N-HYDROXY BENZAMIDES WITH ANT1TUMOUR ACTIVITY

Field of the invention

Object of the present invention are novel N-hydroxybenzamides useful for the treatment of pathologies characterised by abnormal cell proliferation, such as tumours. A further object of the present invention is the use of said N-hydroxybenzamides for the treatment of hyperproliferative pathologies in mammals, in particular in humans, and pharmaceutical compositions containing said compounds.

Background of the invention

Eukaryotic cells respond to exogenous stimuli activating a series of signal transduction cascades having cellular effects like proliferation, differentiation or apoptosis. Further to interaction of growth factors or cytokines, the transmission of the stimulating signals from receptors to the nucleus requires the regulation of a kinase family known as MAPKs (mitogen activated protein kinases) or ERKs (extracellular signal regulated kinases). For example, the ability of growth factors to promote proliferation depends on the activation of tyrosine-kinase receptors which recruit proteins of the Ras family and sequentially activate Raf (MAPK kinase kinase), MEK (MAPK kinase) and ERK (MAPK). The MAP kinase cascade plays an important role in directing the signals from different extra-cellular stimuli and protooncogenes towards the nucleus, where the activation of specific transcription factors results in cellular responses like proliferation, differentiation, survival and apoptosis.

The transduction signal cascade that involves Raf/MEK/ERK is one of the most important and best known as far as human tumours are concerned. Studies on tumour lines and biopsies from human tumours demonstrated that the ERK cascade is constitutively activated in lung, colon, pancreas, kidney

and ovary tumours (Hoshino et al, Oncogene 1999, 18, 813-822). It has been demonstrated that the activation of the Raf/MEK/ERK cascade is a necessary and sufficient condition for cell transformation (Mansour et al. Science, 1994, v. 265, pp. 966-970; Cowley et al, Cell, 1994, v. 77, pp. 841- 852; Brunet et al., Oncogene, 1994, v. 9, pp. 3379-3387). Moreover, oncogenic forms of Ras are present in about one third of human tumours, among them 50% of colon tumours, 30% of lung tumours and more than 9 out of 10 pancreas tumours (Bos, J. L. Cancer Res. 1989, 49(17), 4682- 4689). 60% of malignant melanomas and 40-70% of thyroid papillary tumours bear B-raf mutations (Davies, H et al., Nature 2002, 417(6892), 949-954; Cohen, Y et al, Cancer Res. 2003, 63(15), 4561-4567). The functional consequence of these mutations is a constitutively activated cascade of ERK kinase. MEK plays a pivotal role in this intracellular signals cascade and catalyzes the phosphorylation of ERK1 and ERK2 MAP kinases (Anderson et al., Nature 1990, v.343, pp. 651-653) which are the only MEK substrates identified so far (Seger et al., J. Biol. Chem., 1992, v. 267, pp. 14373-14381). Several studies demonstrated that MEK inhibition has potential therapeutic benefits. In particular, some MEK inhibitors arrest the growth of human tumours in experimental models (Sebolt-Leopold et al,, Nat. Med. 1999, 5, 810-816). Some MEK inhibitors, such as ARRY-142886 (Yeh, T. et al., Proc. Am. Assoc. Cancer Res. 2004, 45, Abst. 3888; Yeh, T et al., Proc. Am. Assoc. Cancer Res. 2004, 45, Abst. 3889; Lee, P. et al., Proc. Am. Assoc. Cancer Res. 2004, 45, Abst. 3890; Wallace, E. et al, Proceedings of the American Association of Cancer Research 2004, 45, Abs 3891; Winkler, J. D. et al.,16th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, Geneva, Switzerland, Sept 27-Oct 1 , 2004, Abst. 342), PD- 0325901 (Tecle, H et al., 29th American Chemical Society Medicinal Chemistry Symposium, Madison, Wl, June 27-JuIy 1 , 2004; Kaufman, M. D.

et ai., Proc. Am. Assoc. Cancer Res. 2004, 45, Abst. 2477) and CI-1040 (Waterhouse et al., Proceedings of the American Society for Clinical Oncology 2003, 22, Abs 816) have entered clinical studies as antitumour agents. However, CI-1040, the first MEK inhibitor to enter clinical trials, showed no efficacy in phase Il studies, where responses were not observed and only in few patients stabilisation of the pathological conditions was attained. The absence of clinical activity of CI-1040 is probably due to its low solubility, high metabolic clearance and low bioavailability. There is therefore the need to develop new MEK inhibitors with an improved pharmacological profile.

MEK inhibitors and their use in therapy have been disclosed in a number of patents and patent applications, for example US 5,525,625, WO 98/37881 , WO 99/01421 , WO 99/01426, WO 00/40235, WO 00/40237, WO 00/41505, WO 00/41994, WO 00/42002, WO 00/42003, WO 00/42022, WO 00/42029, WO 00/56706, WO 00/68199, WO 00/68200, WO 00/68201, WO 01/05390, WO 01/05391, WO 01/05392, WO 01/05393, WO 01/68619, WO 01/76570, WO 02/06213, WO 02/06520, WO 02/069960, WO 03/035626, WO 03/047523, WO 03/053960, WO 03/077855, WO 03/077914, WO 03/103717, WO 2004/005284, WO 2004/045617, WO 2004/056789, WO 2005/000818, WO 2005/007616, WO 2005/009975, WO 2005/011466, WO 2005/023759, WO 2005/023251 , WO 2005/028426, WO 2005/046665, WO 2005/051300, WO 2005/051301 , WO 2005/051302, WO 2005/051906, WO 2006/056427 e WO 2006/061712.

Description of the invention The present invention provides novel N-hydroxybenzamides, enantiomers and diastereoisomers thereof, as well as pharmaceutically acceptable salts, pro-drugs and solvates able to inhibit MEK kinase and therefore useful for the treatment of tumours.

The compounds of the invention have the following general formula I:

wherein R1 is selected from hydrogen, trifluoromethyl, C1-10 alkyl, C-2-10 alkenyl,

C2-10 alkynyl, C3-10 cycloalkyl, C3-10 cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocycloalkyl, said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups being optionally susbstituted with one to three substituents independently selected from hydroxy, amino, alkyl or cycloalkyl;

R2 is selected from hydrogen, C1-8 alkyl, CO-C1-8 alkyl, aryl, aralkyl, or C3-10 cycloalkyl, wherein the alkyl, alkylcarbonyl, aryl, aralkyl or cycloalkyl groups are optionally substituted with one to three substituents independently selected from hydroxy, amino, alkyl or cycloalkyl; R3, R4 and R5 are independently selected from hydrogen, hydroxy, halogen, trifluoromethyl, C1-8 alkyl, C1-8 alkoxy, nitro, cyano or -(O) _m -CH2)n- R6 and -(NH)m-CH2) _n -R6 groups, wherein m is 0 or 1 , R6 is hydrogen, hydroxy, carboxy or NRaRb wherein Ra and Rb are independently selected from hydrogen and Ci _-4 alkyl or, together with the nitrogen atom they are bound to, form a pyrrolidine, piperidine, morpholine or piperazine ring; X is selected from O, NH, CH ₂ , CO;

