ITPD20110091A1 - USEFUL INHIBITORS FOR RELATED PATHOLOGIES: PHARMACOFORIC MODELS, IDENTIFIED COMPOUNDS BY THESE MODELS, METHODS FOR THEIR PREPARATION, THEIR FORMULATION AND THEIR THERAPEUTIC USE. - Google Patents

Description

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to: the discovery and generation of a three-dimensional pharmacophore for the identification of multiple tyrosine kinase inhibitors; the synthesis of the compounds identifiable by this pharmacophore; to the applications in the pharmaceutical and / or clinical field of the same compounds identified. The present invention therefore also relates to the preparation of pharmaceutically acceptable salts of the molecules object of the invention and to pharmaceutical preparations containing at least one of the molecules object of the invention.

STATE OF THE ART:

Each bibliographic reference cited here must be understood as a set of the reference itself and all the bibliographic references cited therein.

Alterations in cell cycle regulation systems can lead to the onset of hyperproliferative diseases such as cancer [Evan G.I., Vousden K.H. "Proliferation, celi cycle and apoptosis in cancer", Nature, 2001, 411, 342-348]. Among the various proteins involved in the reception, transduction and amplification of cellular signals, a role of fundamental importance is played by tyrosine kinases, hereinafter referred to as TC. These enzymatic proteins catalyze the transfer of a phosphate residue from an adenosine triphosphate (ATP) molecule to a tyrosine residue present in their protein target. In this way, the phosphorylated protein is activated and is in turn able to phosphorylate an additional target protein, or to activate transcription mechanisms of deoxyribonucleic acid (DNA), leading to cell replication and arrest of death processes. programmed cell (or apoptosis) [Lenninger A.L.

et al, "Principles of Biochemistry", ed. Zanichelli]. Both membrane (or receptorial, hereinafter referred to as TCR) and cytoplasmic CT (hereinafter referred to as TCC) have been identified. Among the TCRs, the receptor for the epidermal growth factor (EGFR), the receptor for the growth factor of fibroblasts type 1 (Fibroblast Growth Factor Receptor-1, FGFR-1), were particularly important. Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and Platelet-Derived Growth Factor Receptor-beta , PDGFRP). Among the TCCs, abl and src were particularly important. TCRs are transmembrane receptors whose activation occurs following binding to specific protein structures, called growth factors [Hubbard S.R., Miller W.T, "Receptor tyrosine kinases: mechanism of activation and signaling", Curr. Opin. Cell Biol. 2007, 19, 117-123]. The activation of TCRs causes the dimerization of the TCRs themselves and their autophosphorylation. Following autophosphorylation, dimerized TCRs are able to transduce the proliferative signal, mainly by phosphorylation of a TCC. In cells under physiological conditions (i.e. not subject to aberrant genetic mutations or in non-pathological conditions), the activation of TCRs is tightly and finely regulated. In this way, the correct balance between cell proliferation and apoptosis is maintained. On the contrary, in almost all tumor forms, both solid and liquid, an overexpression or hyperactivity of different CTs was found [Baselga J., Arribas J., "Treating cancer's kinase 'addiction'", Nat. Med., 2004, 10, 786-787]. Over the last few years, therefore, we have witnessed the study and development of various techniques for the inhibition of CT. In particular, three different therapeutic strategies have been identified potentially applicable in the treatment of those pathologies involving tyrosine kinase hyperactivity, such as cancer, metastatic phenomena, diseases associated with hypervascularization, rheumatoid arthritis, and other pathologies [Mendelsohn J. , "Targeting the Epidermal Growth Factor Receptor for cancer therapy", J. Clin. Oncol., 2002, 20, ls-13s]:

to. use of antisense oligonucleotides and ribozymes, aimed at inhibiting the cell's production of new CTs;

b. use of monoclonal antibodies, directed to the inhibition of the binding between the growth factor and the relative TCR;

c. use of small organic ATP-mimetic molecules (TC inhibitors, hereinafter referred to as ITC), aimed at inhibiting the catalytic activity of TC.

The considerable attention paid to ITCs is demonstrated by the considerable number of patents filed concerning the use of the same ITCs. By way of example, EP0795556 describes pyrrolopyrimidines as ITC; WO97 / 34876 describes cinnolines as inhibitors of VEGFR in its various isoforms; US5747498 discloses 4-anilinoquinazoline derivatives as EGFR inhibitors; WO001 / 00207 describes pyrimidines as ITC; WO1 / 55114 describes aminonicotinamides as inhibitors of VEGFR in its various isoforms; WO02 / 22598 describes quinoline derivatives as inhibitors of VEGFR in its various isoforms; WO04 / 005281 discloses pyrimidinylaminobenzamides as ITC; WO 08/016192 discloses condensed heterocyclic derivatives such as ITC.

Since the activation of multiple oncogenic pathways are normally involved in the development of tumor diseases, the use of highly selective drugs (monoclonal antibodies and highly selective ITCs) can lead to the rapid onset of drug resistance and selection phenomena. of mutated cell strains [Petrelli A. and Giordano S., "From single- to multi-target drugs in cancer therapy: when aspecificity becomes an advantage", Curr. Med. Chem., 2008, 15, 422-432]. Consequently, therefore, the effectiveness of the chemotherapeutic agent used is no longer effective. The onset of drug resistance leads to a significant reduction in the patient's life expectancy. A considerable increase in the efficacy of the anti-cancer therapeutic protocol can, therefore, be achieved only through the use in therapy of combinations of drugs with different mechanisms of action or through multiple inhibitors, i.e. chemotherapeutic agents capable of simultaneously inhibiting the action of several protein or nucleic structures involved in cellular hyperproliferation. Compared to combined therapy with multiple drugs with specific action, the same result could be achieved with the use of multiple inhibitors, but with a lower incidence of side effects. Due to their intrinsic mechanism of action, monoclonal antibodies cannot act as multi-target drugs, and therefore can only be used in combination therapies. Furthermore, only monoclonal antibodies active against TCRs, but not TCCs, can be detected. Finally, being drugs of a protein nature, monoclonal antibodies cannot be taken orally, and therefore these are currently administered only by injection in a hospital setting, with a significant decrease in patient compliance. For this reason, it is evident the importance of identifying multiple-action ITCs (hereinafter referred to as IMTC), i.e. molecules capable of simultaneously and effectively inhibiting multiple TCs, both RTC and TCC, which can be administered, depending on the case, both via oral and injecting. To date, some dual-acting inhibitors have been identified, such as Vandetanib [Wedge S.R. et al, "ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following orai administration", Cancer Res., 2002, 62, 4645-4655], Imatinib [Buchdunger E. et al, "Abl protein-tyrosine kinase inhibitor STI517 inhibits in vitro signal transduction mediated by c-kìt and platelet-derived growth factor receptors, J. Pharmacol. Exp. Ther., 2000, 295, 139-145] and Sunitinib [Abrams T.J. et al, "SU11248 inhibits KIT and platelet -derived growth factor receptor beta in preclinical models of human small celi lung cancer ", Mol. Cancer. Ther., 2003, 2, 471-478]. These molecules have shown a markedly higher therapeutic efficacy than selective ITCs (ie capable of inhibiting only one CT), such as erlotinib and gefitinib.

Multiple kinase inhibitors are reported, for example, in the patents W02007 / 018137, which presents diarylureide derivatives capable of inhibiting Raf and / or a CT involved in angiogenesis, and WO2009 / 015368, which presents macrolide derivatives as inhibitors of src and MEK , but in both cases they are inhibitors that only act on two kinases. However, the number of IMTCs developed so far is significantly lower than the number of selective ITCs. This is mainly due to the fact that so far only chance has led to the discovery of molecules endowed with multi-tyrosine kinase inhibitory activity.

The substantial technical problem is, therefore, to date the lack of an adequate tool that allows to rationally design multithyrosinkinase inhibitors. The scientific community and the whole community feel the need to make increasingly effective pharmaceutical compositions available for the therapeutic treatment of all pathological states or disorders that can be treated with molecules having inhibitory activity against several tyrosine kinases, which also do not present the negative effects of selective tyrosine kinase inhibitors currently in use in therapy.

