ITMI20082336A1 - COMPOUNDS IRREVERSIBLE EGFR INHIBITORS WITH ANTI-PROLIFERATIVE ACTIVITY - Google Patents

Description

Patent application for industrial invention entitled:

IRREVERSIBLE EGFR INHIBITOR COMPOUNDS WITH FERATIVE ANTI-PROLIS ACTIVITIES

FIELD OF THE INVENTION

The present invention relates to the synthesis and pharmacological activity of new compounds derived from 4-anilinoquinazoline that can be used as irreversible inhibitors of the epidermal growth factor receptor (EGFR).

STATE OF THE TECHNIQUE

The family of epidermal growth factor receptors includes four transmembrane receptors (erbB1 / EGFR, erbB2 / FIER2, erbB3 / HER3 and erbB4 / HER4) consisting of an extracellular portion that binds the Iigand (except erbB2), a transmembrane domain and an intracellular domain with tyrosine kinase activity (except erbB3). The ligands of EGFR, erbB3 and erbB4, mainly the epidermal growth factor (EGF) and the transforming growth factor a (TGF-a), bind the extracellular portion of the receptor, activating the dimerization process and the consequent phosphorylation of specific tyrosine residues in the intracellular domain. Activation of the receptor leads to a cascade of signals that mediate cell growth, proliferation, motility and survival (Wells, A. EGF receptor. Int. J. Biochem. Celi Biol. 1999, 31, 637-643) . EGFR is overexpressed or present in constitutively activated form in several solid tumors of epithelial origin, such as non-small cell lung cancer (NSCLC), and its involvement in tumor onset and progression is often associated with a poor prognosis (Voldborg, B. R .; Damstrup, L .; Spng-Thomsen, M .; Poulsen, H. S. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann. Oncol. 1997, 8, 1197-1206; Ritter, C. A .; Artega, C. L. The epidermal growth factor receptor tyrosine kinase: a promising therapeutic target in solid tumors. Semin. Oncol. 2003, 30, 993-1011; Raymond, E .; Faivre, S .; Armand, J. P. Epidermal growth factor receptor tyrosine kinase as a target for anticancer therapy. Drugs, 2000, 60, 15-23; Salomon, D. S .; Brandt, R .; Ciardiello, F .; Normanno, N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit. Rev. Oncoi. Hematol. 1995, 19, 183-232).

Gefitinib (Iressa®; AstraZeneca) and erlotinib (Tarceva®; Genenthec), approved for the treatment of lung cancer (NSCLC), are 4-anilinoquinazoline compounds active as reversible inhibitors of EGFR, the formula of which is shown in Figure 1a. 4-anilinoquinazoline derivatives potently and selectively inhibit the tyrosine kinase activity of EGFR by binding the site for ATP in the intracellular portion of the receptor (Wissner, A .; Berger, D. M .; Boschelli, H. D .; Floyd M. B .; Greenberger, L. M .; Gruber, B. C .; Johnson, B. D .; Mamuya, N .; Nilakantan, R .; Reich, M. F .; Shen, R .; Tsou, H. R .; Wang, Y. F .; Wu, B .; Ye, F .; Zhang, N. 4 -Anilino-6,7-dialkoxyquinoline-3-carbonitrile Inhibitors of Epidermal Growth Factor Receptor Kinase and Their Bioisosteric Relationship to the 4-Anilino-6,7-dialkoxyquinazoline Inhibitors. J. Med. Chem. 2000, 43, 3244; Bridges, A. J .; Zhou, H .; Cody, D. R .; Rewcastle, G. W .; McMichael, A .; Showalter, H. D. H .; Fry, D. W .; Kraker, A. J .; Denny, W. A. Tyrosine Kinase Inhibitors. 8. An Unusually Steep Structure-Activity Relationship for Analogues of 4- (3-Bromoanilino) -6,7-dimethoxyquinazoline (PD 153035), a Potent Inhibitor of the Epidermal Growth Factor Receptor. J. Med. Chem. 1996, 39, 267; Traxler, P; Bold, G .; Frei, J .; Lang, M .; Lydon, N .; Mett, H .; Buchdunger, E .; Meyer, T .; Mueller, M .; Furet, P. Use of a Pharmacophore Model for the Design of EGF-R Tyrosine Kinase Inhibitors: 4- (PhenyIamino) pyrazolo [3,4-d] pyrimidines. J. Med. Chem. 1997, 40, 3601). Although gefitinib and erlotinib have proved very useful in the treatment of specific groups of patients (Asian women, non-smokers, carriers of specific EGFR mutations), clinical practice has shown phenomena of primary and acquired resistance that limit their usefulness. (Fukuoka, M .; Yano, S .; Giaccone, G .; Tamura, T .; Nakagawa, K .; Douillard, J .; Nishiwaki, Y .; Vansteenkiste, J .; Kudoh, S .; Rischin, D. ; Eek, R .; Horai, T .; Noda, K; Takata, I .; Smit, E .; Averbuch, S .; Macleod, A .; Feyereislova, A; Dong, R .; Baselga, J. Multi- institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J. CUn. Oncol. 2003, 12, 2237-2246; Pérez-Soler, R .; Chachoua, A .; Hammond, L. A. ; Rowinsky, E. K .; Huberman, M .; Karp, D .; Rigas, J .; Clark, G. M .; Santabàrbara, P .; Bonomi, P. Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer. J. Clin. Oncol. 2004, 22, 3 238-3247). In an effort to overcome this type of resistance, a line of chemical-pharmaceutical research has recently turned to the development of irreversible inhibitors, capable of covalently binding to amino acids close to the ATP binding site. To this class belong, for example, the irreversible inhibitors PD168393, compound 5a represented in Figure 1 b (Fry, D. W .; Briges, A. J .; Denny, W. A .; Doerthy, A .; Greis, K. D .; Hicks, J. L; Hook, K. E .; Keller, P. R .; Leopold, W. R .; Loo, J. A .; McNamara, D. J .; Nelson, J. M .; Sherwood, V .; Smail, J. B .; Trumpp-Kallmeyer, S .; Dobrusin, E. M. Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc. Nati. Acad. Sci. U.S.A. 1998, 95, 12022-12027) and canertinib (CI-1033, Pfizer, shown in Figure 1b) (Smalli, J. B .; Rewcastle , G. W .; Loo, J. A .; Greis, K. D .; Chan, O. H .; Reyner, E. L .; Lipka, E .; Showalter, H. D. H .; Vincent, P. W .; Elliott, W. L .; Denny, W. A. Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4- (phenylamino) quinazoline- and 4- (phenylamino) pyrido [3,2-d] pyrimidine-6-acrylamides hearing additional solubilizing functions. J. Med. C hem. 2000, 43, 1380-1397; Alien, L. F .; Eiseman, I. A., Fry, D. W .; Lenehan, P. F. CI-1033, an irreversible pan-erbB receptor inhibitor and its potential application for the treatment of breast cancer. Semin. Oncol. 2003, 30, 65-78), the latter in phase II of development for the treatment of NSCLC, which present an acrylamide group capable of forming a chemically stable bond with Cys773 in the active site of EGFR (Blair, J. A .; Rauh, D .; Kung, C .; Yun, C.-H .; Fan, Q.-W .; Rode, H .; Zhang, C .; Eck, M. J .; Weiss, W. A .; Shokat, K. M .; Structure-guided development of affinity probes for tyrosine kinases using Chemical genetics. Nature Chem. Biol. 2007, 3, 229-238).