A is an aromatic or aliphatic 5 to 6-membered heterocycle, containing one to three nitrogen, oxygen or sulphur atoms and optionally substituted on the carbon atoms with 1 , 2 or 3 R7 groups or, on the nitrogen atoms, with Ci- ₁₀ alkyl, aryl, arylalkyl groups or with oxygen atoms to give N-oxides;

R7 is independently selected from hydrogen, C1-20 alkyl, C2-20 alkenyl, Oz-ZQ alkynyl, -OR7a, -SR7a, -CN, nitro, azido, halogen, haloalkyl, -NH2, - NH(Ci-io alkyl), -N(Ci-io alkyl) ₂ , -NHC(=0)0-Ci-io alkyl, -NHC(=O)Ci-io alkyl, -COOH, -C(=O)O-Ci-io alkyl, -C(=O)Ci _-10 alkyl, -C(O)H, -S(=O)-Ci-io alkyl, - S(=O)2-Ci-io alkyl, -S(=O)~aryl, -S(=O)2-aryl, C3-10 cycloalkyl optionally substituted with 1 , 2 or 3 R8 groups, heterocyclyl optionally substituted with 1 , 2 or 3 R8 groups;

R7a is selected from H, trifluoromethyl, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-10 cycloalkyl, C3-10 cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocycloalkyl, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups are optionally substituted with one to three substituents independently selected from hydroxy, amino, alkyl or cycloalkyl;

R8 is independently selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, phenyl, halogen, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, carboxy, Ci _-4 alkyl-OC(=O)-, C1-4 alkyl-C(=O)-, aryl-OC(=O)-,

C _1-4 alkyl-OC(=O)NH-, aryl-OC(=O)NH-, Ci _-4 alkyl-C(=O)NH-, Ci _-4 alkyl-

C(=O)O-, (C1-4 alkyl-O)r- C1-4 alkyl, HO-(Ci _-4 alkyl-O)r- C1-4 alkyl-, -OH, -SH, -

CN, -N ₃ , -CNO, -CNS, Ci _-4 alkyl-S(=O)-, C1-4 alkyl-S(=O) ₂ -, H2NS(=O)- and H ₂ NS(=O) ₂ , wherein r is 1 or 2.

Preferred R1 groups are hydrogen, methyl, ethyl, tert-butyl, 2- hydroxyethyl, cyclopropylmethyl, 2-propenyl, carboxymethyl, (R)-2,3- dihydroxypropyl, (S)-2,3-dihydroxypropyl.

Preferred R2 groups are hydrogen, methyl and ethyl. Preferred R3, R4 and R5 groups are hydrogen, methyl, fluorine, chlorine, bromine, iodine.

Preferred A heterocycles containing from one to three nitrogen, oxygen, sulphur atoms are: pyrrole, furan, thiophene, pyrazole, thiazole,

oxazole, imidazole, isothiazole, isoxazole, 1 ,2,3-triazole, 1 ,2,4-triazole, 1 ,2,4- oxadiazole, 1 ,3,4-oxadiazole, 1 ,2,5-oxadiazolθ, 1 ,2,5-thiadiazole, 1,3,4- thiadiazole, tetrazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,4- triazine, 1,2,3-triazine, 1 ,2,4-triazine, 1,3,5-triazine, quinoline, and isoquinoline; even more preferred are pyrrole, furan, thiophene, quinoline and pyridine. Examples of pyridine rings are the pyridin-2-yl and the pyridin- 3-yl ring.

Preferred R7 groups are hydrogen, methyl, trifluoromethyl, fluorine, chlorine, bromine, iodine. Among X groups, the NH group is preferred when A is a pyridine ring, in particular a or quinoline ring and the -CH2- and -CO- groups are preferred when A is a thiophene ring.

Particularly preferred are compounds of formula (I) wherein X is NH and A is a pyridine ring, in particular a pyridin-2-yl and a pyridin-3-yl ring, more particularly a pyridin-3-yl ring, such as the compounds disclosed in examples 1-82, 84-127, 129-156, 158-185, and 187-219. Preferred are also the compounds of examples 83, 128 and 157, wherein A is a pyrazine ring, the compounds of Examples 8-10 and of Examples 220-222 wherein A is a quinoline ring, and also the compounds of examples 223-225, wherein A is a thiophene ring.

The compounds of the invention can be prepared from commercially available starting materials using synthetic methods well known to those skilled in the art.

A useful synthetic procedure for the preparation of the compounds of the invention wherein X is NH comprises the reaction of a heterocyclic amine of formula Il

A-NH ₂ (II) wherein A is as defined above

with a benzoic acid of formula III

wherein R3, R4 and R5 are as defined above and L is a leaving group, such as halogen or an activated hydroxy group, for example diethylphosphate, trimethylsilyloxy, p-nitrophenoxy, p-toluenesulphonyloxy or methanesulphonyloxy to give a heteroarylamino-benzoic acid of formula IV

(IV) wherein A, R3, R4 and R5 are as defined above, followed by treatment of the heteroarylamino-benzoic acid IV with a hydroxylamino derivative V

(V) to give the compounds of formula I wherein X is NH.

The reaction of the heterocyclic amine Il with benzoic acid III is carried out mixing benzoic acid III with equimolar amounts or with a slight excess of the heterocyclic amine III in an organic solvent such as tetrahydrofuran or toluene, in the presence of a base such as, for example, lithium diisopropylamide, n-butyl lithium, lithium hexamethyldisilazane, sodium hydride, sodium amide and the like. The reaction is usually carried out at a temperature comprised between about -78°C and about 80 ⁰ C and is usually

complete in a time comprised between four hours and four days.

Heteroarylamino benzoic acid IV is then reacted with hydroxylamine V in the presence of a condensing agent such as, 2-ethoxy-1-ethoxycarbonyl- 1 ,2-dihydroquinoline (EEDQ), I .S-dicyclohexylcarbodiimide (DCC), bromo-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), (benzotriazolyloxy)trispyrrolidinophosphonium hexafluorophosphate

(PyBOP), HOBt, EDCI, diphenylphosphinic chloride. The compound of formula IV and the compound of formula V are usually mixed in equimolar amounts, optionally in the presence of a base such as triethylamine or diisopropylamine, in an organic solvent such as dichloromethane, tetrahydrofuran, chloroform, xylene, followed by addition of an equimolar amount of the condensing agent. The reaction is usually complete in a time period ranging from ten minutes to two hours.