A first object of the present invention is to provide an adequate instrument (i.e. a three-dimensional pharmacophore) which describes the steric and electronic characteristics of the different parts of a molecule and their relative positions in space necessary for the molecule itself to inhibit more efficiently. of a tyrosine kinase involved in tumor pathology, preferably chosen from the following: EGFR, VEGFR-2, FGFR-1, PDGFRβ, abl and src. Another object of the present invention is to identify chemical structures attributable to this three-dimensional pharmacophore, and therefore endowed with IMTC activity, and to describe their synthesis. It is a further object of the following invention to describe for some compounds object of the present invention, taken as an example, the inhibitory capacity towards a CT panel. It is another object of the present invention to describe for the compounds, taken as an example, the efficacy as inhibitors of angiogenesis. Finally, a further object of the present invention is to make available new chemotherapeutic agents, their pharmacologically acceptable salts and the pharmaceutical forms obtainable therefrom, for use in pathologies associated with alterations of tyrosine kinase activity, such as preferentially but not exclusively cancer.

DEFINITIONS:

Pharmacophore: set of zones of space and relative distances between the zones themselves, characterized by certain steric and electronic properties, necessary for an optimal interaction with a specific biological target, such as to evoke a biological response.

IC50: concentration of inhibitor at which there is a 50% reduction in enzymatic activity compared to the maximum activity of the enzymatic system itself, placed in the same conditions but in the absence of inhibitors.

EC50: concentration of inhibitor at which there is a 50% reduction in cell viability with respect to the maximum activity of the cellular system itself, placed in the same conditions but in the absence of inhibitors.

Multiple inhibitors: compounds capable of simultaneously inhibiting biological targets different in function, and / or location, and / or structure. Inhibition, indicated by the value of IC50, must occur at concentrations less than or equal to 1 μΜ.

Multityrosine kinase inhibitors: compounds capable of simultaneously inhibiting different types of tyrosine kinases. In particular, compounds capable of inhibiting at least 4 tyrosine kinases among: EGFR, VEGFR-2, FGFR-1, PDGFRp, sre and abl are considered multityrosine kinase inhibitors. Inhibition, indicated by the value of IC50, must occur at concentrations less than or equal to 1 μΜ.

Virtual screening: any process or set of chemoinformatics processes capable of selecting compounds with specific biological activity from molecular databases.

Rational design: any technique not based on chance that allows to identify molecules with fundamental structural requirements to impart a specific biological activity.

OBJECT OF THE INVENTION:

In a first aspect, the present invention relates to a method for the generation of a pharmacophore and its use for the identification of multityrosinkinase inhibitors, defined in accordance with what is reported in the "definitions" section of the corresponding item. This discovery responds to the increasingly pressing need for an effective method to be made available to the community for the identification of new compounds capable of simultaneously and effectively inhibiting several tyrosine kinases. Thanks, in fact, to the use of the compounds according to the invention, it will be possible to prepare pharmaceutical compositions useful in the treatment of pathologies associated with alterations of the tyrosine kinase activity, including preferentially but not exclusively tumor pathologies, with a reduced probability of incurring in phenomena of drug resistance during the course of treatment.

The pharmacophore described in the present invention was unexpectedly generated by the inventors by means of a chemoinformatic process consisting of two sequential steps, using the UCSF Chimera version 1.5.2 and AutoDock version 4.2 software, identifying the set of steric and electronic characteristics that a molecule must possess to be able to efficiently insert within the pocket for ΙΆΤΡ of tyrosine kinases such as EGFR, VEGFR-2, PDGFRP, FGFR-1, abl and sre, simultaneously in at least four of them.

The pharmacophore, represented in Figure 1, is made up of:

to. an area intended to allocate an aromatic portion, not substituted or variously substituted, and called Ari;

b. an area intended to allocate an aromatic portion, not substituted or variously substituted, and called Ar2, the center of which is located at a distance from the center of Ari between 4.5 and 6.5 A;

c. an area intended to allocate a carbocyclic or heterocyclic ring, not substituted or variously substituted, and called Ar3, whose center is located at a distance from the center of Ari between 6.5 and 8.5 A and which at the same time is at a distance from the center of Ar2 between 4.0 and 5.0 A;

d. a zone for acceptor nitrogen atoms of hydrogen bridges, which constitutes a structural part of Ari, and indicated as NA;

And. a spacer, referred to as a linker, which joins Ari and Ar2 and which determines the distance reported above between Ari and Ar2;

f. a zone X for functional groups containing at least one electron-rich atom, the center of which is at a distance from the center of Ari between 4.5 and 6.5 A and which at the same time is at a distance from the center of Ar2 between 6.5 and 8.5 A and which at the same time it is at a distance from the center of Ar3 between 10.5 and 12.5 A.

In a preferential aspect of the invention, Ari is represented by an unsubstituted or variously substituted aromatic hexatomic ring, containing at least one hydrogen bridge acceptor nitrogen atom.

In another preferential aspect of the invention, Ar2 consists of a carbocyclic or heterocyclic aromatic pentatomic or hexatomic ring, unsubstituted or variously substituted.

In a further preferential aspect of the invention, the linker consists of a chain of 1 or 2 atoms chosen from nitrogen, oxygen, sulfur, selenium, silicon, phosphorus or carbon or combinations thereof.

In another preferential aspect of the invention X contains at least one electron-rich atom selected from oxygen, sulfur and nitrogen.

In a further preferential aspect of the invention, Ar3 consists of a pentatomic or hexatomic carbocyclic or heterocyclic ring, not substituted or variously substituted. Once the pharmacophore has been generated, it can be used as an input for querying a three-dimensional molecular database, in which each compound is contained as a set of plausible conformers, whose energies are between the minimum energy value and 20 Kcal / mole in more than the energy minimum itself [Smelile G. et al. "Analysis of Conformational Coverage. 1. Validation and Estimation of Coverage", J.ChemJnf.Comput.Sci., 1995, 35, 285-294; Smelile G. et al. "Analysis of Conformational Coverage. 2. Application of Conformational Models", J.ChemJnf.Comput.Sci., 1995, 35, 295-304]. For the screening of the database, and therefore for the identification of the molecules that reflect the characteristics described by the pharmacophore, it will be possible to use any commercially available or specially created software capable of supporting pharmacophoric research and the screening of molecular databases.

Alternatively, the pharmacophore coincidence analysis can be performed with simple molecular design software following the following procedure:

the. The molecular structure of interest is drawn and its three-dimensional molecular structure is generated at an energetic minimum;

ii. For each molecule a sufficient number of conformers is generated to guarantee the exploration of the conformational space, whose energy is between that of the structure at the minimum energy and 20 Kcal / mol more than the minimum energy itself;

iii. In each conformer the potential characteristics in common with the pharmacophore are identified;

iv. The distances between the areas highlighted in point iii) are measured using the measuring instruments made available by the software itself.

A further preferential object of the present invention are the compounds identified through the use of the pharmacophore, which can be summarized in the general formula (1),

(1)

in which:

- A is independently selected from a CH group or an oxygen atom or a sulfur atom or a selenium atom or a nitrogen atom or an NH group or an NRi group, where Ri is independently selected from an alkyl or cycloalkyl residue or acyl or aromatic or heteroaromatic, where the heteroatom is another nitrogen atom;

B and D are independently selected from a carbon atom or a nitrogen atom;

E can be absent and, if present, has the same meaning as defined for A;

F and G have the same meaning defined for B and D;

- X and Y are independently selected from an electron-rich atom such as an oxygen atom or a sulfur atom or a nitrogen atom;

R2 and R3 have the same meaning defined for Ri and can be joined together forming a further ring that includes the structural elements indicated with X-F-G-Y.

- All bonds between atoms or groups A, B, D, E, F, G, X, Y, R2 / R3 can be independently single or double.

Some of the possible compounds meeting the requirements of the general formula (1) are preferentially specified in the following list, and can be independently understood both as free bases and as corresponding pharmaceutically acceptable salts:

([li3] dioxol [4i5-g] quinazolin-8-yl) - (m-biphenyl-3'-yl) amine (I).

(7,8-dihydro [li4] dioxine [2i3-g] quinazolin-4-yl) - (/ T7-biphenyl-3'-yl) amine (II).

(8i9-dihydro-7H- [li4] dioxepino [2i3-g] quinazolin-4-yl) - (/ T7-biphenyl-3'-yl) amine (III).