The irreversible binding to EGFR confers to this class of inhibitors considerable advantages over the reversible inhibitors gefitinib and erlotinib: (a) they maintain a prolonged effect over time, once the irreversible inhibitor is covalently linked to the enzyme the effect persists until new resynthesis of EGFR; (b) exhibit better selectivity of action, irreversible inhibitors covalently bind a cysteine residue located near the entrance to the ATP binding site (Cys773 in EGFR, Cys784 in erbB2, and Cys778 in erbB4), which characterizes in unambiguously the erbB family with respect to the other kinases; (c) are able to overcome secondary resistance to gefitinib and erlotinib treatment. Irreversible inhibition of EGFR, therefore, represents a very important approach for the development of anticancer drugs (Carter, T. A .; Wodicka, L. M .; Shah, N. P .; Velasco, A. M .; Fabian, M. A .; Treiber, D. K .; Milanov, Z. V .; Atteridge , C. E .; Biggs III, W. H .; Edeen, P. T .; Floyd, M .; Ford, J. M .; Grotzfeld, R. M .; Herrgard, S .; Insko, D. E .; Mehta, S. A .; Patel, H. K .; Pao, W .; Sawyers, C. L. ; Varmus, H .; Zarrinkar, P. P .; Lockhart, D. J. Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases. Proc. Nati. Acad. Sci. U.S.A. 2005, 102, 11011-11016).

The irreversible inhibitors of EGFR described in the literature are therefore characterized by a 4-anilinoquinazoIine or 4-anilinoquinolino-3-carbonitrile nucleus to which the reactive group is bound, capable of establishing the covalent bond with the molecular target by means of addition reactions 1,4 Michael-type, or nucleophilic substitution, with cysteine residues present in the catalytic site of the target protein. The Michael acceptor groups, present in the known irreversible inhibitors, e.g. acrylamides, propargylamides and vinylsulfonamides, have an intrinsic tendency to react with some amino acids (preferably cysteines); even if the remaining portion of the molecule should confer reaction selectivity, that is, it should make reactions that give adducts with proteins other than EGFR much less likely, it is possible that side effects due to undesired reactions may occur. For this reason, it is important that a new therapeutic strategy is not entrusted to compounds with the same chemical characteristics, although promising as canertinib, but is supported by studies on the possibility of modulating the chemical structure of the inhibitor, in order to know the structure-activity relationships. and have alternatives to possible problems that emerge from clinical studies.

The object of the present invention is therefore to provide compounds which can be used as irreversible inhibitors with high reaction selectivity.

SUMMARY OF THE INVENTION

This purpose was achieved by means of a 4-anilinoquinazoline derivative of formula (A)

wherein X represents hydrogen, bromine, chlorine, fluorine, ethynyl or methyl;

A is a residue chosen from the group consisting of

the)

in which

Y represents -CO- or -CHR-i-;

Z represents -OR2, -SR2 or -NR2R3;

R-i, R2 and R3 represent, independently of each other, hydrogen, (Ci_ 6) alkyl, (CH2) n-COOR4, (CH2) n-CONR4R5, (CH2) n-NR4R5 or (CH2) n-morpholine;

R4 and R5 independently represent hydrogen or (C_6) alkyl;

n can be an integer chosen from 0, 1, 2, 3, 4, 5, and 6.

ii)

in which

R6 represents hydrogen or (C 1-6) alkyl;

R7, Re, R9, R10 and R11 represent, independently of each other, hydrogen, chlorine, fluorine, the radical CF3, -N02 or -OR12;

R12 represents hydrogen, (Ci-6) alkyl, (CH2) n-COOR13, (CH2) n-CONR13R14, (CH2) n-NRi3Ri4 or (CH2) n-morpholine;

RI3 and R14 represent, independently of each other, hydrogen or (C- |.

6) alkyl; and n is an integer chosen from 0, 1, 2, 3, 4, 5, and 6.

iii)

in which

R15, represents hydrogen, (C1-6) alkyl, (CH2) n-COOR18j (CH2) n-CONR18Rig, (CH2) n-NRi8Rig or (CH2) n-morpholine;

Ri6, Ri7, Rie and R19 represent, independently of each other, hydrogen or (Ci-8) alkyl;

iv)

Re represents hydrogen or (C1-6) alkyl;

W represents -C = or -N =;

R20, R21. R22 and R23 represent, independently of each other, hydrogen, chlorine, fluorine, the radical -CF3, -N02, (Ci-6) alkyl.

v)

in which

R24 represents (C1-6) alkyl, (CFl2) n-COOR251 (CH2) n-CONR25R26, (CH2) n-NR25R26 or (CH2) n-morpholine;

R25 and R26 represent, independently of each other, hydrogen or (C-i-

6) alkyl;

you)

in which

R6 represents hydrogen or (C-6) alkyl;

Q represents an aryl or heteroaryl group, optionally substituted with one or more substituents selected, independently of each other, from hydrogen, chlorine, fluorine, the radical CF3, -N02 or -OR12;

R12 represents hydrogen, (Ci-6) alkyl, (ChhVCOOR ^, (CH2) n-CONR'i3R14, (CH2) n-NRi3Ri4 or (CH2) n-morphoIine;

RI3 and R14 represent, independently of each other, hydrogen or (C-i.

6) alkyl; and n is an integer chosen from 0, 1, 2, 3, 4, 5, and 6.

vii)

R27 and R28 can represent hydrogen or (C1-6) alkyl; -NR28R29 can be morpholine;

The derivative of the invention can be used as an irreversible inhibitor of the tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR).

The invention therefore concerns a derivative of Formula (A) chosen from the group consisting of:

- a compound in which the substituent (A) is the substituent i), i.e. a derivative of Formula (I),

Formula (I)

in which;

X, Y and Z have the meaning indicated for the formula (A);

- a compound in which the substituent (A) is the substituent ii), i.e. a derivative of Formula (II)

Formula (II)

where X, R6, R7, Re, Rg, R-ιο and R11 have the meaning indicated in formula (A);

- a compound in which the substituent (A) is the substituent iii), i.e. a derivative of Formula (III)

Formula (III) wherein X, R-15, R16, R17, Rie and R-ig have the meaning indicated in formula (A);

- a compound in which the substituent (A) is the substituent iv), i.e. a derivative of Formula (IV)

Formula (IV)

wherein X, Re, W, R20, R21, R22 and R23 have the meaning indicated in formula (A);

- a compound in which the substituent (A) is the substituent v), i.e. a derivative of Formula (V)

Formula (V)

wherein X, R241R25 and R26 have the meaning indicated in formula (A);

- a compound in which the substituent (A) is the substituent vi), i.e. a derivative of Formula (VI)

Formula (VI)

in which X, R6, Q have the meaning indicated in formula (A);

- a compound in which the substituent (A) is the substituent vii), i.e. a derivative of Formula (VII)

Formula (VII)

wherein X, R6, R27 and R28 have the meaning indicated in formula (A);

These compounds are able to interact covalently with cysteine residues present in the catalytic site of EGFR and other tyrosine kinase receptors of the same family, giving the compounds the ability to maintain a prolonged inhibition over time and to overcome resistance. of cancer cells to reversible EGFR inhibitors. The compounds object of the present invention consist of groups reactive towards cysteines, in particular epoxides, aryloxymethylcarbonyls, isothiazolinones, benzisothiazolinones, 1, 2,4-thiadiazoles or carbamates, joined by amide bond to a 6-amino-4-anilinoquinazole nucleus, responsible for the interaction with the molecular target. Amides deriving from β-aminocarboxyls are also the object of the present invention, since, following a β-elimination reaction, they can give rise to acrylamide derivatives. The present invention relates to their preparation and their use as anticancer drugs in the treatment of solid tumors of epithelial origin such as non-small cell lung cancer (NSCLC) with particular reference to tumors that have developed secondary resistance to reversible EGFR inhibitors such as gefitinib. and erlotinib.