Alternatively, the heteroarylamino benzoic acid IV can be converted, with conventional methods, into the corresponding acyl chloride, mixed anhydride, esters such as, for example, the methyl ester, or activated esters such as, for example, the N-hydroxysuccinimido ester, p-nitrophenyl ester, pentafluorophenyl ester or a thioester, followed by addition of the compound of formula V. An alternative process for the preparation of the compounds of formula

I in which X is NH comprises the reaction of a compound of formula III with a compound of formula V, to give a hydroxamic derivative Vl

(VI) in which, R1 , R2, R3, R4, R5 and L are as defined above, followed by

reaction of the compound of formula Vl with a heterocyclic amine of formula Il to give compounds of formula I in which X is NH.

The reaction of compounds of formula III with compounds of formula V can be carried out according to the methods described above for the reaction of the compounds of formula IV with the compounds of formula V. The reaction of the compounds of formula Vl with the compounds of formula Il can be carried out according to the methods described above for the reaction between the compounds of formula Il with the compounds of formula IV.

A process useful for the preparation of certain compounds of formula I wherein one or more nitrogen atoms of the heterocyclic ring A are substituted with an oxygen atom to give an N-oxide group consists in the reaction of a heteroarylamino-benzoic acid of formula IV

(IV) wherein R3, R4 and R5 are as defined above and A' has the same meanings as A with the exception that A' does not contain N-oxide groups, with a reagent capable of converting one or more nitrogen atoms of A' into N- oxides, to give a compound of formula IV which is then converted in a N- oxide containing compound of formula I as described above. Still another process useful for the preparation of certain compounds of formula I wherein one or more nitrogen atoms of the heterocyclic ring A are substituted with an oxygen atom to give an N-oxide group consists in the conversion of an heteroarylamino-benzoic acid of formula IV into the corresponding methyl ester using standard esterification procedures to give a compound of formula IV"

(IV") wherein A', R3, R4 and R5 are as defined above, with a reagent capable of converting one or more nitrogen atoms of A' into N-oxides, to give a compound of formula IV"

(IV ¹ ") wherein A, R3, R4, and R5 are as defined above, which is then converted in a N-oxide containing compound of formula I by reaction with from one to three molar equivalents of a hydroxylamino derivative V in the presence of up to six molar equivalents of a trialkyl aluminium reagent such as, for example, trimethyl aluminium. The reaction is preferably performed in an aromatic solvent such as toluene at a temperature between 50 and 80 ⁰ C, preferably at 65 ⁰ C.

In some cases, the compound of formula V used in the condensation reaction with the compounds of formula IV or III may contain a conventional protective group. For example, protective groups than can be advantageously used for the protection of a hydroxy group are vinyl ethers, silyl groups, acetals, acetonides and carbamates. In this case the protective group can be removed using conventional methods known in organic chemistry.

The compounds of formula Il and III are known and are commercially available or can be prepared with known methods. The hydroxylamino derivatives of formula V are known or can be prepared with known methods, for example according to the procedures disclosed in WO 02/06213.

Thanks to their ability to selectively inhibit protein-kinases MEK1 and MEK2, the compounds of the invention are useful for the treatment of tumour

pathologies and other proliferative disorders. The compounds of the invention have been evaluated in biological tests conventionally used for evaluating the inhibition of protein kinases and measuring the cellular effects due to this inhibition. For example, the ability of the compounds of the invention to inhibit

MEK can be determined with the procedure disclosed in WO 2005/051906 or using a commercially available kit, for example the MEK Activity Assay Kit (Sigma, product code CS0490).

MEK1 kinase activity of the compounds of the invention can also be monitored by an enzyme-linked immunosorbent assay (ELISA) method essentially as described in R. Mallon et al. Anal. Biochem. 294, 48 (2001).

In these tests, the compounds of the invention show an IC _SO lower than 50 μM for the inhibition of MEK.

The antiproliferative effects of the compounds of the invention on human tumours lines from different cell histotypes (for example, colon, melanoma, lung, prostate, ovary, breast, leukemia, lymphoma, myeloma) can be evaluated with a number of methods well known to those skilled in the art, for example incubating the cells with the compounds for 144 hours and then determining their cytotoxicity with the MTT assay (Mosman, T., J. Immunolog. Methods, (1983), 65, 66; Green, L.M., J. Immunol. Methods, (1984), 70. 257-268). In this test the compounds of the invention showed IC50 values (compound concentration necessary to induce 50% cell death) lower than 50 μM.

As an alternative, tumour cells are incubated with the compounds for 96 hours and after this time cell proliferation is determined using the ATP lite cell proliferation kit (Packard).

The compounds of the invention have IC50 values (compound concentration necessary to induce 50% cell death) lower than 50 μM.

A further object of the invention are pharmaceutical compositions for

the treatment of hyperproliferative pathologies in a mammal, the compositions comprising a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, pro-drug or hydrate thereof, and one or more pharmaceutically acceptable excipients. These pharmaceutical compositions are particularly useful for the treatment of brain, lung, squamous cells, bladder, stomach, pancreas, breast, head, neck, kidney, ovary, prostate, colon-rectum, esophagus, testicles, thyroid tumours, gynaecological tumours and melanoma. According to a further aspect, the compositions can contain, further to a compound of formula I, another chemotherapeutic agent in such an amount to effectively inhibit, together with the compound of formula I, anomalous cell proliferation. Chemotherapeutic agents that can be effectively combined with the compounds of the invention are for example mitosis inhibitors, alkylating agents, antimetabolites, intercalating agents, antibiotics, growth factors inhibitors, cell cycle inhibitors, topoisomerase inhibitors, anti-hormonal and angiogenesis inhibitors. The compositions of the invention can be prepared with excipients and methods known to those skilled in the art, for example those described in Remington's Pharmaceutical Sciences Handbook, Mack Pub., N.Y., U.S.A..

A further object of the invention is a method for the treatment of hyperproliferative disorders in a mammal, the method comprising administering to said mammal a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, a pro-drug or a hydrate thereof. In an aspect, the method relates to the treatment of tumour pathologies of the brain, lung, squamous cells, bladder, stomach, pancreas, breast, head, neck, kidney, ovary, prostate, colon-rectum, esophagus, testicles, and thyroid, or for the treatment of gynaecological tumours.

The invention is illustrated in greater detail in the following experimental section.

EXPERIMENTAL SECTION

PREPARATION OF ACIDS OF FORMULA IV

Preparation 1a: 3,4-Difluoro-2-(3,5-dichloro-2-pyridylamino)benzoic acid

Under nitrogen atmosphere, a solution of 3,5-dichloro-2-pyridylamine

(1.63 g, 10 mmol; Aldrich, code 135925) and 2,3,4-trifluorobenzoic acid (1.76 g, 10 mmol; Aldrich, code 333824) in anhydrous THF (15 ml) cooled to -78°C is added drop by drop with a solution of 1.06 M of LiHMDS (lithium hexamethyldisilazane) in THF (28.4 ml, 30 mmol). At the end of the addition the reaction mixture is allowed to warm to room temperature and left under stirring for 24 hours. After evaporation of the solvent under reduced pressure, the residue is divided and taken up in HCI 1 M (50 ml) and ethyl acetate (2 x 50 ml). The organic phases are pooled and washed in sequence with water (50 ml), with a saturated NaCI solution (50 ml) and anhydrified over Na2SO ₄ . After evaporation of the solvent the residue is purified by chromatography on silica gel eluting with ethyl acetate and with a 9/1 ethyl acetate/methanol mixture. The product-containing fractions are pooled and the solvent is evaporated off to give 3,4-difluoro-2-(3,5-dichloro-2- pyridylamino)benzoic acid.