In another aspect, the present invention relates to the synthesis of structures deriving from the use of the pharmacophore to identify multiple tyrosine kinase inhibitors. The compounds identified can be synthesized using the general methods reported in the literature. In particular, the derivatives with a quinazoline structure can be preferably synthesized according to the strategy reported by Marzaro G. et al. ["A novel approach to quinazolin-4 (3W) -one via quinazoline oxidation: an improved synthesis of 4-anilinoquinazolines", Tetrahedron, 2010, 66, 962-968].

To verify the inhibitory capacity on the tyrosine kinase activity of the compounds identified in the present invention, their biological evaluation was carried out on: i) a panel of 6 isolated kinases (EGFR, VEGFR-2, FGFR-1, PDGFR3, sre and abl), determining the ability to counteract the phosphorylation of a synthetic polytyrosine peptide;

ii) two cell lines, A431 (EGFR overexpressing) and NIH3T3 (non EGFR expressing), determining the cytotoxic activity and the reversibility of the cytotoxic effect;

iii) human umbilical cord vein endothelial cells (HUVEC) verifying the antiangiogenic effect in vitro,

iv) Matrigel plugs injected into C57 / BL6 mice, verifying the antiangiogenic effect in vivo. The results obtained showed that compounds I, II and III are capable of simultaneously inhibiting the activity of various isolated tyrosine kinases, evaluated as inhibition of phosphoric action.

They also demonstrate significant cytotoxicity, in the order of the sub-micromolar, on specific cell lines such as A431, which overexpress EGFR, and NHI3T3, which do not express EGFR; by using the Celi Death Detection ELISA <PLUS> kit (Roche) it was also possible to highlight that the antiproliferative effect is linked to a proapoptotic action.

Advantageously, in addition to the cytotoxic effect, the compounds have a notable antiangiogenic effect, inhibiting in vitro the proliferation of HUVEC, the release of FGF-2 and the formation of capillary-like structures on Matrigel.

The antiangiogenic activity is also maintained in vivo: in fact, the histological analysis of the Matrigel plugs implanted in mice revealed a reduction in the hemoglobin content and in the number and size of neo-vessels, compared to the control sample, in the fabrics treated with compounds I, II and III in the form of hydrochlorides.

A further aspect of the present invention relates to the use of the compounds obtainable by means of the invention itself for the treatment of pathologies associated with alterations of the tyrosine kinase activity, such as: brain, lung, squamous cell, pancreatic, pancreatic cancer. kidney, spleen, stomach, liver, esophagus, prostate, colorectal, neck, head, thyroid, ovary, breast, uterus, testicles, lymphoma and leukemia and their combinations; in the treatment of pathologies related to angiogenesis.

In accordance with what has been reported up to now, the present invention should also be understood as relating also to all the pharmaceutical compositions containing at least one of the compounds object of the invention and their pharmaceutically acceptable salts, for the treatment of the disorders listed above. The pharmaceutical compositions can be used as they are or in association with drugs or pharmaceutical preparations suitable for the treatment of the aforementioned pathologies. The obtainable pharmaceutical compositions may be suitable for oral, sublingual, intramuscular, subcutaneous, intravenous, epidural, topical, transdermal, rectal, intranasal administration. The pharmaceutical compositions that can be obtained may be immediate release, modified release, deposit, delayed release.

DESCRIPTION OF THE FIGURES

Figure 1. The figure describes the schematic structure of the three-dimensional pharmacophore object of the present invention.

Figure 2. The figure shows the distances detected in the compound (I) between the different areas of coincidence with those described in the pharmacophore.

Figure 3. The figure shows the pro-apoptotic effect of I, II, III hydrochlorides on A431 cells.

After 24 hours of treatment with the compounds (10 μΜ), the cells were used and the amount of oligo- and mono-nucleosomes was determined using an enzyme immunoassay. The results, mean of 3 experiments and expressed as percent change from untreated control cultures (considered as 0), are reported as mean ± standard deviation. * = p <0.05 vs control (untreated cultures); Student's t-test.

Figure 4. The figure shows the effects of I, II, III hydrochlorides on the release of FGF-2 into the culture medium by enzyme immunoassay. After 72 hours of treatment of the HUVEC cells with the compounds at a concentration of 0.1 μΜ, the culture medium was withdrawn and the cytokine concentration was determined. The results, mean of 3 experiments, were expressed as pg / mL of FGF-2 * = p <0.05 vs control; Student's t-test.

Figure 5. The figure describes the morphogenesis on HUVEC Matrigel treated for 18 h with the compounds at non-cytotoxic concentration (Ο.ΙμΜ) under analysis. The results, mean of 3 experiments, were expressed as mean ± standard deviation. * = p <0.05 vs control Student's t test.

Figure 6. The figure describes the morphogenesis on HUVEC Matrigel treated for 18 h with 50 ng / mL of FGF-2. The results, mean of 3 experiments, were expressed as mean ± standard deviation. * = p <0.05 vs control Student's t test.

Figure 7. The figure describes the morphogenesis on HUVEC Matrigel treated for 18 h with the compounds under analysis in concentration Ο.ΙμΜ and 50 ng / mL of FGF-2. The results, mean of 3 experiments, were expressed as mean ± standard deviation. * = p <0.05 vs control Student's t test.

Figure 8. The figure describes the morphogenesis on HUVEC Matrigel treated for 18 h with 50 ng / mL of VEGF. The results, mean of 3 experiments, were expressed as mean ± standard deviation. * = p <0.05 vs control Student's t test.

Figure 9. The figure describes the morphogenesis on HUVEC Matrigel treated for 18 h with the compounds under analysis in concentration Ο.ΙμΜ and 20ng / mL of VEGF. The results, mean of 3 experiments, were expressed as mean ± standard deviation. * = p <0.05 vs control Student's t test.

Figure IO. The figure shows the determination of the hemoglobin concentration present in the Matrigel plugs containing 200 ng / mL of FGF-2 and in the presence or absence of compounds I, II and III in a concentration equal to 1 μΜ. The plugs were removed seven days after implantation and the hemoglobin concentration was determined by a colorimetric assay using the Drabkin's Reagent kit. The results, mean of 3 experiments and expressed as mg / mL of hemoglobin, are reported as mean ± standard deviation. * = p <0.05 vs FGF-2; Student's t-test.

Figure 11. The figure shows the histological sections of the Matrigel plugs after explantation, stained with hematoxylin and eosin: only Matrigel (A), Matrigel added with: 200 ng / mL FGF-2 (B), FGF-2 and I 1.0 μΜ (C), FGF-2 and II 1.0 μΜ (D), FGF-2 and III 1.0 μΜ (E). Magnification x200.

DETAILED DESCRIPTION OF SOME PREFERENTIAL FORMS OF REALIZATION OF THE INVENTION

Purely by way of example, the inventors consider it useful to report a number of examples sufficient to demonstrate utility, applicability and advantages of the invention itself. These examples are intended only as specifications of the invention, without actually limiting the invention to them only. The person skilled in the art can reproduce the invention in its entirety in the light of what is described in the following sections, illustrated merely by way of non-limiting example of the invention itself.

Naturally, the invention is susceptible of modifications and variations, all of which are within the scope of the inventive concept expressed in the attached claims. All the phases, the characteristics, the compounds and the compositions can be replaced in an equivalent way, and the materials used can be different according to the requirements, without departing from the scope of protection of the invention itself.

Even if the invention is described with particular reference to the present detailed description of some preferred embodiments, this is intended for the sole purpose of improving the understanding of the invention itself and must not constitute any limitation to the scope of protection claimed.