DESCRIPTION OF THE FIGURES

The invention will now be described in detail with reference to some figures in which:

Figures 1a and 1b show the structures of EGFR inhibitors known in the literature, reversible (gefitinib and erlotinib, Figure 1a) and irreversible (PD168393 and canertinib, Figurei b);

Figures 2a and 2b show the effect of Compound 7a (UPR1135) of the invention on the levels of EGFR autophosphorylation in A431 cells. The cells are incubated for 1 h with various concentrations of Compound 7a, and subsequently stimulated with EGF immediately after incubation (Figure 2a) or 8 h after removal of the compound (Figure 2b);

Figure 3 shows the antiproliferative effect of the reversible inhibitor gefitinib (Iressa, solid bars) and Compound 7a (UPR1135, empty bars) on the H1975 cell line. Cells are incubated with various concentrations of inhibitor for 72 h. Cell proliferation is determined by the MTT assay. Results are reported as mean standard deviation (SD) for two independent experiments; Figure 4 shows the effect of gefitinib (Iressa), PD187376 and Compound 7a (UPR1135) on the autophosphorylation levels of EGFR and erbB2 in H1975 cells. The cells are incubated for 1 h with various concentrations of inhibitor, stimulated with EGF and subsequently subjected to analysis by Western blot.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore concerns a derivative of 4-anilinoquinazoline of formula (A)

wherein X represents hydrogen, bromine, chlorine, fluorine, ethynyl or methyl;

A is a residue chosen from the group consisting of

the)

in which

Y represents -CO- or -CHR1-;

Z represents -OR2, -SR2 or -NR2R3;

R-i, R2 and R3 represent, independently of each other, hydrogen, (C-i.

6) alkyl, (CH2) n-COOR4, (CH2) n-CONR4R5, (CH2) n-NR4R5 or (CH2) n-morpholine;

R4 and R5 independently represent hydrogen or (C1-6) alkyl;

n can be an integer chosen from 0, 1, 2, 3, 4, 5, and 6.

ii)

in which

Re represents hydrogen or (C) alkyl;

R7IRB, R9, R10 and Rn represent, independently of each other, hydrogen, chlorine, fluorine, the radical CF3, -N02 or -OR12;

It represents hydrogen, (Ci-6) alkyl, (CH2) n-COOR13, (CH2) n-CONRi3R14, (CH2) n-NRi3R14 or (CH2) n-morpholine;

R-I3 and RM represent, independently of each other, hydrogen or (C-i-6) alkyl; and n is an integer chosen from 0, 1, 2, 3, 4, 5, and 6.

iii)

in which

R15, represents hydrogen, (Ci-6) alkyl, (CH2) n-COOR18, (CH2) n-CONR18Ri9, (CH2) n-NR18R19 or (CH2) n-morpholine;

R16. R17, Rie and R19 represent, independently of each other, hydrogen or (C1-6) alkyl;

iv)

R6 represents hydrogen or (C 1-6) alkyl;

W represents -C = or -N =;

R20, R21, R22 and R23 represent, independently of each other, hydrogen, chlorine, fluorine, the radical -CF3, -N02, (C1-6) alkyl.

v)

in which

R24 represents (C1-6) alkyl, (CH2) n-COOR25, (CH2) n-CONR25R26, (CH2) n-NR25R26 or (CH2) n-morpholine;

R25 and R26 represent, independently of each other, hydrogen or (C-i.

6) alkyl;

you)

in which

R6 represents hydrogen or (C 1-6) alkyl;

Q represents an aryl or heteroaryl group, optionally substituted with one or more substituents selected, independently of each other, from hydrogen, chlorine, fluorine, the radical CF3, -N02 or -OR- | 2;

RI2 represents hydrogen, (C1-6) alkyl, (CH2) n-COOR13, (CH2) n-CONR13R14, (CH2) n-NRi3Ri4 or (CH2) n-morpholine;

R13 and R-I4 represent, independently of each other, hydrogen or (C-ie) alkyl; and n is an integer chosen from 0, 1, 2, 3, 4, 5, and 6.

vii)

R27 and R28 can represent hydrogen or (C1-6) alkyl; -NR28R29 can be morpholine.

In this description when using the term

- "aryl group" means a fused monocyclic or polycyclic aromatic ring composed only of carbon atoms with a completely conjugated π-electron system;

- "heteroaryl group" means a fused monocyclic or polycyclic system having one or more eternatoms in the ring, such as for example nitrogen, oxygen, sulfur and characterized by a completely conjugated π-electron system.

The derivative of the invention can be used as an irreversible inhibitor of the tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR).

Preferably the compound of Formula (A), is a compound in which the substituent A is selected from the group consisting of:

vii)

More preferably, the derivative of the invention of formula (A) has as substituent A the substituent i), more preferably the derivative of the invention is ethyl ester of trans- (2R, 3R) -3- (4- (3 -bromoanilino) quinazolin-6-iI-carbamoyl) -2,3-epoxybutyric.

The compounds object of the present invention, characterized by the presence of groups capable of establishing covalent chemical bonds with bio-nucleophiles, such as cysteine residues, linked by amide bond to a 4-anilinoquinazolinic portion, are able to selectively ensure the interaction between the compounds described and EGFR. The association of the two halves makes it unlikely reactions that give covalent adducts with proteins other than EGFR, leading to the selective inhibition of enzymes that simultaneously recognize the recognition portion with high affinity and present in sterically favorable position an amino acid capable of reacting with the reactive portion.

In Formulas (A), (l) - (VII):

- the term (C-6) alkyl is preferably an alkyl selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl and tert-butyl, npentyl, n-hexyl;

- the terms (CH2) n-COOR, (CH2) n-CONRiRj, (CH2) n-NRjRj where i-j are respectively R4, R5; Ri3, Ri4 ;, Ri8, Ri9; R2, R26 in the above formulas or (CH2) n-morpholine preferably represent linear methylene, ethylene, propylene, butylene, pentylene or hexylene alkyl chains which function as alkyl spacers and link carboxylic, amide, or amino groups on the terminal carbon.

The compounds object of the present invention include carboxylic derivatives of groups reactive towards nucleophiles, such as epoxides (Formula I), aryloxymethylcarbonyls (Formula 11), isothiazolinones (Formula III), benzisothiazolinones (Formula IV), 1, 2,4-thiadiazoles (Formula V), and carbamates (Formula VI). The β-aminocarboxylic derivatives (Formula VII), in the presence of a base that stabilizes the enol form of the amide, can undergo β-elimination reaction with the formation of the corresponding acrylamide derivative. (Maresso, A. W .; Wu, R .; Kern, J. W .; Zhang, R .; Janik, D .; Missiakas, D. M .; Duban, M. E .; Joachimiak, A; Schneewind, O. Activation of inhibitors by sortase triggers irreversible modification of the active site. J. Biol. Chem. 2007, 282, 23129-23139). All the groups considered are linked by amide bond to the 6-amino-4-anilinoquinazoIine portion, responsible for the recognition with the molecular target.