Preparation 1b: 3,4-Difluoro-2-(5-bromo-2-pyridylamino)benzoic acid

A solution of 2-amino-5-bromopyridine (980 mg, 5.68 mmol) in freshly distilled THF (7 ml) was cooled at -75"C and added dropwise over a 15 min period with a litium diisopropylamide solution (2M in THF/ept/ethylbenzene, 4.25 ml, 8.51 mmol). The reaction mixture was stirred at -75"C for 1 h and added dropwise over a 10 min period with a solution of 2,3,4-trifluorobenzoic acid (500 mg, 2.84 mmol) in THF (2 ml). The mixture was stirred at -70"C for 1 h, than reached room temperature over a 2 h period and than stirred at room temperature for 18 h. The reaction mixture was cooled at 0°C added with NH ₄ CI sat (3 ml), diluted with brine and extracted with ethyl acetate. The collected organic phase was washed twice with citric acid 2.5% solution, dried over anhydrous Na2SO4, filtered and evaporated from the solvent under reduced pressure. The crude solid was triturated with CH2CI2, filtered and the solid washed once with a CHaCb/petrol mixture and once with petrol ether. The expected product was obtained as brown solid (430 mg, 49% yield). ¹ H-NMR (d6-DMSO) 13.31 (s,1H,CONH); 9.19 (s,1 H ₁ Ph-NH-Ph); 8.12

(d,1 H,Py); 7.77 (m,1 H,Py); 7.70 (m,1 H,H ⁶ -Ph); 7.23 (m,1 H,H ⁵ -Ph); 6.93 (d,1 H ₍ J=8.80,Py).

HPLC-UV Rt = 6.76 m, 98.9% purity by UV 220 nm. MS [M-1] = 328 Preparation 1c: 3,4-Difluoro-2-(5-methyl-2-pyridylamino)benzoic acid

The compound was prepared and purified using the same procedure described in Preparation 1b using 2-amino-5-methylpyridine (614 mg, 5.68 mmol) as aminopyridine. The product was obtained as a white solid (480 mg, 75% yield).

¹ H-NMR (d6-DMSO) 13.4 (s,1H,CONH); 9.12 (s,1 H ₁ Ph-NH-Ph); 7.91 (d, 1 H ₁ Py); 7.72 (m,1H,H ⁶ -Ph); 7.46 (m,1H,Py); 7.14 (m,1H,H ⁵ -Ph); 6.87 (d,1H,J=8.55,Py); 2.17 (s,3H,CH ₃ ).

HPLC-UV Rt = 4.05 m, 98% purity by UV 220 nm.

MS [M-1] = 263

Preparation 1d: 3,4-Difluoro-2-(3-methy(-2-pyridylamlno)benzoic acid

The compound was prepared and purified using the same procedure described in Preparation 1 b using 2-amino-3-methylpyridine (614 mg, 5.68 mmol) as aminopyridine. The product was obtained as a white solid (405 mg, 54% yield).

¹ H-NMR (d6-DMSO) 13.7 (s,1H,CONH); 9.10 (s,1H, Ph-NH-Ph); 7.96 (d,1 H,Py); 7.78 (m,1 H,H ⁶ -Ph); 7.52 (m,1 H,Py); 7.18 (m.i H.H^-Ph); 6.84 (m ₎ 1H,Py); 2.27 (s,3H _J CH ₃ ).

Preparation Ie: 3,4-Difluoro-2-(6-trifluoromethyl-3- pyridylamino) benzoic acid

Step 1 : Preparation of 3-amino-6-trifluoromethylpyridine

N,N-dimethylformamide (300 ml), 3-aminopyridine (4.8 g, 51 mmol), and zinc powder (5.3 g, 81.5mmol) were placed in a flask equipped with a dry-ice condenser, and the mixture was stirred at ~60°C under a Ar

atmosphere. Then sulfur dioxide (1O g, 15 mmol) and trifluoroiodomethane (50 g, 255 mmol) were liquefied into the flask. The required amounts of these reagents were determined by weight differences of the small commercial cylinders containing the reagents. The resultant mixture was allowed to reach 23°C within 4 hours and then stirred under Ar atmosphere for an additional 16 hours. Workup involved acidification with aq. hydrochloric acid (20 ml, 20 mmol), removal of N,N-dimethylformamide and water on a rotary evaporator, neutralization of the resultant slurry with solid sodium carbonate, and then extraction with diethyl ether (3x100 ml). Concentration of the extract was followed by silica gel chromatography with petroleum ether/ethyl acetate (3:1) to afford 3-amino-6-trifluoromethylpyridine as n yellow oil (180 mg, yield: 2.2%), which was characterized by LC-MS.

Step 2: 3,4-Difluoro-2-(6-trifluoromethyl-3-pyridylamino)benzoic acid Into a three-neck-round-bottomed flask equipped with a magnetic stirrer and under Ar atmosphere was placed 2,3,4-trifluorobenzoic acid (195 mg, 1.11 mmol, 1eq.), 3-amino-6-trifluoromethylpyridine of step 1 (180 mg, 1.11 mmol, 1eq.), and dry THF (10 ml). After cooling this solution to -70 ⁰ C in a dry ice-acetone bath, LiHMDS (3.3 ml of a 1M solution in THF, 3.3 mmol, 3eq.) was added via syringe. The resulting solution was stirred at this temperature for 15 min. Then, the resulting mixture was allowed to reach ambient temperature overnight. The reaction mixture solution was quenched with 1N aqueous HCI to pH=1 and extracted with diethylether (3 ^χ 30 ml). The combined organic extracts were washed with brine and dried over MgSO4. The solvent was removed to afford yellow solid (50 mg, yield: 14%) and the crude product was directly used in the next step.

Preparation 1f: 3,4-Difluoro-2-(5~methyl-3-pyridylamino)benzoic acid

Step 1: 3-amino-5-methylpyridine A solution of 2-chloro-3-nitro-5-methylpyridine (1 g, 5.81 mmol, from

Alfa) was hydrogenated under a hydrogen balloon in methanol (10 ml) with 10% Pd/C (50 mg) overnight. The reaction mixture was filtered and concentrated. The residue was treated with DCM saturated with Et3N. The resultant slurry was filtered and concentrated. The crude product was directly used in the next step.