METHODS

Example 1. Generation of the pharmacophore

The three-dimensional structures used for the first step of the pharmacophore generation and related to 57 non-covalent cocrystallized ligands with non-mutated forms of EGFR, VEGFR-2, FGFR-1, sre and abl were found to be available in the Protein Data Bank (identification codes of the structures : 1FPU, 1IEP, 1M52, 10PJ, 2GQG, 2HIW, 2HYY, 2HZN, 2QOH, 3CS9, 3GVU, 3HMI, 3IK3, 1M17, 1XKK, 2ITW, 2ITY, 2RGP, 3BEL, 3C4F, 1YQ8, 2BDHI, , 3EL7, 3EL8, 3EN4, 3EN5, 3EN6, 3EN7, 3F3T, 3F3U, 3F3V, 3F6X, 3G6G, 1Y6A, 1Y6B, 1YWN, 20H4, 2P2H, 2P2I, 2QU5, 2QU6, 2RL5, 3B8CQ2, 3BE8, 3B8CQ, 3B , 3CJG, 3CP9, 3CPB, 3CPC, 3DTW, 3EFL, 3EWH), at the internet address: http: //www.rcsb.ora/Ddb/home/home.do. From the crystallographic structures containing several identical chains, all but one chains have been eliminated, chosen independently from among all the chains containing the molecular structure of the inhibitor reported in the bibliographic reference relating to the crystallographic structure itself. For this purpose the UCSF Chimera software version 1.5.2 was used. With the same software, all the crystallographic structures were superimposed, following the instructions given in the user manual of the software itself. In particular, the alignment was obtained by applying the following options available in the "Tools" menu, "StructureComparison" submenu, "MatchMaker" submenu:

- Reference structure: any of the above structures;

- Structurefsl to match: all the above structures, with the exception of the structure used as a reference;

- Chain pairina: "Best-aligning pair of chains between reference and match structure"; - Alianment aloorithm: "Needleman-Wunsch";

- Matrix: "BLOSUM-62";

- Gap extension penalty: 1;

- Include secondarv structure score: option activated;

- compute secondarv structure assianments: option activated;

- show pairwise alianment (s): option disabled;

- iterate bv prunina Iona atom pairs untll no pair exceeds: 2.0 À.

After the superimposition of the reference structures, all the atoms with partial formal charge between -0.1 and -0.6 common to at least 80 ° / o of the ligand structures were selected, with a tolerance of 2.0 À. This selection operation was performed using the "Select" menu, "Render / Select by Attribute" submenu, using the following options:

- Models: all the above structures;

- choose attribute: formai charge;

- value: values indicated in the description above.

Then, all carbon atoms common to at least 80 <o> / o of the ligand structures were selected and added to the previous selection, with a tolerance of 2.0 À. This selection operation was performed using the "Select" menu, "Chemistry" submenu, "Element" submenu, "C" option, making sure that the "Selection Mode" option of the "Select" menu was set to "append" . Through visual inspection the pharmacophoric zones indicated in Figure 1 as Ari, Ar2, NA and linker were individualized. In a second step, using the AutoDock version 4.2 software, the positioning of the ligands contained in the above crystallographic structures were redesigned, following the general methodology reported in the tutorial of the program itself, available at the website http: //autodock.scripps. edu / faqs-help / manual / autodock-4-2-userauide / AutoDock4.2 UserGuide.pdf. In particular, for each ligand-tyrosine kinase pair a docking grid with dimensions of 20 A was generated in each direction of space and centered on the ligand itself. The grid spacing value was then set to 0.375 A. The "AutoGrid" component of the software was then made to calculate the relative default grids and the grids relative to the following atom types: carbon, aromatic carbon, nitrogen, bridge acceptor nitrogen hydrogen, oxygen. The Lemarkian Genetic Algorithm has been set as the docking protocol used by the "AutoDock" component with the default options selected. Once the molecular docking was performed, the affinity grids for carbon, aromatic carbon, nitrogen acceptor of hydrogen and oxygen bridges were visualized by means of the sequence of operations described in the program manual itself in the chapter "STEP 4: Evaluating the Results of a Docking ". All the grids calculated with energies between -0.4 and -0.8 Kcal / mole inside the catalytic pockets of EGFR, VEGFR-2, FGFR-1, sre and abl previously used were loaded within the same working section, highlighting the interaction zones of a potential ligand in common to all kinases. The two further pharmacophoric zones indicated in Figure 1 as X and Ar3 were then identified by visual analysis.

Example 2. Identification of multitlroslnchinaslcl inhibitors through the pharmacophore, without the aid of specific software for pharmacophoric research

The molecular structure of ([1,3] dioxol [4,5-g] quinazolin-8-yl) - (m-biphenyl-3'-yl) amine (I) was designed using the graphical interface of the MarvinSketch software version 5.3.5. The structure at absolute minimum energy was obtained using the "Calculate lowest Energy conformer" option, available in the "Tools" menu, "Conformation" submenu, "Conformers" submenu. Then, all conformers having energy between that of the structure at minimum energy and 20 Kcal / mol more were determined, using the "Tools" menu, "Conformation" submenu, "Conformers" submenu, using the following options:

- Store conformer Information in propertv field: option enabled:

- Calculate lowest enerav conformer: option disabled:

- Maximum number of conformers: 1000;

- Diversity limit: 0.1;

- Timelimit f yes: 900;

- Prehydrooenize: option activated:

- Hvperfine: option enabled.

All conformers with energy between that of the structure at minimum energy and 20 Kcal / mol more have been selected and introduced into a molecular database, then saved in ".mol2" format, ordering the molecular structures in increasing order of energy, expressed in Kcal / mole. Each molecular structure, therefore, was independently loaded into the MarvinSpace software version 5.3.5. Through visual analysis, the areas of the molecule potentially coinciding with those described in the pharmacophore were identified. Then, through the option "Place a pharmacophoric point on a selected atom", virtual spheres have been added to the molecular structure, representing the geometric center of the areas of the molecule potentially coinciding with those described in the pharmacophore. In the case of the areas called Ari, Ar2 and Ar3, after selecting the corresponding sphere, the positioning of the sphere itself was corrected using the "translate a ligand in the space" option, so that the sphere was found in the geometric center of the aromatic ring. By means of the "Measure distance of two atoms" option and selecting the pair of pharmacophore characteristics each time, the relative distances between them were determined (Figure 2). Since the distances already highlighted in the lower energy conformation were found to be consistent with what is reported in the pharmacophore object of the invention, the molecule (I) was selected as a multityrosinkinase inhibitor.

Example 3. Synthesis of (ri.3idlossolor4.S-a1chlnazolin-8-yl) - (m-blfenll-3'-11) amine (IL In the form of hydrochloride.

Ethyl chloroformate (6.9 ml, 72.9 mmol) was added to a solution of 5-aminobenzo [1.3] dioxolane (5.0 g, 36.5 mmol) in anhydrous tetrahydrofuran (440 ml) and triethylamine (10.2 ml, 72.9 mmol). The reaction mixture was left under stirring at room temperature for 30 minutes (TLC: chloroform / methanol, 9/1). The triethylamine hydrochloride formed was removed by filtration and the filtrate was concentrated to dryness, obtaining ethyl (benzo [1,3] dioxolan-5-yl) carbamate (IV) (quantitative yield) of oily consistency. ^ -IMMR (DMSO-c / 6): 9.47 (s all, 1 H, IMH); 7.11 (s, 1H, 4-H); 6.87-6.78 (m, 2H, 6-H and 7-H); 5.95 (s, 2H, OChUO); 4.09 (q, J = 7.1, 2H, COOCH, CH, V. 1.22 (t, J = 7.1, 3H, COOCH2CH3).

(IV)

A solution of ethyl (benzo [1,3] dioxolan-5-yl) carbamate (IV) (7.2 g, 34.0 mmol) and urotropine (4.2 g, 34.0 mmol) in trifluoroacetic acid (90 ml) was microwave irradiated at 80 W, reaching a temperature of 110 ° C in 30 seconds. This temperature was kept constant for 10 minutes by automatic regulation of power and cooling (TLC: chloroform / methanol, 9/1). After cooling, the reaction mixture was taken up with a 10% potassium hydroxide solution in 1/1 ethanol / water (1200 ml). Potassium hexacyanoferrate (III) (89.5 g, 272.0 mmol) was added to the mixture (pH> 10) and the suspension was refluxed for 4 hours (TLC: chloroform / methanol, 9/1). The reaction mixture was cooled, poured into water (1200ml) and extracted thoroughly with ethyl acetate (3 x 700ml). The organic phase was dried with anhydrous sodium sulphate and concentrated to dryness, obtaining [1.3] dioxol [4,5-g] quinazoline (V) (5.0 g, 83% yield) with m.p. 158 ° C. ^ -NM R (CDCI3): 9.12 (s, 1H, 8-H); 9.10 (s, 1H, 6-H); 7.29 (s, 1H, 4-H or 9-H); 7.12 (s, 1 H, 4-H 0 9-H); 6.17 (s, 2H, OCH20).