The compounds belonging to the series described showed irreversible inhibitory activity on EGFR autophosphorylation and antiproliferative activity both on a cell line sensitive to treatment with reversible EGFR inhibitors (A431, human ovarian cancer, overexpressing EGFR), and on a treatment resistant cell line. with reversible EGFR inhibitors (H1975, human lung cancer, carrying the T790M mutation in the intracellular portion of the receptor responsible for resistance to gefitinib treatment). Furthermore, the compounds of the invention showed inhibitory activity on both EGFR and erbB2 autophosphorylation in the H1975 cell line.

The compounds of the invention can be prepared with conventional methods or techniques of organic chemistry. Some ways of synthesis will be described below (schemes 1-8). The average person skilled in the art will be able to choose the suitable starting compounds and the corresponding reactants and reaction conditions as a function of the desired product in the context of Formulas (I), (II), (III), (IV), ( V), (VI) and (VII) above.

The synthesis pathways differ according to the reactive portion present in position 6 on the 4-anilinoquinazoline nucleus, and each reactive portion differs according to the substituent present in the beta position on the α, β-epoxy propionamide (Formula I), on the aromatic ring (Formulas II, IV and VI), in position 3 on the 1, 2,4-thiadiazole ring (Formula V), or on the nitrogen atom in position β with respect to the amide function (Formula VII).

The common intermediate used for the synthesis of the compounds object of the present invention is 4-anilinoquinazoline (5) having in position 6 an amino group - NH2 and in position 3 'an X substituent, i.e. a substituent chosen from the group consisting of hydrogen, bromine , chlorine, fluorine and methyl). The intermediate (5) was synthesized according to the method described in the scheme 1 0 as reported in the literature. (Rewcastle, G. W .; Denny, W. A .; Bridges, A. J .; Zhou, H .; Cody, D. R .; McMichael, A .; Fry, D. W. Tyrosine kinase inhibitors: synthesis and structureactivity relationships for 4 - [(phenylmethyl) amino] - and 4 - [(phenylamino) quinazolines as potent adenosine 5'-triphosphate binding site inhibitors of the tyrosine kinase domain of the epidermal growth factor receptor. J. Med. Chem. 1995, 38, 3482-3487 ;. Domarkas, J .; Dudouit, F .; Williams, C; Qiyu, Q .; Banerjee, R .; Brahimi, F .; Jean-Claude, B. J. The combi-targeting conceived: synthesis of stable nitrosureas designed to inhibit the epidermal growth factor receptor (EGFR). J . Med. Chem. 2006, 49, 3444-3552). The starting product 5-nitroantranilonitrile (1) is cyclized with reflux heating in concentrated formic acid / sulfuric acid to obtain 6-nitroquinazoIin-4-one (2), which can be chlorinated in position 4 with thionyl chloride. The resulting 4-chloro-6-nitroquinazoline (3) is treated with 3-substituted aniline to obtain (4), wherein the nitro group at position 6 can be reduced with iron / acetic acid to obtain product (5).

Scheme 1

Reagents: in step (i): H2SO4 / formic acid, reflux; in phase (ii): SOCI2, dioxane, reflux; in step (iii): 3-substituted aniline, 2-propanol, 60 ° C; in phase (iv): Fe / acetic acid / ethanol / H20, reflux.

The synthesis of the compounds (7) of Formula (I) is carried out as described in scheme 2, by reaction of α, β-epoxy carboxylic derivatives (6) with amine (5), following the procedure described by Miki et al. for epoxyamide derivatives (Miki, T; Kori, M .; Tozawa, R .; Nakamura, M .; Sugiyama, Y .; Yukimasa, H. Synthesis of (4,1-benzoxazepine-3-yIidene) acetic acid derivatives and their inhibition of squalene synthase. Chem, Pharm. Bull. 2002, 50, 53-58).

Scheme 2

Reagents: in step (i) dichloromethylenedimethyliminium chloride, NaHC03, dichloromethane, 0 ° C.

The compounds (9) of Formula (II) are synthesized as described in scheme 3. The amine (5) is acylated through the corresponding β-phenoxyacetic acid (8) in the presence of PCI5.

Scheme 3

Reagents in step (i) PCI5, dichloromethane, reflux.

The ethyl ester of the (3-oxoisothiazol-2 (3 / - /) - il) acetic acid (10) (Lewis, S. N .; Miller, G. A .; Hausman, M .; Szamborski, E. C. 4-isothiazolin-3-ones . A generai synthesis from 3,3'-dithiodipropionamides. J. Heterocycl. Chem. 1971, 8, 571-580) is hydrolyzed with trifluoroacetic acid (1 M solution) to obtain the free acid (11), which has been condensed with the amine (5) to obtain the derivatives (12) of Formula (II), scheme 4.

Scheme 4

Reagents, in step (i): trifluoroacetic acid solution 1M, reflux; in step (ii): dicyclohexylcarbodiimide (DCC), dimethylformamide, from 0 ° C to room temperature.

The compounds (14) with Formula (IV) are synthesized as described in scheme 5. The amine (5) is treated with the acyl chloride of the appropriate (3-oxo-1,2-benzisothiazoIin-2-yl) acetic acid (13). The acids with formula (13) have been synthesized with procedures described in the literature (Bordi, F .; Catellani, P. L; Morini, G .; Piazzi, P. V .; Silva, C .; Barocelli, E .; Chiavarini, M. 4- (3-Oxo-1, 2-benzisothiazolin-2-yl) alkanoic, -phenylalkanoic, and -phenoxyalkanoic acids: synthesis and antiinflammatory, analgesie and antipyretic properties. Farmaco 1989, 44, 795-807).

Scheme 5

Reagents: in step (i): PCI5, dichloromethane, reflux.

The ester of 1, 2,4-thiadiazoI-5-carboxylic acid (15) (Howe, R. K .; Franz, J. E. Nitrile suphides. Synthesis of 1,2,4-thiadiazoles. J. Org. Chem. 1974, 39 , 962-964) reacts with the amine (5) in the presence of potassium tert.-butoxide (1M solution) with the formation of the products (16) having general formula (V). The reaction is carried out with microwave heating (scheme 6).

Scheme 6

Reagents: (i) potassium tert.-butoxide, dimethylformamide, microwave, 100 ° C. The / \ / - (phenoxycarbonyl) glycine derivative (17) and the amine (5) react in the presence of dicyclohexylcarbodiimide (DCC) with the formation of compounds (18) of Formula (VI), as described in scheme 7.

Scheme 7

Reagents ·, in step (i): dicyclohexylcarbodiimide (DCC), dimethylformamide, from 0 ° C to room temperature.

The compounds (21) of Formula (VII) are synthesized as described in scheme 8. The amine (5) is acylated with 3-chloropropionyl chloride (19) to obtain the intermediate 3-chloropropionamide (20). Subsequently, the treatment with the appropriate secondary amine leads to the product 3-aminopropionamide (21) in good yields.

Reagents ·, in phase (i): reflux; in phase (ii): NHR27R28, Nal, ethanol, reflux. The compounds of the invention can contain stereogenic centers and therefore can be prepared and / or isolated both as single stereoisomers and as their enantiomeric or diastereoisomeric mixtures. Both the aforementioned single stereoisomers and their enantiomeric or diastereoisomeric mixtures therefore fall within the scope of the invention claimed herein.

In some cases, the compounds of the invention contain a basic -NH- group which can form addition salts with acids, in particular with pharmaceutically acceptable acids.

The present invention relates to new compounds derived from 4-anilinoquinazoline that can be used as irreversible inhibitors of EGFR with antiproliferative activity that can be used in the treatment of disorders associated with an altered activity of the EGF receptor. In particular, the compounds of the present invention can be used as therapeutic agents for the treatment of pathologies, such as, for example, various tumor forms of epithelial origin, in which there is an alteration in the functioning of receptors with tyrosine kinase activity of the family by EGFR.

