old C57/BL6 mice were anesthetized with isoflurane (2-3%) via nose cone and 100% oxygen used as the carrier gas; the back was shaved and disinfected with ethyl alcohol. A total of 0.5 mL of Matrigel, mixed with 12 U.I. heparin (Vister) with or without 200 ng/plug FGF-2 [Sigma] and ΙμΜ Examples 1, 2 and 3, was injected subcutaneously on dorsal area. After injection, the Matrigel polymerized forming a plug. After 7 days, the animals were sacrificed and the plugs were carefully removed.

Some plugs were fixed with 4% formaldehyde [Sigma] and then dehydrated through exposition to ethanol (70%, 80%, and 90% for 2 h, 95% overnight, 100% for 2 h). Samples were treated with xilene/100% ethanol (1:1, v/v) for 2 h, xilene for 2 h, and embedded in paraffin at 60°C for 2 h. Paraffin blocks were cut with a microtome [Histoslide 2000, Reichert-Jung] at 5 microm. Sections were mounted onto SuperFrost Plus [Menzel-Glaser] slides and deparaffinized through 2 changes of Histochoice Clearing Agent IX, 5 min each. After hydratation in 100%, 95%, 80%, 70%, 50%, 30%, and 10% ethanol for 5 min each and rinsing in distilled water, samples were stained with haematoxylin and eosin [Merck]. Sections were treated with hematoxylin for 2.5 min, washed with distilled water, treated with tap water for 2 min and then with eosin for 10 sec. After rinsing with distilled water, dehydratation in 95% and absolute alcohols, and clearing Histochoice Clearing Agent IX, sections were mounted with Histochoice Mounting Media. The micrographs, shown in Figure 6, were taken using an optical microscope (DM2000, Leica] connected with a digital camera.

Alternatively, plugs were steeped in 300 μί/ρΚ¾ Brij-35 0.1% solution in PBS at 4°C overnight. Haemoglobin concentration was analyzed using Drabkin's Reagent kit (Sigma). A calibration curve was made to determine haemoglobin content of plugs. A cyanmethemoglobin standard solution (10 mg/mL) was prepared in Drabkin's Solution (1 vial of Drabkin's Reagent in 1 L distilled water containing 0.5 mL of 30% Brij-3S Solution]. Then, seven dilute cyanmethemoglobin standard solutions (0,25; 0,5; 1; 2; 4; 8; 10 mg/mL) were obtained. The optical density was read at 540 nm using a Microplate autoreader EL 13. A calibration curve of absorbance values versus the cyanmethemoglobin concentration (mg/mL) was plotted and used to determine the values of plugs. The results, mean of three plugs, were expressed as mg/mL haemoglobin. The statistical analysis was performed by analysis of variance, followed by Student's t-test. The results are shown in Figure 7. Histological analysis revealed the decrease in number and size of new vessels in plugs containing the Examples 1, 2 and 3 compared to those with only FGF-2. Consistent with these observations, also the haemoglobin concentration was lowered by the Examples 1, 2 and 3. These results indicate that the Examples 1, 2 and 3 are able to counteract FGF-2 -induced angiogenesis.

Claims

CLAIMS:

1. A compound of formula (I)

wherein

R is hydrogen or C^.4 alkyl;

n is an integer from 1 to 5;

X and Y are each independently an oxygen or a sulphur atom;

R^ is trifluoromethyl, a alkyl, a alkoxy, a trifluoromethoxy or a halogen group;

m is zero or an integer from 1 to 3; or salts thereof.

2. A compound of formula (I) as claimed in claim 1, in the form of a pharmaceutically acceptable salt thereof.

3. A compound of formula (I) as claimed claim 1 or 2, wherein R is hydrogen; n is an integer from 1 to 3; X and Y are oxygen; m is zero.

4. A compound of formula (I) which is:

([l,3]Dioxolo[4,5-g]quinazolin-8-yl)-(biphenyl-3'-yl)amine hydrochloride;

(7,8-Dihydro[l,4]dioxino[2,3-,g]quinazolin-4-yl)-(biphenyl-3'-yl)amine

hydrochloride;

(8,9-Dihydro-7H-[l,4]dioxepino[2,3-g]quinazolin-4-yl)-(biphenyl-3'-yl)amine hydrochloride.

5. A pharmaceutical composition comprising a compound of formula (I) as claimed in any one of claims 1 to 4, or a pharmaceutically acceptable salt, and one or more pharmaceutically acceptable excipients.

6. A compound of formula (I) as claimed in any of claims 1 to 4 or a pharmaceutically acceptable salt, for use in therapy.

7. A compound of formula (I) as claimed in any of claims 1 to 4, or a pharmaceutically acceptable salt, for use in treating or preventing a disease or condition mediated by tyrosine kinases.

8. Use of a compound of formula (I) as claimed in any of claims 1 to 4, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of conditions mediated by tyrosine kinases.

9. A method of treatment or prophylaxis of conditions mediated by tyrosine kinases in mammals including humans, which comprises administering to the sufferer a therapeutically effective amount of a compound of formula (I) as claimed in any of claims 1 to 4, or a pharmaceutically acceptable salt thereof.

10 A process for preparing a compound of formula (I) as claimed in any of claims 1 to 4, which com rises

(III) reacting a compound of formula (II), wherein W is a suitable leaving group such as chloro, X, Y and n are as defined in formula(I), with a phenyl aniline derivative of formula (III), wherein R, Rl and n have the meanings defined in formula(I), in a suitable solvent such as alcohol at a temperature ranging from 20-100°C, to obtain compounds of formula(I), followed where desired by isolation of the compound as salt thereof.