(V)

A solution of cerium ( IV) ammonium nitrate (6.4 g, 11.6 mmol) in water (13 ml). At the end of the addition (TLC: chloroform / methanol, 9/1), the precipitate formed was collected by filtration and washed with acetic acid, resulting in [1.3] dioxol [4,5-g] quinazolin -8 (7 ") - one (VI) (0.4 g, 68% yield), with m.p. > 300 ° C. ^ -NMR (DMSO-c / 6): 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,

To a suspension of [1.3] dioxol [4,5-g] quinazolin-8 (7 / Y) -one (VI) (0.8 g, 4.3 mmol) in phosphorus oxychloride (9.0 ml, 96.5 mmol) was added triethylamine (3 ml). The reaction mixture was refluxed for 3 hours (TLC: chloroform / methanol, 9/1). After cooling, the solution was concentrated to dryness and the solid residue was taken up with ethyl acetate (100 ml) and washed with a saturated sodium bicarbonate solution (100 ml). The organic phase was anhydrified with anhydrous sodium sulphate and concentrated to dryness, obtaining S-chloro-Hl ^ 1 dioxol ^ S-glquinazoline (VII) (0.6 g, yield 72%) with m.p. 168 ° C. ^ -NMR (CDCI3): 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, 0CH20).

(VII)

To a solution of 8-chloro - ([1,3] dioxol [4,5-g] quinazoline (VII) (0.1 g, 0.5 mmol) in isopropanol (5 ml) was added 3-phenylaniline (81 mg, 0.5 mmol). The reaction mixture was irradiated with microwaves at 60 W, reaching a temperature of 80 ° C in 30 seconds. This temperature was kept constant for 15 minutes by automatic regulation of power and cooling (TLC: chloroform / methanol, 9/1). After cooling, the reaction mixture was filtered to obtain ([1/3] dioxol [4/5-g] quinazolln-8-yl) - (m-biphenyl-3'-yl) amine hydrochloride (I) (0.2 g, yield 85%) with m.p.> 300 ° C. ^ -NMR (DMS 0-d6): 10.66 (s, 1 H, NH); 8.77 (s, 1 H, 6-H ); 8.18 (s, 1 H, 4-H or 9-H); 8.03 (s, 1 H, Harom); 7.78-7.67 (m, 3 H, Harom); 7.60-7.47 (m, 4 H, Harem ); 7.45-7.37 (m, 1 H, Harom); 7.30 (s, 1 H, 4-H or 9-H); 6.37 (s, 2 H, 0CH20).

Example 4. Synthesis of (7.8-dihydror 1.4 dioxinor2.3-a lchlnazolin-4-yl) -f m-biphenyl-3'-lliamine filaments. In the form of hydrochloride.

(Π)

Ethyl chloroformate (2.4 ml, 25.0 mmol) was added to a solution of 6-aminobenzo [1,4] dioxane (1.9 g, 12.5 mmol) in non-anhydrous tetrahydrofide (150 ml) and triethylamine (3.5 ml, 25.0 mmol). . The reaction mixture was left under stirring at room temperature for 30 minutes (TLC: chloroform / methanol, 9/1). The formed triethylamine hydrochloride was removed by filtration and the filtrate was concentrated to dryness. The solid residue was taken up with ethyl acetate and filtered under heat. The filtrate was concentrated to dryness, obtaining ethyl (benzo [l / 4] dioxane-6-yl) carbamate (VIII) (2.2 g, yield 77%) with m.p. 117 ° C. ^ -NMR (DMSO-ri6): 9.38 (s all, 1H, NH); 7.03 (d, J = 2.1, 1H, 5-H); 6.85 (dd, J = 8.7, J = 2.1, 1H, 7-H); 6.74 (d, J = 8.7, 1H, 8-H); 4.22-4.14 (m, 4H, OChbCH20); 4.08 (q, J = 7.1, 2H, COOCH2CH3); 1-21 (t, J = 7.1, 3H, COOCH, CHA

(Vili)

A solution of ethyl (benzo [1,4] dioxane-6-yl) carbamate (VIII) (5.0 g, 22.4 mmol) and urotropine (3.1 g, 22.4 mmol) in trifluoroacetic acid (70 ml) was irradiated with microwaves at 80 W, reaching a temperature of 110 ° C in 30 seconds. This temperature was kept constant for 10 minutes by automatic regulation of power and cooling (TLC: chloroform / methanol, 9/1). After cooling the reaction mixture was taken up with a solution of 10% potassium hydroxide in ethanol / water 1: 1 (800 ml). Potassium hexacyanoferrate (III) (59.0 g, 179.2 mmol) was added to the mixture (pH> 10) and the suspension was refluxed for 4 hours (TLC: chloroform / methanol, 9/1). The reaction mixture was cooled, poured into water (800 ml) and extracted thoroughly with ethyl acetate. The organic phase was anhydrified with anhydrous sodium sulphate and concentrated to dryness, obtaining 7,8-dihydro [1,4] dioxine [2,3-gichlnazoline (IX) (3.6 g, yield 86%) with m.p. 109 ° C. ^ -NMR (DMSO-tf6): 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, OCH2CH2O).

To a solution of 7,8-dihydro [1,4] dioxine [2,3-g] quinazoline (IX) (1.7 g, 9.1 mmol) in acetic acid (4 ml) was added slowly and under stirring at room temperature a solution of cerium (IV) ammonium nitrate (20.0 g, 36.4 mmol) in water (48 ml). At the end of the addition (TLC: chloroform / methanol, 9/1), the precipitate formed was collected by filtration and washed with acetic acid, resulting in 7,8-dihydro [1,4] dioxin [2, 3-g] quinazolin-4 (3 / f) -one (X) (1.1 g, yield 65%), with m.p. 282 ° C. ^ -NMR (DMSO-d6): 12.01 (s all, 1 H, 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, OCHzChbO).

OR

A suspension of 7,8-dihydro [1,4] dioxine [2,3-g] quinazolin-4 (3Ay) -one (X) (0.2 g, 1.0 mmol) in phosphorus oxychloride (1.4 ml, 10.6 mmol) is was irradiated with a microwave at 50W, reaching a temperature of 65 ° C in 30 seconds. This temperature was kept constant for 5 minutes by automatic regulation of power and cooling (TLC: chloroform / methanol, 9/1). After cooling, further phosphorus oxychloride (0.5 ml, 5.4 mmol) was added by subjecting the reaction mixture to another cycle of irradiation for 5 minutes (TLC: chloroform / methanol, 9/1). After cooling, the reaction mixture was taken up with a saturated sodium bicarbonate solution and thoroughly extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulphate and concentrated to dryness, obtaining 4-chloro-7,8-dihydro [1,4] dioxine [2,3-g] quinazoline (XI) (0.2 g, yield 70% ) with M.P. 216 ° C. ^ -NMR (CDCI3): 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, 4 H, OCH, CH, OT Cl

N (XI)

3-phenylaniline ( 76 mg, 0.5 mmol). The reaction mixture was irradiated with microwave at 60 W, reaching the temperature of 80 ° C in 30 seconds. This temperature was kept constant for 15 minutes by automatic regulation of power and cooling (TLC: chloroform / methanol, 9/1). After cooling the reaction mixture was filtered, obtaining (7,8-dihydro [1,4] dioxylno [2,3- <7] quinazolln-4-yl) - (m-biphenyl-3'-flamniine hydrochloride ( ZI) (0.2 g, yield 88%) with m.p.> 300 ° C. ^ -NMR (DMSO-d6): 11.01 (s, 1 H, NH); 8.82 (s, 1 H, 2-H); 8.34 (s, 1 H, 5-H or 10-H); 8.03 (s, 1 H, Haram); 7.7-7.66 (m, 3 H, Haram); 7.64-7.47 (m, 4 H, Harom); 7.44 -7.37 (m, 1 H, Harom); 7.31 (s, 1 H, 5-H or 10-H); 4.55-4.41 (m, 4 H, OChhChbO).

Example 5. Synthesis of ΐ 8.9-dihydra-7H-T 1.4 IdlasseainaT 2.3-a Tchinazaiin-4-ii) - (mbifenii-3'-ii) amine strands h in the form of hydrochloride.