The invention therefore concerns the use of these compounds in the treatment of pathologies in which there is an alteration in the functioning of receptors with tyrosine kinase activity of the EGFR family. Preferably, the invention therefore concerns the use of such compounds for the treatment of various tumor forms of epithelial origin. More preferably, the invention also relates to the use of such compounds in the treatment and prevention of cell proliferation disorders such as papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytoma , pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma.

Without being bound to any theory, it is believed that the ability of the reactive groups considered to covalently bind Cys773 of EGFR and Cys784 of erbB2 within the binding site for ATP, makes the compounds considered irreversible inhibitors. The reactive groups considered can give reactions of addition (I, III, IV, V), or of nucleophilic substitution (II, VI) with the cysteine residues present in the catalytic site of the target protein. The β-aminocarboxylic derivatives (VII), following β elimination and formation of the corresponding acrylamide derivative, can interact with nucleophiles by Michael addition reactions 1, 4.

The following examples illustrate in a non-limiting way the preparation of the compounds of the invention according to the procedures described above

Experimental part

Example 1:

Preparation of the ethyl ester of trans- (2R, 3R) -3- (4- (3-bromoanilino) quinazolin-6-yl-carbamoyl) -2.3-epoxybutyric acid (7a)

A solution of mono ethyl ester of the acid between /? S- (2R, 3R) -2,3-epoxy-epoxy (Korn, A .; Rudolph-Bòhner, S; Moroder, L. A convenient synthesis of optically pure (2 R , 3R) -2,3-epoxysuccinyl-dipeptides. Tetrahedron, 1994, 50, 8381-8392) (350 mg, 2.19 mmol) and dichloromethylenedimethyl iminium chloride (356 mg, 2.19 mmol) in CH2CI2 (10 mL) was stirred for 1 hour at 0 ° C, and subsequently added with 6-amino-4- (3-bromoanilino) quinazoline (5a) (458 mg, 1.46 mmol) and NaFICOa (613 mg, 7.3 mmol). The mixture was stirred for 1 hour at 0 ° C, concentrated by distillation under reduced pressure, and the residue was diluted with a mixture of ethyl acetate and water. The organic phase was separated, anhydrified (Na2S04) and the solvent distilled under reduced pressure. The solid obtained was purified by washing with hot methanol. It appeared as a white solid; yield 60%; p. f. > 230 ° C dee .; MS (APCI): m / z 458.3, 459.2; <1> H NMR (DMSO-c / 6, 300 MHz) δ 1.26 (t, J = 7.1 Hz, 3H), 3.82 (d, J = 1.7 Hz, 1 H), 3.93 (d, J = 1.7 Hz, 1 H), 4.23 (q, J = 7.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 1 H), 7.35 (t, J = 7.9 Hz, 1 H), 7.92-7.82 (m, 3H), 8.16 (b s, 1H), 8.60 (s, 1 H), 8.72 (d, J = 1.3 Hz, 1 H), 9.93 (b s, 1H), 10.80 ( b s, 1 H).

Example 2:

Preparation of the ethyl ester of trans- (2S, 3S) -3- (4- (3-bromoanilino) quinazolin-6-yl-carbamoyl) -2.3-epoxybutyric acid (7b)

Following the procedure indicated in Example 1, but starting from the monoethyl ester of the frans- (2S, 3S) -2,3-epoxy-epoxy-epoxy-succinic acid, the title compound was obtained (Tamai, M .; Yokoo, C .; Murata , M .; Oguma, K .; Sota, K .; Sato, E .; Kanaoka, Y. Efficient synthetic method for ethyl (+) - (2S, 3S) -3 - [(S) -3-methyl-1 - (3-methylbutylcarbamoyl) butylcarbamoyl] -2-oxiranecarboxylate (EST), a new inhibitor of cysteine proteinase. Chem. Pharm. Bull. 1987, 35, 1098-1104. Hebicidal (1, 2,4) thiadiazoles. European Patent EP0726260 A1 of 08/02/1995.). The residue was purified by chromatography (Si02, eluent CH2CI2 / CH3OH from 99: 1 to 97: 3). White solid; yield 66%; p. f. > 230 ° C; MS (APCI): m / z 457.9, 459.3; <1> H NMR (DMSO-c / 6, 300 MHz) δ 1.26 (t, J = 7.2 Hz, 3H), 3.81 (d, J = 1.7 Hz, 1H ), 3.92 (d, J = 1.7 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 1 H), 7.35 (t, J = 7.8 Hz, 1 H ), 7.80-7.87 (m, 2H), 7.89 (dd, J = 9.0, 2.2 Hz, 1H), 8.16 (s, 1 H), 8.60 (s, 1 H), 8.72 (s, 1 H), 9.94 (br s, 1 H), 10.80 (br s, 1 H).

Example 3:

Preparation of compound A / - (4- (3-Bromoanilino) quinazolin-6-yl) -2-phenoxyacetamide (9a)

Phenoxyacetic acid (360 mg, 2.37 mmol) was added to a suspension of PCI5 (490 mg, 2.37 mmol) in CH2CI2 (15 mL) kept stirred at room temperature. The mixture was heated under reflux for 30 min and, after cooling to room temperature, 6-amino-4- (3-bromoanilino) quinazoline (5a) (500 mg, 1.59 mmol) was added in portions in 10 minutes. The reaction mixture was then refluxed for 2 hours, cooled on ice and 2.5 mL of water was added. After 30 minutes, the mixture was neutralized by adding a solution of Na2C03- The precipitated product was separated by filtration, purified by chromatography (S1O2, eluent CH2CI2 / CH3OH from 99: 1 to 95: 5), and crystallized from ethanol /water. White crystals were obtained; yield 77%; p. f. 212 ° C; MS (APCI): m / z 449.0, 451.0; <1> H NMR (DMSO-c / 6, 300 MHz) δ 4.79 (s, 2H), 6.99 (t, J = 7.4 Hz, 1H), 7.05 (d , J = 7.8 Hz, 2H), 7.27-7.36 (m, 4H), 7.80 (d, J = 8.9 Hz, 1 H), 7.85 (d, J = 7.9 Hz, 1 H), 7.95 (dd, J = 9.0, 2.1 Hz, 1H), 8.16 (s, 1H), 8.58 (s, 1H), 8.74 (d, J = 1.7 Hz, 1H), 9.93 (s, 1H), 10.44 (s, 1H) .

Example 4:

Preparation of compound A / - (4- (3-Bromoanilino) quinazolin-6-yl) -2- (4-fluorophenoxyOacetamide (9b)

Following the procedure indicated in example 4, the title product was obtained, which was purified by chromatography (YO2, eluent CH2CI2 / CH3OH from 99: 1 to 97: 3), and crystallized from ethanol / water. It appeared as white crystals; yield 67%; p. f. 224 ° C; MS (APCI): m / z 467.3, 469.1; <1> H NMR (DMSO-d6, 300 MHz) δ 4.78 (s, 2H), 7.07 (dd, J = 9.2, 4.3 Hz, 2H), 7.15-7.21 (m, 2H), 7.27-7.37 (m, 2H), 7.85 (m, 2H), 7.95 (dd, J = 8.2, 1.4 Hz, 1H), 8.16 (b s, 1H), 8.59 (s, 1H), 8.74 (b s, 1H), 9.93 (b s, 1 H), 10.44 (b s, 1 H).