Step 2: 3,4-difluoro-2-(5-methyl-3-pyridylamino)benzoic acid Flask A: Into a three-neck-round-bottomed flask equipped with a magnetic stirrer and under Ar atmosphere was placed 2,3,4-trifluorobenzoic acid (374 mg, 2.1 mmol, 1eq.) and dry THF (10 ml). After cooling this solution to -70 ⁰ C in a dry ice-acetone bath, LiHMDS (2.1 ml of a 1 M solution in THF, 2.1 mmol, 1eq.) was added via syringe. The resulting suspension was stirred at this temperature for 15 min.

Flask B: Into a three-neck-round-bottomed flask equipped with a magnetic stirrer and under Ar atmosphere was placed 3-amino-5- methylpyridine of step 1 (230 mg, 2.1 mmol, 1eq.) and dry THF (10 ml). After cooling this solution to -70 ⁰ C in a dry ice-acetone bath, LiHMDS (4.2 ml of a

1M solution in THF, 4.2 mmol, 2eq.) was added via syringe. The resulting suspension was stirred at this temperature for 15 min. Then, the contents of

Flask A were transferred by cannula into Flask B and the resulting mixture was allowed to reach ambient temperature overnight. The brown solution was quenched with 1N aqueous HCI to pH=1 and extracted with diethylether

(3x50 ml). The combined organic extracts were washed with brine and dried over MgSO4. The solvent was removed to afford 3,4~difluoro~2-(5-methyl-3- pyridylamino)benzoic acid as a yellow solid (500 mg, yield: 90%) which was directly used in the next step. Preparation 1g: 3,4-Difluoro-2-(4-methyl-3-pyridylamiπo)benzoic acid

This product was obtained following the procedure described in Step 2 of Preparation 1f using as starting materials 2,3,4-trifluorobenzoic acid (1.6 g, 9.2mmol, 1eq.) and 3-amino-4-methylpyridine (1.0 g, 9.2mmol, 1eq.).

3,4-Difluoro-2-(4-methyl-3-pyridylamino)benzoic acid was obtained as a yellow solid (2 g, yield: 82%) and the crude product was directly used in the next step.

Preparation 1h: 3,4-Difluoro-2-(5-bromo-3-pyridylamino)benzoic acid

This product was obtained following the procedure described in Step 2 of Preparation 1f using as starting materials 2,3,4-trifluorobenzoic acid (1.01 g, 5.78 mmol, 1eq.) and 3-amino-5-bromopyridine (1.0 g, 5.78 mmol, 1eq; from Aldrich.). 3,4-Difluoro-2-(5-bromo-3-pyridylamino)benzoic acid was obtained as a yellow solid (1.8 g, yield: 95%) and the crude product was directly used in the next step

Preparation 1i: 3,4-Difluoro-2-(3-quinolylamino)benzoic acid

This product was obtained following the procedure described in Step 2 of Preparation 1f using as starting materials 2,3,4-trifluorobenzoic acid (500 mg, 2.84 mmol, 1 eq.) and 3-aminoquinoline (410 mg, 2.84 mmol, 1 eq; from Aldrich.). 3,4-Difluoro-2-(3-quinolylamino)benzoic acid was obtained as a brown solid (138 mg, yield: 16%) and the crude product was directly used in the next step.

Preparation 11: 3,4-Difluoro-2-(4-methyl-3-quinolylamino)benzoic acid

Step 1 : 2-amimoacetophenone

To a solution of 2-nitroacetophenone (1.0 g, 6mmol; from Alfa) in water (30 ml) and ethanol (15 ml) were added NH4CI (3.1 g, 58mmol) and Fe (1.3 g, 23 mmol). The reaction mixture was stirred at 8O ⁰ C for 30 min before cooling to ambient temperature, then filtered and the filtrate was extracted with CH2CI2 (40 mlx3). The organic phase was dried (Na2SO4) and concentrated to a yellow oil that was used directly in the next step.

Step 2: 1-[2-[2-nitroethylideneamino]phenylethanone

To a freshly prepared solution of sodium hydroxide (12 g, 300 mmol) in water (25 ml) which had cooled to 47°C was added dropwise nitromethane (12 g, 196 mmol) with stirring and with the temperature kept between 47°C to

50°C. After addition was complete the temperature was allowed to rise freely (57°C) for 15mins. The solution was cooled to 5°C and it was neutralized by slow addition of concentrated hydrochloric acid while the temperature was maintained below 10 ⁰ C. About 26 ml of acid was used to make the solution just acidic to Congo red paper. The light orange methazonic acid was filtered and the crude methanzonic acid was characterized by 1 H-NMR and used immediately for the following preparation.

To a solution of 2-aminoacetophenone of Step 1 (0.81 g, 6 mmol) in water (20 ml) and concentrated hydrochloric acid (1.4 ml) was added slowly crude methazonic acid. Toward the end of the addition, a yellow precipitate formed. An additional amount of concentrated hydrochloric acid (2 ml) was added, and the thick mixture was stirred for 10 min and then cooled in the refrigerator overnight. The precipitate was filtered and washed with water until the filtrate was neutral to litmus. The solid was dried. 900 mg yellow solid of 1- [2-[2-nitroethylideneamino]phenylethanone was obtained. Yield was 73%. Step 3: 4-methyl-3-nitroquinoline

To a solution of 1-[2-[2-nitroethylideneamino]phenylethanone of Step 2 (900 mg, 4.37 mmol) in warm acetone (80 ml) was added alumina (8.1 g, 9 times the weight of 1-[2-[2-nitroethylideneamino]phenylethanone, activated by heating at 120 ⁰ C for 2 hr.) within IOmins with vigorous stirring. Stirring was continued for additional 20mins and the light yellow mixture was allowed to stand at room temperature for 12 hours. The alumina was removed by filtration and the clear filtrate was concentrated to obtain yellow solid (740 mg). Yield was 90%. The product was characterized by 1 H-NMR. Step 4: 4-methyl-3-aminoquinoline

To a solution of 4-methyl-3-nitroquinoline of Step 3 (740 mg, 3.9 mmol) in water (20 ml) and ethanol (10 ml) were added NH4CI (1.86 g, 35.1 mmol) and Fe (0.87 g, 15.6 mmol). The reaction mixture was stirred at 80 ⁰ C

for 30 min, cooled to ambient temperature, then it was filtered and the filtrate was extracted with CH2CI2 (30 ml x 3). The organic phase was dried (Na∑SO-t) and concentrated to a yellow oil of 4-methyl-3-aminoquinoIine that was characterized by LC-MS and used directly in the next step Step 5: 3,4-Difluoro-2-(4-methyl-3-quinolylamino)benzoic acid

This product was obtained following the procedure described in Step 2 of Preparation 1e using as starting materials 2,3,4-trifluorobenzoic acid (245 mg, 1.4 mmol, 1eq.) and 4-methyl-3-aminoquinoline of Step 4 (220 mg, 1.4 mmol, 1eq.). 3,4-Difluoro-2-(4~methyl-3-quinolylamino)benzoic acid was obtained as a yellow solid (400 mg, yield: 80%) and the crude product was directly used in the next step.