Ethyl chloroformate ( 2.7 ml, 27.8 mmol). The reaction mixture was left under stirring at room temperature for 30 minutes (TLC: chloroform / methanol, 9/1). The formed triethylamine hydrochloride was removed by filtration and the filtrate was concentrated to dryness, obtaining ethyl (3,4-dihydro-2 // - 1,5-benzodioxepin-7-yl) carbamate (XII) (3.3 g, quantitative yield), with an oily consistency. ^ -NMR (DMSO -d6) \ 9.48 (s all., 1 H, NH); 7.12 (d, J = 2.4, 1H, 6-H); 7.00 (dd, J = 8.7, J = 2.4, 1H, 8-H); 6.86 (d, J = 8.7, 1H, 9-H); 4.13-4.01 (m, 6H, 2'-CH2, 4'-CH2 and COOCH2CH3); 2.05 (quint, J = 5.3, 2H, 3'-CH2); I.22 (t, J = 7.1, 3H, COOCH2CH3).

A solution of ethyl (3,4-dihydro-2Ay-1,5-benzodioxepin-7-yl) carbamate (XII) (3.3 g, 13.9 mmol) and urotropine (1.9 g, 13.9 mmol) in trifluoroacetic acid (42 ml) was irradiated with a microwave at 80 W, reaching a temperature of 110 ° C in 30 seconds. This temperature was kept constant for 10 minutes by automatic regulation of power and cooling (TLC: chloroform / methanol, 9/1). After cooling, the reaction mixture was taken up with a 10% potassium hydroxide solution in 1/1 ethanol / water (420 ml). Potassium hexacyanoferrate (III) (41.2 g, 125.1 mmoles) was added to the mixture (pH> 10) and the suspension was refluxed for 20 hours (TLC: ethyl acetate). The reaction mixture was cooled, poured into water (450ml) and extracted thoroughly with ethyl acetate (800ml). The organic phase was dried with anhydrous sodium sulphate and concentrated to dryness, obtaining 8,9-dihydro-7 / f- [1,4] dioxepine [2,3- <7] quinazoline (XIII) (2.5 g, yield 89%) of oily consistency. NMR (DMSO-cfe): 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 (t, J = 5.7, 2H, 7'-CH2 or 9'-CH2); 4.31 (t, J = 5.7, 2H, 7'-CH2 or 9'-CH2); 2.23 (quint, J = 5.7, 2 H, 8'-CH2).

To a solution of 8,9-dihydro-7Ay- [1,4] dioxepine [2,3-g] quinazoline (XIII) (2.5 g, 12.3 mmol) in acetic acid (5 ml) was added slowly and under stirring at room temperature a solution of cerium (IV) ammonium nitrate (27.0 g, 49.2 mmol) in water (60 ml).

At the end of the addition (TLC: chloroform / methanol, 9/1), the precipitate formed was collected by filtration and washed with acetic acid, resulting in the obtainment of 8,9-dlhydro-7H- [1,4] dioxepine [2,3-g] quinazolin-4 (3H) -one (XIV) (2.0 g, yield 73%), with m.p. 180 ° C. ^ -NMR (DMSO-c / e): 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, J = 5.6, 2H, 7'-CH2 or 9'-CH2); 4.22 (t, J = 5.6, 2H, 7'-CH2 or 9'-CH2); 2.17 (quint, J = 5.6, 2 H, 8'-CH2).

To a suspension of 8,9-dihydro-7 / V- [1,4] dioxepine [2,3-g] quinazolin-4 (3W) -one (XIV) (0.8 g, 3.7 mmol) in phosphorus oxychloride (7 ml) triethylamine (1.9 ml) was added. The mixture was refluxed for 3 hours (TLC: chloroform / methanol, 9/1). After cooling, the solution was concentrated to dryness and the solid residue was taken up with ethyl acetate (100 ml) and washed first with a saturated sodium bicarbonate solution (100 ml) and then with water (100 ml). The organic phase was dried with anhydrous sodium sulphate and concentrated to dryness, obtaining 4-chloro-8,9-dihydro-7 / Y- [1,4] dioxepine [2,3-gr] quinazoline (XV) (0.8 g, yield 93%), with m.p. 194 ° C. ^ -NMR (DMSO-c / e): 8.92 (s, 1H, 2-H); 7.68 (s, 1H, 5-H or 11-H); 7.56 (s, 1H, 5-H or 11-H); 4.42 (t, J = 5.8, 2 H, 7'-CH2O 9'-CH2); 4.36 (t, J = 5.8, 2H, 7'-CH2 or 9'-CH2); 2.26 (quint, J = 5.8, 2 H, 8'-CH2).

To a solution of 4-chloro-8,9-dihydro-7 / V- [1,4] dioxepine [2,3-g] quinazoline (XV) (0.1 g, 0.5 mmol) in isopropanol (5 ml) was 3-phenylaniline addition (71 mg, 0.5 mmol). The reaction mixture was irradiated with microwave at 60 W, reaching the temperature of 80 ° C in 30 seconds. This temperature was kept constant for 15 minutes by automatic regulation of power and cooling (TLC: chloroform / methanol, 9/1). After cooling, the reaction mixture was filtered to obtain (8,9-dihydro-7 / Y- [1,4] dioxepino [2,3-g] quinazolin-4-yl) - (m-blphenyl-3'- II) amine hydrochloride (III) (0.2 g, yield 89%) with m.p. > 300 ° C. ^ -NMR (DMSO -d6) \ 10.73 (s, 1H, NH); 8.78 (s, 1H, 2-H); 8.39 (s, 1H, 5-H or 11-H); 8.06 (s, 1H, Harom); 7.82-7.66 (m, 3H, Harom); 7.59-7.46 (m, 4 H, Harom) /7.4-7.37 m, 1 H, Harom); 7.35 (s, 1H, 5-H or 11-H); 4.42 (t, J = 5.9, 2H, 7'-CH: or 9'-CH2); 4.35 (t, J = 5.9, 2H, 7'-CH2 or 9'-CH2); 2.27 (quint, J = 5.4, 8'-CH2).

Example 6. Determination of the inhibition of phosphorylation on solated tyrosine kinases

In vitro inhibition of the kinase activity of hydrochloride compounds I, II and III at a concentration of 0.1 μΜ was evaluated on six different tyrosine kinases (EGFR, FGFR-1, VEGFR-2, PDGFRP, sre and abl). The intracellular catalytic domains of the kinases were dissolved in a dilution buffer (5mM MOPS pH 7.2, 2.5 mM glycerol 2-phosphate, 5 mM MgCI2, 0.4 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT, 0.5 mM BSA). The reactions were carried out in previously cooled microcentrifuge tubes. To the different kinases (final concentration of 200 ng / ml), in addition to the hydrochlorides of compounds I, II and III, 0.2 mg / ml of substrate (Myelin Basic Protein, Sigma), 0.05 mM ATP and 0.25 μθ of [γ - <32> Ρ] ΑΤΡ [PerkinElmer, Monza, MI) (final volume 25pL). In the negative control the phosphorylatable substrate was replaced with water. In the positive control, water replaced the test compounds. The reactions were carried out at 30 ° C for 20 minutes, and subsequently blocked with a loading buffer containing 0.25 mM β-mercaptoethanol. The samples were then analyzed by electrophoretic running on 10% w / v polyacrylamide gel. The phosphorylated substrate was identified by autoradiography. VersaDoc Quantity One (BioRad) software was used for densitometric analysis. The data are collected in Table 1.

Isolated tyrosine kinase assays *

Composed

EGFR FGFR-1 VEGFR-2 PDGFRP sre abl

1 N M M H M M

II (-) M (-) H M M

III M ω (-) H N (+)

Table 1. Inhibitory capacity of hydrochlorides of compounds I, II and III on isolated tyrosine kinases. <a> (+) = IC50 equal to or less than 1.0 μΜ; (-) = IC50 greater than 1.0 μΜ

Example 7. Effects on cell proliferation

The cell viability assay was conducted by verifying the metabolic activity of cultured cells through a colorimetric assay using 3- [4,5-dimethylthiazole-2-i |] -2,5-diphenyltetrazolobromide, known as MTT [Denizot F., Lang R., "Rapid colorimetry assay for celi growth and servivai. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability", J. Immunol. Methods, 1986, 89, 271-277; Wemme H. et al., "Measurement of lymphocyte proliferation: criti analysis of radioactive and photometric methods", Immunobiology, 1992, 185, 78-89].