Example 5:

Preparation of the compound /V-(4-(3-Bromoanilino)chinazolin-6-il)-2-(2.3.4.5.6-pentafluorophenoxy)acetamide (9c)

Following the procedure indicated in example 4, the title product was obtained, which was purified by chromatography (Si02, eluent CH2CI2 / CH3OH from 99: 1 to 97: 3), and crystallized from ethanol / water. The product appeared as white crystals; yield 50%; p. f. 220-221 ° C; MS (APCI): m / z 539.1, 541.1; <1> H NMR (DMSO-c / 6, 300 MHz) δ 4.99 (s, 2H), 7.24-7.34 (m, 2H), 7.75-7.87 m, 3H ), 8.11 (s, 1H), 8.55 (s, 1H), 8.66 (s, 1H), 9.91 (b s, 1H), 10.50 (b s, 1H).

Example 6:

Preparation of compound A / - (4- (3-Bromoanilino) quinazolin-6-yl) -2- (3-oxoisotiazol-2 (3 / - /) - yl) acetamide (12a)

a) Preparation of (3-oxoisothiazol-2 (3 / - /) - il) acetic acid (11a)

A solution of (3-oxoisotiazol-2 (3H) -yl) acetic acid (10) ethyl ester (200 mg, 1.07 mmol) in 1M trifluoroacetic acid (27 mL, 27 mmol) was refluxed for 12 hours . The solvent was evaporated by distillation under reduced pressure and the residue (11) was used in the subsequent reaction without further purification. Quantitative yield; <1> H NMR (DMSO-c / 6, 300 Mz) δ 4.41 (s, 2H), 6.19 (d, J = 6.2 Hz, 1 H), 8.51 (d, J = 6.2 Hz, 1 H ).

b) A / - (4- (3-Bromoanilino) quinazolin-6-yl) -2- (3-oxoisotiazol-2 (3 / - /) - yl) acetamide (12a)

To a solution of 6-amino-4- (3-bromoane) quinazoline (5a) (200 mg, 0.64 mmol) in dimethylformamide (3 mL) cooled in an ice-water bath, acid was added (11) (152 mg, 0.96 mmol) and dicyclohexylcarbodimide (DCC) (208 mg, 1.02 mmol). The mixture was then stirred at room temperature for 16 hours. The suspended solid was removed by filtration, and the solution distilled under reduced pressure to obtain the crude product 13a, which was purified by chromatography (SiO2, eluent ethyl acetate, then ethyl acetate / CH30H at 90:10), and crystallized from ethanol / water. The product appeared as white crystals; yield 40%; p. f. > 230 ° C; MS (APCI): m / z 456.1, 458.3; <1> H NMR (DMSO-c / 6, 300 MHz) δ 4.65 (s, 2H), 6.24 (d, J = 6.2 Hz, 1H), 7.28 (d , J = 8.0 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.79-7.85 (m, 3H), 8.10 (s, 1H), 8.55 (d, J = 6.2 Hz, 1H), 8.55 (s, 1 H), 8.71 (s, 1 H), 9.94 (b s, 1 H), 10.69 (b s, 1 H).

Example 7:

Preparation of compound A / - (4- (3-bromoanilino) quinazolin-6-ivD-2- (3-oxo-1, 2-benzoisothiazolin-2 (3 / - /) - il) acetamide (14a)

Starting from 6-amino-4- (3-bromoanilino) quinazoline (5a) and acid (13), following the procedure indicated in example 4, the title product was obtained, which was purified by chromatography (Si02 , eluent ethyl acetate / n-hexane 99: 1), and crystallized from methanol. White crystals; yield 45%; p. f. 238 ° C; MS (APCI): m / z 506.1, 508.1; <1> H NMR (DMSO-c / 6, 300 MHz) δ 4.75 (s, 2H), 7.25-7.31 (m, 2H), 7.43 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.76-7.83 (m, 3H), 7.88 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H) , 8.09 (s, 1H), 8.54 (s, 1H), 8.72 (s, 1H), 9.89 (b s, 1H), 10.66 (b s, 1H).

Example 8:

Preparation of compound A / - (4- (3-Bromoanilino) quinazolin-6-yl) -3-isopropyl-1.2,4-thiadiazol-5-carboxamide (16a)

To a mixture of 6-amino-4- (3-bromoane) quinazoline (5a) (0.235 g, 0.75 mmol) and ethyl ester of 3-isopropyl-1,2,4-thiodiazol-5-carboxylic acid (15) (0.150 g, 0.75 mmol) (Hebicidal (1,2,4) thiadiazoles. European Patent EP0726260 A1 dated 08/02/1995) in dimethylformamide (1 mL) 0.75 ml_ of a 1M solution of tert.-potassium butoxide are added in tetrahydrofuran (0.75 mmol). The reaction is heated to 100 ° C for 10 min. with microwave heating at 150 watts. The mixture is then diluted with water and extracted with ethyl acetate. The organic phase is washed with water, brine, anhydrified (Na2S04) and the distilled solvent at reduced pressure. The product is purified by chromatography (Si02, eluent CH2CI2 / CH3OH 98: 2), and crystallized from methylene chloride. Yellow crystals; yield 40%; p. f. 224 ° C; MS (APCI): m / z 469.0, 471.0. <1> H NMR (DMSO -d6, 300 MHz) δ 1.47 (d, J = 6.9 Hz, 6H), 3.43 (sep, J = 6.9 Hz, 1H), 7.34 (dt, J = 8.2, 1.5 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.93 (d, J = 7.9Hz, 1H), 8.20-8.23 (m, 2H), 8.68 (s, 1H), 8.93 (d, J = 1.8 Hz, 1H), 10.01 (s, 1H), 11.33 (brs, 1 H).

Example 9:

Preparation of the compound phenyl 2- (4- (3-bromoanilino) quinazolin-6-ylamino) -2-oxoethylDcarbamate (18a)

Starting from 6-amino-4- (3-bromoanilino) quinazoline (5a) and acid (17), following the procedure indicated in example 6, the title product was obtained, which was purified by chromatography (S1O2, eluent ethyl acetate), and crystallized from ethanol / water. The product appeared as a white solid; yield 55%; p. f. . > 250 ° C (dee,); MS (APCI): m / z 492.1. <1> H NMR (DMSO-c / 6, 300 MHz): δ 4.20 (s, 2H), 6.73-6.78 (m, 3H), 7.15 (t, J = 8.6 Hz, 2H), 7.32 (dt, J = 8.1, 1.6 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.82-7.93 (m, 3H), 8.21 (s, 1H), 8.50 (s , 1H), 8.57 (d, J = 1.7 Hz, 1H), 8.70 (s, 1H), 9.32 (s, 1H), 9.98 (b s, 1H).