Preparation 1 m: S^-Difluoro-Z-CT-chloro-S-quinolylaminoJbenzoic acid

Step 1: 7-chloro-4-hydroxy-3-nitroquinoline

7-chloro-4-hydroxyquinoline (6 g, 33 mmol; from Aldrich) was added to nitric acid (60 ml, 1.6mol) preheated to 85°C, then the resulting solution was heated to 100 ⁰ C for 2 hours. The solution was poured into boiling water, then filtered, the filter cake washed with water and acetone until the washings were colorless. 3.6 g of 7-chloro-4-hydroxy-3-nitroquinoline as yellow solid was obtained. Yield is 47%.

Step 2: 4,7-dichloro-3-nitroquinoline

7-chloro-4-hydroxy-3-nitroquinoline of Step 1 (3.6 g, 16mmol) was added to POCI ₃ (24 ml, 0.24mol). The mixture was stirred at 105 ⁰ C with refluxing for 3hours, then it was poured into ice with stirring and filtered

immediately. The solid dissolved in DCM, washed once with ice-cold sodium hydroxide, dried and evaporated to give 4,7-dichloro-3-nitroquinoline as a yellow solid: 3 g. Yield is 77%.

Step 3: 3-amino-7-chloroquinoline A solution of 4,7-dichloro-3-nitroquinoline of Step 2 (3.0 g, 12mmol) in

MeOH (50 ml) with Raney Ni (3 g) was hydrogenated under a hydrogen balloon overnight. Then KOH (1.5 g) was added. After 3 hours, the reaction was filtered, evaporated and purified by column chromatography to afford 3-amino-7-chloroquinoline as a yellow solid: 600 mg. Yield is 27%. The product was characterized by LC-MS.

Step 4: 3,4-Difluoro-2-(7-chloro-3-quinolylamino)benzoic acid

This product was obtained following the procedure described in Step 2 of Preparation 1f using as starting materials 2,3,4-trifluorobenzoic acid (591 mg, 3.4 mmol) and 3-amino-7-chloroquinoline of Step 3 (600 mg, 3.4 mmol). 3,4-Difluoro-2-(7-chloro-3-quinolylamino)benzoic acid was obtained as a solid (500 mg, yield: 45%) and the crude product was directly used in the next step.

Preparation 2}, Methyl 3,4-Difluoro-2-(5-bromo-2- pyridylamino)benzoate

A heterogeneous solution of 3,4-Difluoro-2-(5-bromo-2- pyridylamino)benzoic acid of Preparation 1b (415 mg, 0.835 mmol) in toluene (10 ml) was added at O ⁰ C with methanol (0.2 ml, 5 mmol) and trimethylsilyldiazomethane (2.5 ml, 5 mmol), The resulting homogeneous solution was stirred 30 m at 0 ⁰ C than checked by TLC (CH ₂ Cl ₂ /MeOH/acetic

acid 90/10/1) showing complete conversion. After diluition with brine, the mixture was extracted with ethyl acetate and the collected organic phase was dried over anhydrous Na2SO ₄ , filtered and evaporated from the solvent under reduced pressure. The solid was triturated with petrol ether to give the expected product as white solid (370 mg, 85% yield).

Analysis:

¹ H-NMR (CDCI ₃ ) 9-12 (s,1 H,Ph-*NH-Py); 8.27 (m,1H,Py); 7.79 (m,1 H,H ⁶ -Ph); 7.65 7.26 (s,1 H,Py); 6.90 (m,1 H,H ⁵ -Ph); 6.70 (m,1H;Py); 3.89 (s,3H, ^* CH ₃ -Py). HPLC-UV: Rt = 7.57 m, 96% purity by UV 220 nm

MS [M+1] = 344

Preparation 3: 3,4-Difluoro-2-(3-methyl-1-oxy-2-pyridylamino)benzoic acid

A solution of 3,4-difluoro-2-(3-methyl-2-pyridylamino)benzoic acid of

Preparation 1d (400 mg, 1.51 mmol) in CHCI3 (10 ml) and MeOH (2 ml) was added with m-chloroperbenzoic acid (400 mg, 77% pure, 1.8 mmol). The reaction mixture was stirred 18 h than evaporated from the solvent under reduced pressure. The solid was triturated with ethyl acetate filtered and washed with petrol ether to give 3,4-difluoro-2-(3-methyl-1-oxy-2- pyridylamino)benzoic acid as white solid (370 mg, 85% yield).

¹ H-NMR (d6-DMSO) 13.5 (s,1H,COOH); 10.0 (s,1 H,Ph-*NH-Py); 8.11 (m,1 H,Py); 7.73 (m,1 H,H ⁶ -Ph); 7.23 (m,1 H ₁ Py); 7.04 (m,1 H ₁ H ₅ -Ph); 6.99 (m,1 H;Py); 2.15 (s,3H,*CH3-Py).

Preparation 4: 3,4-Difluoro-2-(5-methyl-1 -oxy-2-pyridylamSno)benzoic acid

The compound was prepared and purified using the same procedure described for 3,4-difluoro-2-(3-methyl-1-oxy-2-pyridylamino)benzoic acid of

Preparation 3 starting from 3,4-difluoro-2~(5-methyl-2-pyridylamino)benzoic acid of Preparation 1c (295 mg, 1.11 mmol) as starting material. The product was obtained as a white solid (215 mg, 68% yield).

¹ H-NMR (d6-DMSO) 13.5 (s,1H,COOH); 10.0 (s,1H,Ph-*NH-Py); 8.16 (m,1H,Py); 7.82 (m,1H,H ⁶ -Ph); 7.33 (m,1H,Py); 7.13 (m,1 H,H ⁵ -Ph); 6.82 (m,1H;Py); 2.18 (s,3H,*CH ₃ -Py).

HPLC-UV:Rt = 5.40 m; 95% purity by UV 220 nm MS [M+1] = 281

Preparation 5: Methyl 3,4-difluoro-2-(5-bromo-1-oxy-2- pyridylamino)benzoate

The compound was prepared using the same procedure described for 3,4-difluoro-2-(3-methyl-1-oxy-2-pyridylamino)benzoic acid of Preparation 3 starting from methyl 3,4-difluoro-2-(5-bromo-2-pyridylamino)benzoate of Preparation 2 (260 mg, 0.70 mmol) as starting material. Purification was performed by chromatography using a MeGa Varian Bong colomn and eluting with a petrol ether/ethyl acetate solvent. The product was obtained as a white solid (111 mg, 44% yield).

¹ H-NMR (CDCI ₃ ) 10.3 (s,1H,Ph-*NH-Py); 8.40 (m,1H,Py); 7.87 (m,1H,H ⁶ - Ph); 7.30 (m,1 H ₁ Py); 7.06 (m,1H,H ⁵ -Ph); 6.57 (m,1H;Py); 3.94 (s,3H,*CH ₃ ).

HPLC-UV:Rt = 5.13 m; 100% purity by UV 220 nm.