A431 or NIH3T3 cells were seeded at a density of 5 x 10 <3> cells / well in 96-well plates (Becton Dickinson Falcon) in the presence of complete medium (150 μΙ per well). HUVEC cells were seeded at a density of 3.5 x 10 <3> cells / well in 96-well plates (Becton Dickinson Falcon) in the presence of complete medium (150 μίρβΓ well). After 24 hours of incubation at 37 ° C, a wash was carried out with PBS, to eliminate any dead cells and soil residues, and the hydrochlorides of compounds I, II and III dissolved in complete medium (final volume 200 pL) were added. at various concentrations (from 0.01 to 10 μΜ). The control consisted of untreated cells in complete medium. After 68 hours of incubation at 37 ° C with the substances, 20 pL of MTT [Sigma) (5 mg / mL in PBS) was added to the wells and a further 4 hours was waited for the cells to metabolize the composed. The formed formazan crystals were solubilized with 75 µL of acidic isopropanol [Carlo Erba) and the plates were placed under gentle mechanical stirring for 20 minutes in the dark. The reading of the absorbency values was performed with Microplate autoreader EL 13 at 570 nm. The results were expressed as EC50, concentration of the compound capable of reducing cell viability by 50% compared to the untreated control, and reported in Table 2.

Sa] »already on cell lines

Composed

A431 NIH3T3 HUVEC

1 0.81 ± 0.06 0.60 ± 0.02 0.75 ± 0.01

II 0.08 ± 0.01 0.60 ± 0.20 0.08 ± 0.17

Ili 0.66 ± 0.08 0.90 ± 0.06 0.91 ± 0.06

Table 2. Cytotoxicity of hydrochlorides of compounds I, II and III measured on cell lines A431, NIH3T3 and HUVEC. Values are expressed as EC50 (μΜ) ± S.D.

Example 8. Determination of the aooDtosI rate in the cell line A431

The Celi Death Detection ELISA <PLUS> kit [Roche) was used to evaluate the rate of apoptosis in cultures treated with hydrochloride compounds I, II and III at a concentration of 10 pM. The assay was conducted according to the instructions given by the supplier company. The results, averaged over at least 3 experiments, were expressed as a percentage change with respect to the control. Statistical analysis was performed with Student's t-test. The data obtained are shown in Figure 3.

Example 9. Determination of the amount of FGF-2 released into the culture medium by HUVEC cells

The inhibition of FGF-2 release by I, II and III hydrochlorides at a concentration of 0.1 μΜ, was evaluated on HUVEC cells with the Human FGF basic Immunoassay [Quantikiné) kit, according to the indications given by the supplier company. The results, averaged from at least 3 experiments, were expressed as pg / mL of FGF-2. Statistical analysis was performed with Student's t-test.

The data obtained are shown in Figure 4.

Example IO. Evaluation of the variations of morphohaenesis on Matriael.

Each well of a plate of 24 pcs [Falcon) was conditioned with a layer of Normal Matrigel (50 pL / pc) [B&D Biosciences), previously thawed overnight at 4 ° C on ice, with the foresight to use material (tips , pipettes, plates) pre-cooled in the freezer and that the plastic cell culture holder remained on ice throughout the conditioning procedure. The plate was then incubated at 37 ° C for at least 1 h to induce gelation of the glycoprotein matrix. In each conditioned well, 7.5xl0 <4> cells suspended in 1 mL of MV2base medium containing 1% FCS (Promocell) and compounds I, II, III at 0.1 μΜ concentration were seeded, shown to be non-cytotoxic by cell viability assay. After 18 h of incubation at 37 ° C and washing in PBS, the cells were fixed with a 2% solution of glutaraldehyde {Merck) in 0.1 M sodium cacodylate buffer {Sigma) [Albini A. et al, "The ' chemoinvasion assay ': a tool to study tumor and endothelial celi invasion of basement membranes ", Int. J. Dev. BioL, 2004, 48, 563-571]. For each well 5 fields were then photographed (x50 magnification), following a cross pattern. The images thus obtained were analyzed using the ImageJ-Matrigel Assay Software, evaluating both dimensional parameters, such as the percentage of the area covered by the cells, the total length of the network formed by the HUVECs for each field and the percentage area enclosed. inside the meshes, and topological parameters, such as the number of closed meshes and branching points per field. The results, averaged from at least 3 experiments, were expressed as percentage changes with respect to the untreated sample. Statistical analysis was performed with Student's t-test. The assay was also performed with Matrigel reduced in growth factors by seeding the cells in 1 mL of base medium containing 1% FCS, 50 ng / mL FGF-2 or 20 ng / mL VEGF alone or with compounds I , II, III, at non-cytotoxic concentration. The results, averaged from at least 3 experiments, were expressed as percentage changes with respect to the untreated sample. Statistical analysis was performed with Student's t-test.

Effect on morphohaenesis in vitro. To evaluate the effect of 4-aminoquinazoline derivatives on morphogenesis in vitro, HUVECs were seeded on Matrigel. The cultures were treated for 18 h with the compounds used at a non-cytotoxic concentration of 0.1 μΜ, as demonstrated by the results of the MTT assay (Figure 5) carried out following exposure for 18 h.

Effects on in vitro morphohaenesis induced by exogenous cytokines. The assay was conducted using reduced growth factor Matrigel by seeding the cells in 1 mL of base medium containing 1% FCS, FGF-2 at a concentration of 50 ng / mL or VEGF at a concentration of 20 ng / mL alone. (Figures 6 and 8) or with the compounds under analysis at non-cytotoxic concentration (Figures 7 and 9). The results, averaged over at least 3 experiments, were expressed as percentage changes with respect to the control cultures treated with FGF-2 and VEGF, respectively. Statistical analysis was performed with Student's t-test. The non-cytotoxic concentration of the compounds was previously identified with an MTT cell viability assay (data not shown) by incubating the cells for 18 h with the test compounds alone or with 50 ng / mL of FGF-2 or 20 ng / mL of VEGF.

Example 11. Evaluation of the anti-flocking effect In vivo

Implantation of matrigel plugs. For each animal (CD57 / BL6 mice), 500 μΙ_ of Matrigel (B&D Biosciences), previously thawed at 4 ° C overnight, was used. The material to be used for the procedure (tips, eppendorf) was pre-cooled in the freezer. 200 ng / mL of FGF-2 (Sigma) and 12 I.U./plug of heparin (Vister) were then mixed with Matrigel; subsequently the hydrochlorides of compounds I, II and III were added at a concentration equal to 1 μΜ. The mice were anesthetized with isoflurane and needles and syringes previously cooled in the freezer were used at the time of the subcutaneous injection. Mice treated with Matrigel containing only heparin represent the negative controls, while those treated with Matrigel containing heparin and FGF-2 the positive controls.

Determination of the hemoglobin content. Seven days after the injection, the animals were sacrificed by inhalation of carbon dioxide. The plugs were removed, immersed in a buffer consisting of 300 μί of 0.1% Brij-35 in PBS per plug, and placed at 4 ° C overnight. They were then centrifuged for 5 minutes at 13500 g / min and the hemoglobin content was determined in the supernatant, using Drabkin's Reagent, containing sodium bicarbonate, potassium ferricyanide and potassium cyanide. To calculate the concentration of hemoglobin extracted from the plug, a calibration line was constructed by preparing a standard hemoglobin solution (10 mg / mL) in Drabkin's Solution (1 way! Of Drabkin's Reagent in 1 L of distilled water added with 0.5 mL of Brij-35 Solution 30%). Seven cyanomethaemoglobin standards diluted with Drabkin's Solution at a known concentration in a range between 0 and 10 mg / mL (0.25; 0.5; 1; 2; 4; 8; 10 mg / mL were prepared with this stock solution. ). The absorbance of the standards was read at 540 nm. With the straight line obtained, the cyanomethaemoglobin concentrations (mg / ml) relative to the samples treated with compounds I, II and III were calculated. The results, averaged from at least 3 animals, were expressed as mg / mL of hemoglobin. Statistical analysis was performed with Student's t-test. The data obtained are shown in Figure 10.