Example 10:

Preparation of compound A / - (4- (3-bromoanilino) quinazolin-6-yl) -3- (dimethylamino) propionamide (21 a)

a) Preparation of compound A / - (4- (3-bromoanilino) quinazolin-6-iD-3-chloropropanamide (20a)

A suspension of amine (5) (300 mg, 0.95 mmol) and 3-chloropropionyl chloride (3 mL, 31.28 mmol) was refluxed and stirred for 16 hours. After cooling to room temperature, the solid was collected by filtration, washed with ethyl ether and dried under vacuum. The product was then purified by chromatography (Si02, eluent CH2CI2 / CH3OH from 98: 2 to 95: 5). A light yellow solid was obtained; yield 80%; MS (APCI): m / z 409.3, 407.4. <1> H NMR (CDCI3, 300 MHz): δ 2.96 (t, J = 6.2 Hz, 2H), 3.95 (t, J = 6.2 Hz, 2H), 7.31 -7.33 (m, 2H), 7.78 (m, 3H), 8.14 (s, 1 H), 8.55 (s, 1 H), 8.72 (s, 1 H).

b) Preparation of compound A / - (4- (3-bromoanilino) quinazolin-6-yl) -3- (dimethylamino) propanamide (21 a)

Dimethylamine (1.6 mL of a 33% v / v solution in absolute EtOH) was added in 15 min to a suspension of 3-chloropropionamide (20a) (290 mg, 0.714 mmol) and Nal (75 mg, 0.5 mmol) in 20 mL of absolute EtOH stirred under reflux. After 8 hours under reflux, the reaction mixture was cooled to 0 ° C, basified by the addition of KOH (1.48 g, 26.42 mmol), stirred at 0 ° C for 1 hour, and the solvent evaporated by distillation under reduced pressure. The solid was then extracted with ethyl acetate, the organic phase washed with brine, anhydrified (Na2SO4) and the solvent distilled under reduced pressure. The product was purified by chromatography (S1O2, eluent CH2CI2 / CH3OH from 100: 0 to 70:30), and crystallized from ethyl acetate / hexane. A yellow solid product was obtained; yield 86%; p. f.

172 ° C; MS (APCI): m / z 414.4, 416.4. <1> H NMR (DMSO-d6, 300 MHz) δ 2.35, (s, 6H), 2.65 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 6.5 Hz, 2H), 7.29-7.31 (m, 2H), 7.74 (m, 3H), 8.12 (b s, 1 H), 8.53 (s, 1 H), 8.66 (b s, 1 H).

Example of inhibition

The following compounds were used for the inhibition test:

Preparation of compounds. Compounds 7a, 9c and 21a were prepared in Examples 1, 5,10, respectively. Compounds 5a, PD168393 and gefitinib were used as known derivatives and were synthesized as described in the literature. For all the assays, the compounds were dissolved in DMSO at a concentration of 10 mM and subsequently diluted in the culture medium to obtain final concentrations of DMSO always lower than 0.1% (v / v). Control samples are treated with an equal amount of DMSO.

Cell cultures. Human carcinoma cell lines A431 (cervix) and H1975 (lung cancer) were cultured in Dulbecco's modified Minimum Essential Medium (D-MEM) 4.5 g / l glucose, and RPMI, supplemented with 10% serum, respectively. of fetal calf (FCS) and supplemented with the antibiotics Penicillin (100 Ul / ml) and Streptomycin (100 pg / ml). A431 cells express high levels of unmutated EGFR, while H1975 cells carry the T790M mutation in the intracellular portion of the receptor, responsible for decreased sensitivity to gefitinib treatment. The cultures were kept in a thermostat at 37 ° C in air saturated with water vapor and enriched with 5% of CO2.

Autophosphorylation inhibition test. The inhibition of EGFR autophosphorylation to erbB2 in A431 and H1975 whole cells was determined using specific anti-tyrosine phosphorylated antibodies by Western blot technique. Cells were incubated 1 h with the indicated inhibitor concentrations, before being stimulated with EGF (0.1 pg / ml) for 5 min. Equal aliquots of cellular iisate were analyzed by Western blot using specific anti-phosphorylated tyrosine antibodies (Cavazzoni, A .; Petronini, P.G .; Gaietti, M .; Roz, L; Andriani, F .; Carbognani, P .; Rusca, M .; Fumarola, C .; Alfieri, R .; Sozzi, G. Dose-dependent effect of FHIT-inducible expression in Calu-1 lung cancer celi line. Oncogene 2004, 23, 8439-46). To evaluate the irreversible effect, a second set of cells was incubated 1 h with the indicated concentrations of compound, then the inhibitor was removed from the medium and the cells were subjected to a series of washes for 8 h before being stimulated. with EGFR (0.1 pg / ml) for 5 min. The cells were then analyzed by Western blot, as previously described.

Cell proliferation inhibition test. The viability of the cell cultures was assayed using the MTT (3 (4,5-dimethylthiazol-2-yl) -diphenyltetrazolium bromide in formazan; Sigma, Dorset, UK) viability assay, as previously described (Cavazzoni, A .; Petronini, P.G .; Gaietti, M .; Roz, L .; Andriani, F .; Carbognani, P .; Rusca, M .; Fumarola, C .; Alfieri, R .; Sozzi, G. Dosedependent effect of FHIT -inducible expression in Calu-1 lung cancer celi line. Oncogene 2004, 23, 8439-46), after the cells were incubated 72 h with the indicated concentrations of compound.

The compounds object of the present invention showed inhibitory activity on EGFR autophosphorylation, both on a cell line sensitive to gefitib treatment (A431 cells) and on a cell line resistant to gefitinib treatment (H1975 cells). Furthermore, the compounds of the invention showed antiproliferative activity in the considered models. Finally, in the lung tumor model used, the compounds of the invention showed inhibitory activity on the autophosphorylation of erbB2. By way of example, the results obtained for some compounds object of the present invention are reported in Table 1 and Figures 2-4. The results were compared with those obtained for the reference compounds 5a and PD168393 in the same assays.

Table 1: IC50 values (μΜ) of inhibition of EGFR autophosphorylation and proliferation of A431 and H1975 cells.

Concentration inhibiting 50% EGFR autophosphorylation activity in A431 cells incubated for 1 h with various concentrations of compound and, immediately thereafter, stimulated with EGF.

<b> Concentration that inhibits 50% of the EGFR autophosphorylation activity in A431 cells incubated for 1 hour with various concentrations of compound and stimulated with EGF 8 hours after the removal of the inhibitor from the medium.

Concentration that inhibits proliferation of A431 cells by 50%. Cell proliferation is determined by means of the MTT viability assay after treatment for 72 h with compound concentrations between 0.01 and 10 μΜ. <d> Concentration that inhibits the proliferation of H1973 cells by 50%. Cell proliferation is determined by the MTT viability assay after treatment for 72 h with compound concentrations between 0.01 and 20 μΜ.

The effect of compound 7a on EGFR autophosphorylation in A431 cells was reported in Figure 2. When cells are treated with the reported concentrations of compound 7a for 1 h and subsequently stimulated with EGF, dose-dependent inhibition was achieved. deH'autophosphorylation of EGFR with IC5014 nM (Figure 2a and Table 1). As a demonstration of the irreversible effect, when the A431 cells were incubated 1 h with compound 7a and subsequently washed for 8 h, before being stimulated with EGF, a dose-dependent inhibition of EGFR autophosphorylation with IC50168 was obtained. nM (Figure 2b and Table 1). The results obtained were comparable to those of the acrylamide derivative PD1 68393 obtained under the same conditions (Table 1).

Compound 7a exhibited antiproliferative activity on A431 cells with an IC50 of 1.97 μΜ (Table 1). Compound 7a also showed inhibitory activity on H1975 cell proliferation with IC506.76 μΜ (Figure 3 and Table 1). Finally, in model H1975, 7a inhibited EGFR and erbB2 (Figure 4) proving capable of overcoming the resistance that this cell line develops towards gefitinib.