MS [M+1] = 360

Preparation 6: Methyl 3,4-difluoro-2-(5-methyl-1-oxy-2- pyridylamino)benzoate

A solution of 3,4-difluoro-2-(5-methyl-1-oxy-2-pyridylamino)benzoic acid of Preparation 4: (150 mg, 0.53 mmol) in MeOH (3 ml) was added with

H2SO4 cone (0.3 ml). The reaction mixture was stirred for 3 days at 70 ⁰ C.

After being cooled at room temperature the solution was added with NaHCO3

5% until pH>7 was reached and evaporated from the solvent under reduced pressure. The resulting solid was diluted with brine and ethyl acetate and the collected organic phase was dried over anhydrous Na2SO4, filtered and evaporated from the solvent under reduced pressure. The expected product methyl 3,4-difluoro-2-(5-methyl-1-oxy-2-pyridylamino)benzoate was obtained as pale green solid (155 mg, 99% yield).

¹ H-NMR (CDCI ₃ ) 10.22 (s,1 H,Ph-*NH-Py); 8.13 (m,1H,Py); 7.85 (m,1 H,H ⁶ -Ph); 7.04 (m,2H,Py+H ⁵ -Ph); 6.62 (m,1 H;Py); 3.94 (s,3H,*CH ₃ Py); 2.26 (s,3H,*CH ₃ ).

Preparation 7: Methyl 3,4-difluoro-2-(3-methyl-1-oxy-2- pyridylamino)benzoate

The compound was prepared and purified using the same procedure described for IRT0046456 starting from 3,4~difluoro-2-(3-methyl-1-oxy-2- pyridylamino)benzoic acid of Preparation 3 (200 mg, 0.71 mmol) as starting material. The product was obtained as a white solid (167 mg, 79% yield).

¹ H-NMR (CDCI ₃ ) 9.59 (s,1 H,Ph- ^* NH-Py); 8.12 (m,1H,Py); 7.70 (m,1 H,H ⁶ -Ph); 7.07 (m,1 H,Py); 6.88 (m,1H;H ⁵ -Ph); 6.78 (m,1H;Py); 3.90 (s,3H ₍ ^* CH3Py); 2.17 (s,3H, ^* CH ₃ ). Preparations 8 -86

The compounds of table 1 are prepared following the procedure of preparations 1a-m and using suitable benzoic acids and heterocyclic amines as starting products. Table 1

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Example 1 : 2-(3,5-Dichloro-2-pyridylamino)-3,4-difluoro-N-methoxy-N- methylbeπzamidθ

3,4-Difluoro-2-(3,5-dichloro-2-pyridylamino)benzoic acid from preparation 1 (0.319 g, 1.0 mmol) is dissolved in dichloromethane (5 ml) and N-methylmorpholine (0.6 ml) and the resulting solution is added with N,O-dimethylhydroxy!amine hydrochloride (0.127 g, and 1.30 mmol) and (benzotriazolyloxy)trispyrrolidino phosphonium hexafluorophosphate (PyBOP; 0.52 g, 1.0 mmol). The reaction mixture is stirred for 4 hours at room temperature. A further portion of PyBOP (0.17 g; 0.33 mmol) is added and stirring is continued at room temperature for 24 hours. After evaporation of the reaction solvent under reduced pressure, the residue is dissolved in ethyl acetate (25 ml) and the organic solution is washed in sequence with water (25 ml) and with a saturated solution of NaCI (3 x 25 ml). The organic

phase is anhydrified over Na2SO4, the solvent is removed under reduced pressure and the residue is purified by chromatography on silica gel to afford the product.

Example 2: N-(2-Hydroxyethoxy)-2-(3,5-dichloro-2-pyridylamino)-3,4- difluorobenzamide

Step i

A solution of 3,4-difluoro-2-(3,5-dichloro-2-pyridylamino)benzoic acid of preparation 1 (0.319 g, 1.0 mmol) and HOBt (0.203 g 1.5 mmol) in dichloromethane (5 ml) is added with EDCI (0.233 g, 1.5 mmol), then left under stirring for 2 hours at room temperature and added with O-(2- vinyloxyethyl)hydroxylamine (0.113 g. 1.1 mmol; obtained according to preparation 47 of WO02/06213) and triethylamine (0.17 ml, 1.2 mmol). The reaction mixture is stirred for two hours. The reaction solvent is evaporated off under reduced pressure, the residue is dissolved in ethyl acetate (25 ml) and the organic solution is washed in sequence with an ammonium chloride saturated solution (25 ml), a NaCI saturated solution (25 ml), a NaHCO3 saturated solution (25 ml) and finally with a NaCI saturated solution (25 ml). The organic phase is anhydrified over Na2SO4, the solvent is removed under reduced pressure and the obtained residue of N-(2-vinyloxyethoxy)-2-(3,5- dichloro-2-pyridylamino)-3,4-difluorobenzamide is used for the following step without further purification. Step 2 A solution of the product from step 1 (0.404 g; 1.0 mmol) in THF/EtOH

1/1 is added at room temperature with 1 N HCI (2 ml_; 2.0 mmol). The

reaction mixture is stirred at room temperature for about one hour, then neutralised by addition of a saturated NaHCO3 solution and diluted with ethyl acetate (25 ml). The organic solution is washed with a NaCI saturated solution (25 ml), anhydrified over Na2SO4, the solvent is evaporated off under reduced pressure and the residue is purified by column chromatography to give N-(2-hydroxyethoxy)-2-(3,5-dichloro-2-pyridylamino)-3,4- difluorobenzamide.

Example 3: N-[(R)-2,3-Dihydroxypropoxy]-2-(3,5-dichloro-2- pyridylamino)-3,4-difluorobenzamide

Step i

A solution of 3,4-difluoro-2-(3,5-dichloro-2-pyridylamino)benzoic acid of preparation 1 (0.319 g, 1.0 mmol) in anhydrous THF (5 ml) is added with (R)-O-(2,2-dimethyl-1 ,3-dioxolan-4-yl-methyl)hydroxylamine (0.147 g; 0.1 mmol; prepared according to preparations 70 and 71 of WO 02/06213), followed by N-methylmorpholine (0.28 ml, 2.5 mmol). The solution is cooled to 0 ⁰ C and added with chlorodiphenylphosphine oxide (0.23 ml, 1.2 mmol). The reaction mixture is allowed to warm to room temperature and left under stirring for 24 hours. After removal of the solvent under reduced pressure, the residue is partitioned between water (25 ml) and ethyl acetate (25 ml). The organic phase is separated and washed twice with a 1/1 NaCI/NaHCO3 saturated solution. After anhydrification on Na2SO4, the solvent is removed under reduced pressure and the residue is purified by column chromatography to give N-[(R)-2,2-dimethyl-1 ,3-dioxolan-4-yl-methoxy]-2-

(3,5-dichloro-2-pyridylamino)-3,4-difluorobenzamide.