Histological analysis. Some plugs were fixed for 24 hours in PBS containing 4% formalin (Sigma) and subsequently dehydrated using the ascending scale of ethyl alcohol solutions as follows: 2 hours in 70% ethyl alcohol, 2 hours in ethyl alcohol 80%, 2 hours in 90% ethyl alcohol, one night in 95% ethyl alcohol, 2 hours in 100% ethyl alcohol, 2 hours in xylene / 100% ethyl alcohol (1: 1, v / v) and then in xylene for another 2 hours. This was followed by a passage in paraffin at 60 ° C for 2 hours for the impregnation and then the inclusion in metal trays. At the microtome (Histoslide 2000, Reichert-Jung), 5 µm thick sections were obtained, which were adhered to SuperFrost Plus slides (Menzel-Glaser). Then hematoxylin-eosin staining (Merck) was performed. The sections were previously de-refined through two passages in Histochoice Clearing Agent IX, each lasting 5 minutes, and hydrated through passages of 5 minutes each in descending scale of ethyl alcohol: ethyl alcohol at 100%, ethyl alcohol at 95%, ethyl alcohol at 80%, 70% ethyl alcohol, 50% ethyl alcohol, 30% ethyl alcohol, 10% ethyl alcohol, to finish with a 5 minute passage in distilled water. Then each section was treated with hematoxylin for 2.5 minutes, washed twice quickly in distilled water and contrasted for 2 minutes in spring water, then treated with eosin for 10 seconds and washed again quickly in distilled water. After staining, the preparations were dehydrated by quick passages in 95% alcohol and absolute alcohol, treated with Histochoice Clearing Agent IX and mounted with Histochoice Mounting Media. The observations were made with a DM2000 optical microscope (Le / <'> ca). The results obtained are shown in Figure 11.

Claims (15)

TITLE: Multityrosine kinase inhibitors useful for related diseases: pharmacophoric models, compounds identified through these models, methods for their preparation, their formulation and their therapeutic use CLAIMS: 1. Method for the generation of the three-dimensional pharmacophore, represented in figure 1, which can be used for the identification of compounds active as multityrosinkinase inhibitors with simultaneous inhibitory action on at least 4 kinases including EGFR, VEGFR-2, FGFR-1, PDGFRP, sre and abl . 2. Three-dimensional pharmacophore generated according to claim 1 consisting of: three zones for aromatic portions, not substituted or variously substituted (called Ari, Ar2 and Ar3); a zone for nitrogen atoms (called NA) which is a structural part of Ari; a spacer (called linker) that joins Ari and Ar2; a zone X for functional groups containing at least one electron-rich atom. 3. Three-dimensional pharmacophore according to claim 2, wherein the Ari zone is an unsubstituted or variously substituted aromatic hexatomic ring, containing at least one hydrogen bridge acceptor nitrogen atom; the zones Ar2 and Ar3 are pentatomic or hexatomic, carbocyclic or heterocyclic rings, not substituted or variously substituted; a linker consisting of a chain of 1 or 2 atoms chosen from nitrogen, oxygen, sulfur, selenium, silicon, phosphorus, carbon or their combinations; zone X contains at least one electron-rich atom selected from oxygen, sulfur and nitrogen; the distance between the center of Ari and the center of Ar2 is between 4.5 and 6.5 À; the distance between the center of Ari and the center of Ar3 is between 6.5 and 8.5 À; the distance between the center of Ar2 and the center of Ar3 is between 4.0 and 5.0 À; the distance between the center of X and the center of Ari is between 4.5 and 6.5 À; the distance between the center of X and the center of Ar2 is between 6.5 and 8.5 A; the distance between the center of X and the center of Ar3 is between 10.5 and 12.5 A. 4. Use of the three-dimensional pharmacophore according to claims 2-3 by any software or strategy in order to identify new multithyrosinkinase inhibitors with inhibitory action simultaneously on at least 4 kinases between EGFR, VEGFR-2, FGFR-1, PDGFRP, sre and abl, preferentially through chemoinformatics techniques of Virtual screening or rational design, such as, but not limited to, querying of three-dimensional molecular databases, in which the pharmacophore is the key to research. 5. Compounds identified according to claim 4 having any structure that complies with the structural requirements described by the three-dimensional pharmacophore described in claims 2-3. 6. Compounds identified according to claim 4, having the structural requirements indicated in claim 5 and preferably having a structure corresponding to the general formula (1) in which: - A is independently selected from a CH group or an oxygen atom or a sulfur atom or a selenium atom or a nitrogen atom or an NH group or an NR1 group, where RI is independently selected from an alkyl or cycloalkyl residue or acyl or aromatic or heteroaromatic, where the heteroatom is another nitrogen atom; B and D are independently selected from a carbon atom or a nitrogen atom; E can be absent and, if present, has the same meaning as defined for A; F and G have the same meaning defined for B and D; X and Y are independently selected from an electron-rich atom such as an oxygen atom or a sulfur atom or a nitrogen atom; R2 and R3 have the same meaning defined for RI and can be joined together forming a further ring that includes the structural elements indicated with X-F-G-Y. - All bonds between atoms or groups A, B, D, E, F, G, X, Y, R2, R3 can be independently single or double. 7. Compound identified according to claim 4 characterized in that it consists of ([1,3] dioxol [4,5-g] quinazolin-8-yl) - (m-biphenyl-3'-yl) amine (I) or a pharmaceutically acceptable salt thereof. 8. Compound identified according to claim 4 characterized in that it consists of (7,8-dihydro [1,4] dioxine [2,3-g] quinazolin-4-yl) - (m-biphenyl-3'-yl ) amine (II) or a pharmaceutically acceptable salt thereof. 9. Compound identified according to claim 4 characterized in that it consists of (8,9-dihydro-7H- [1,4] dioxepine [2,3-g] quinazolin-4-yl) - (m-biphenyl-3 '-yl) amine (III) or a pharmaceutically acceptable salt thereof. 10. Method of preparation of compounds according to claims 5-9 represented by the following synthetic way: (A) cyclization of a pyrimidine nucleus on an anilide derivative of formula general (2) (2) where: X and Y are independently selected from an electron-rich atom such as an oxygen atom or a sulfur atom or a nitrogen atom; R2 and R3 are independently selected from an alkyl or cycloalkyl or acyl or aromatic or heteroaromatic residue and can be joined together forming with X and Y a further condensed ring on the benzene core; Z is independently chosen among all the possible protecting groups of the amino functions, in particular the groups not easily hydrolyzable at acid pH such as COOEt [otherwise indicated as C (= 0) OCH2CH3]. (B) oxidation of quinazoline of general formula (3) (3) where X, Y, R2 and R3 have the same meaning as defined in point (A). (C) halogenation of quinazolinone of general formula (4) where X, Y, R2 and R3 have the same meaning as defined in point (A). (D) substitution with 3-phenylaniline of the halogen atom of the aloquinazoline of general formula (5) where X, Y, R2 and R3 have the same meaning defined in point (A) and W is independently chosen between a chlorine atom or a bromine atom or an iodine atom or a fluorine atom. 11. Compounds identified according to claim 4 prepared by any other synthetic method known or reported in the literature. 12. Compounds according to claims 5-9 usable both as multikinase inhibitors and as inhibitors of each kinase individually. 13. Use of the compounds according to claims 5-9 for the preparation of pharmaceutical compositions useful in the treatment of pathologies associated with alterations of tyrosine kinase activity, such as, for example, but not limited to: brain, lung, squamous cell, pancreas, kidney, spleen, stomach, liver, esophagus, prostate, colorectal, neck, head, thyroid, ovary, breast, uterus, testes, lymphoma and leukemias and their combinations; in the treatment of pathologies related to angiogenesis; in the treatment of other related diseases. 14. Pharmaceutical compositions containing at least one of the compounds according to claims 5-9, or their pharmaceutically acceptable salts, and their associations for the treatment of the disorders referred to in point 13, suitable for oral, sublingual, intramuscular, subcutaneous, intravenous, peridural administration , topical, transdermal, rectal, intranasal, both immediate release, and modified release, deposit, delayed release. 15. Use of the compositions according to claim 14 in pharmacological association with other therapeutic agents and their pharmaceutical forms.