Claims (17)

CLAIMS 1. A derivative of 4-anilinoquinazoline of formula (A) wherein X represents hydrogen, bromine, chlorine, fluorine, ethynyl or methyl; A is a residue chosen from the group consisting of the) in which Y represents -CO- or -CHR-i-; Z represents -OR2, -SR2 or -NR2R3; R-i, R2 and R3 represent, independently of each other, hydrogen, (C-i. 6) alkyl, (CH2) n-COOR4, (CH2) n-CONR4R5, (CH2) n-NR4R5 or (CH2) n-morpholine; R4 and R5 independently represent hydrogen or (C1-6) alkyl; n can be an integer chosen from 0, 1, 2, 3, 4, 5, and 6. ii) in which R6 represents hydrogen or (C 1-6) alkyl; R7, Re. R91R10e Rn represent, independently of each other, hydrogen, chlorine, fluorine, the radical CF3, -N02 or -ORI2; RI2 represents hydrogen, (C1-6) alkyl, (CH2) n-COORi3, (CH2) n-CONR13R14, (CH2) n-NRi3R14 or (CH2) n-morpholine; RI3 and R14 represent, independently of each other, hydrogen or (C-i. 6) alkyl; and n is an integer chosen from 0, 1, 2, 3, 4, 5, and 6. iii) in which Ri5> represents hydrogen, (C1-6) alkyl, (CH2) n-COOR18, (CH2) n-CONRi8Ri9, (CH2) n-NRi8Ri9 or (CH2) n-morpholine; R161RI7, Rie and R19 represent, independently of each other, hydrogen or (C1-6) alkyl; iv) R6 represents hydrogen or (C1-6) alkyl; W represents -C = or -N =; R2o, R21. R22 and R23 represent, independently of each other, hydrogen, chlorine, fluorine, the radical -CF3, -N02, (Ci-6) alkyl. v) in which R24 represents (C1-6) alkyl, (CH2) n-COOR25, (CH2) n-CONR25R261 (CH2) n-NR25R26 or (CH2) n-morpholine; R25 and R26 represent, independently of each other, hydrogen or (C-i- 6) alkyl; you) in which R6 represents hydrogen or (C 1 6) alkyl; Q represents an aryl or heteroaryl group, optionally substituted with one or more substituents chosen, independently of each other, from hydrogen, chlorine, fluorine, the radical CF3, -N02 or -OR-I2; RI2 represents hydrogen, (Ci-6) alkyl, (CH2) n-COORi3, (CH2) n-CONRi3R-i4, (CH2) n-NRi3Ri4 or (CH2) n-morpholine; RI3 and R14 represent, independently of each other, hydrogen or (Ci. 6) alkyl; and n is an integer chosen from 0, 1, 2, 3, 4, 5, and 6. vii) R27 and R28 can represent hydrogen or (C1-6) alkyl; -NR28R2gmay be morpholine. 2. The 4-anilinoquinazoline derivative of claim 1, which is a compound in which the substituent (A) is the substituent i), i.e. a derivative of Formula (I), Formula (I) in which: X, Y and Z have the meanings indicated for formula (A). 3. The 4-anilinoquinazoIine derivative of claim 1, which is a compound in which the substituent (A) is the substituent ii), i.e. a derivative of Formula (II) Formula (II) where X, R6, R7, R8, Rg, R-10 and R-n have the meaning indicated in formula (A). 4. The 4-anilinoquinazoIine derivative of claim 1, which is a compound in which the substituent (A) is the substituent iii), i.e. a derivative of Formula (III) Formula (III) where X, R15, R-16, R17, R-ιβ and R-ig have the meaning indicated in formula (A). 5. The 4-anilinoquinazoline derivative of claim 1, which is a compound in which the substituent (A) is the substituent iv), i.e. a derivative of Formula (IV) Formula (IV) where X, R6, W, R2O, R21, R22 and R23 have the meaning indicated in formula (A). 6. The 4-anilinoquinazoline derivative of claim 1, which is a compound in which the substituent (A) is the substituent v), i.e. a derivative of Formula (V) Formula (V) where X, R24, 13⁄45 and R26 have the meaning indicated in formula (A). 7. The 4-anilinoquinazoline derivative of claim 1, which is a compound in which the substituent (A) is the substituent vi), i.e. a derivative of Formula (VI) Formula (VI) where X, R6, Q have the meaning indicated in formula (A). 8. The 4-anilinoquinazoIine derivative of claim 1, which is a compound in which the substituent (A) is the substituent vii), i.e. a derivative of Formula (VII) Formula (VII) in which X, R6, R27 and R28 have the meaning indicated in formula (A). The 4-anilinoquinazoline derivative of claim 1, which is is a compound in which the substituent A is selected from the group consisting of: vii) The 4-anilinoquinazoline derivative of claim 9, wherein the substituent A is the substituent i). 11. The 4-anilinoquinazoline derivative of claim 10 which is the ethyl ester compound of trans- (2RI3R) -3- (4- (3-bromoanilino) quinazolin-6-yl-carbamoyl) -2,3-epoxybutyric acid . The 4-anilinoquinazoline derivative of claim 1, which is a compound selected from the group consisting of: - ethyl ester of trans- (2R, 3R) -3- (4- (3-bromoanilino) quinazolin-6-ylcarbamoyl) -2,3-epoxybutyric acid (7a); - ethyl ester of trans- (2S, 3S) -3- (4- (3-bromoanilino) quinazolin-6-ylcarbamoyl) -2,3-epoxybutyric acid (7b) acid; - N- (4- (3-Bromoanilino) quinazolin-6-yl) -2-phenoxyacetamide (9a); - N- (4- (3-Bromoanilino) quinazolin-6-yl) -2- (4-fluorophenoxy) acetamide (9b) - N- (4- (3-Bromoanilino) quinazolin-6-yl) -2- (2,3,4,5,6-pentafluorophenoxy) acetamide (9c); - N- (4- (3-Bromoanilino) quinazolin-6-yl) -2- (3-oxoisotiazol-2 (3H) -yl) acetamide (12a) -N- (4- (3-bromoanilino) quinazolin-6 -iyl) -2- (3-oxo-1,2-benzoisothiazolin-2 (3H) -yl) acetamide (14a): - N- (4- (3-Bromoanilino) quinazolin-6-yl) -3-isopropyl-1,2,4-thiadiazol-5-carboxamide (16a); - phenyl 2- (4- (3-bromoanilino) quinazolin-6-ylamino) -2-oxoethyl) carbamate (18a): - A / - (4- (3-bromoanilino) quinazolin-6-yl) -3- ( dimethylamino) propionamide (21 a); - A / - (4- (3-bromoanilino) quinazolin-6-yl) -3-chloropropanamide (20a) - A / - (4- (3-bromoanilino) quinazolin-6-yl) -3- (dimethylamino) propanamide (21 a). 13. The 4-anilinoquinazoline derivative of claim 1, wherein (C-6) alkyl is an alkyl selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl and tert- butyl, n-pentyl, n-hexyl. The 4-anilinoquinazoline derivative of claim 1, wherein R4, R5; R13, R14 ;, Ri8> Ri9 '. R25.R26 in formula (A) or the fragment (CH2) n of the substituent (CH2) n-morpholine represent linear methylene, ethylene, propylene, butylene, pentylene or hexylene alkyl chains. 14. A derivative of 4-anilinoquinazoline of formula (A) according to any one of claims 1 to 13 for use as a medicament. 15. Use of a 4-anilinoquinazoline derivative according to any one of claims 1 to 13, for the manufacture of a medicament for the treatment and prevention of pathologies in which there is an alteration in the functioning of receptors with tyrosine kinase activity of the EGFR family. 16. Use according to claim 15, in which the pathologies in which there is an alteration in the functioning of receptors with tyrosine kinase activity of the EGFR family are tumor forms of epithelial origin. Use according to claim 15, wherein the pathology is selected from the group consisting of papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytoma, pancreatic cancer, gastric cancer , hepatocellular carcinoma, leukemia and lymphoma.