Step 2

A mixture of product of step 1 (0.224 mg; 0.5 mmol), p-toluenesulphonic acid monohydrate (0.048 g, 0.25 mmol), methanol (2.5 ml) and water (0.25 ml) is stirred at room temperature for 24 hours. The reaction mixture is concentrated under reduced pressure and the residue is extracted with ethyl acetate (2 x 10 ml). The organic phases are pooled and washed with a NaHCO3 saturated solution and dried over Na2SO4. After evaporation of the solvent the residue is purified by column chromatography to give N-[(R)-2,3-dihydroxypropoxy]-2-(3,5-dichloro-2-pyridylamino) -3,4- difluorobenzamide.

Example 4: N-cyclopropylmethoxy-Z-Cβ-trifluoromethyl-S-pyridylamino)- 3,4-difluorobenzamide

To a solution of 3,4-difluoro-2-(6-trifluoromethyl-3- pyridylamino)benzoic acid of Preparation 1e (50 mg, 0.16 mmol, 1eq.) in dry DMF (2 ml) was added EDCI hydrochloride (43 mg, 0.19 mmol, 1.2eq.) and HOBt (25 mg, 0.19 mmol, 1.2eq.) under Ar atmosphere. The reaction mixture was stirred at room temperature for 2 h. O-(cyclopropylmethyl)hydroxylamine hydrochloride (29 mg, 0.24 mmol, 1.5eq.) and Et3N (32 mg, 0.31 mmol, 2.0eq.) was added, then the reaction mixture was stirred at room temperature overnight. The solvent was removed and the residue was washed with water and extracted with ethyl acetate (3* 15ml). The combined organic phase was washed with brine and dried over anhydrous magnesium sulfate.

Concentration was followed by purification by preparative HPLC to afford N-cyclopropylmethoxy-2-(6-trifluoromethyl-3~pyridylamino)-3, 4- difluorobenzamide as a yellow oil (5 mg, yield: 8.1%), which was characterized by 1 H-MNR and LC-MS.

¹ HNMR (300MHz, CDCI ₃ ):δ 8.87 (s, 1 H), 8.77 (s, 1 H), 8.25 (t, J = 3 Hz, 1 H), 7.55 (d, J = 8.4 Hz, 1 H), 7.28 (m, 1 H), 7.17 (m, 1 H), 6.9 (m, 1 H), 3.8 (d, J = 7.2 Hz, 2 H), 1.15 (m, 1 H), 0.6 (m, 2 H), 0.31 (m, 2 H).

The preparative HPLC conditions were the following:

Instrumentation: SHIMADZU

Pump: LC-8A

UV-Vis detector: SPD-10A VP

Column: SHIMADZU

Mobile phase: A/ B (1 Ommol NH ₄ HCO ₃ /1 L water)/MeCN

Gradient:

Example 5: N-cyclopropylmethoxy^-Cδ-methyl-S-pyridylamiπoj-S^- difluorobenzamide

The compound was prepared and purified following the procedure of

Example 4 using as starting material 3,4-difluoro-2-(5-methyl-3- pyridylamino)benzoic acid of Preparation 1f (745 mg, 2.8 mmol.). N-cyclopropylmethoxy^-Cδ-methyl-S-pyridylaminoJ-S^-difluoro benzamide was obtained as a yellow solid (100 mg, yield: 10.6%), which was characterized by 1 H-MNR and LC-MS.

¹ HNMR (300MHz ₁ CDCI ₃ ):δ 8.97 (s, 1 H), 8.18 (s, 1 H), 8.02 (s, 1 H), 7.49 (s, 1 H), 7.40 (m, 1 H), 7.03 (m, 1 H), 3.75 (d, J = 6.3, 2 H), 2.47 (s, 3 H), 1.08 (m, 1 H), 0.55 (m, 2 H), 0.23 (m, 2 H).

Example 6: N-cyclopropylmethoxy-2-(4-methyl-3^yridylamino)-3,4- difluorobenzamide

The compound was prepared and purified following the procedure of Example 4 using as starting material 3,4-difluoro-2-(4-methyl-3- pyridylamino)benzoic acid of Preparation 1g (600 mg, 2.27 mmol). N-cyclopropylmethoxy-2-(4-methyl-3-pyridylamino)-3,4-difluor obenzamide

was obtained as yellow oil (120 mg, yield: 16%), which was characterized by 1 H-MNR and LC-MS.

¹ HNMR (300MHz, CDCI ₃ ):δ 9.07 (s, 1 H), 8.18 (d, J = 5.7 Hz, 1 H), 8.04 (d, J = 5.7 Hz, 1 H), 7.57 (d, J = 6 Hz, 1 H), 7.4 (m, 1 H), 6.91 (m, 1 H) ₅ 3.78 (d, J= 7.5 Hz, 2 H), 2.60 (s, 3 H), 1.15 (m, 1 H), 0.58 (m, 2 H), 0.26 (m, 2 H).

Example 7: N-cyclopropy[methoxy-2-(5-bromo-3-pyridylamino)-3,4- difluorobenzamide

The compound was prepared following the procedure of Example 4 using as starting material 3,4-difluoro-2-(5-bromo-3-pyridylamino)benzoic acid of Preparation 1h (200 mg, 0.608mmol). Purification was accomplished by preparative TLC (petroleum ether : ethyl acetate 2 : 1). N-cyclopropylmethoxy-2-(5-bromo-3-pyridylamino)-3,4-difluoro benzamide. was obtained as a white powder (100 mg, yield: 41 %). which was characterized by 1 H-MNR and LC-MS.

¹ HNMR (300MHz, DMSO):δ 11.6 (s, 1 H), 8.7 (s, 1 H), 8.1 (m, 2 H), 7.3 (m, 3 H), 3.3 (d, J = 7.5 Hz, 2 H), 1.05 (m, 1 H), 0.50 (m, 2 H), 0.25 (m, 2 H). Example 8: N-cyclopropylmethoxy-2-(3-quinolylamino)-3,4- difluorobenzamide

The compound was prepared and purified following the procedure of

Example 4 using as starting material 3,4-difluoro-2-(3~quinolylamino)benzoic acid of preparation 1i (138 mg, 0.46 mmol). N-cyclopropylmethoxy-2-(3~ quinolylamino)-3,4-difluorobenzamide was obtained as a brown solid (68 mg, yield: 40%), which was characterized by 1 H-MNR and LC-MS.

¹ HNMR (300MHz, CD ₃ OD):δ 8.63 (d, J = 2.4 Hz, 1 H), 7.9 (d, J = 7.8 Hz, 1H), 7.7 (m, 1 H), 7.55 (m, 3 H), 7.46 (m, 1 H), 7.25 (m, 1 H), 3.37 (d, J = 7.5 Hz, 2 H), 0.96 (m, 1 H), 0.42 (m, 2 H), 0.1 (m, 2 H).

Example 9: N-cyclopropylmethoxy-2-(4-methyl-3-quinolylamino)-3,4- difluorobenzamide

The compound was prepared and purified following the procedure of Example 4 using as starting material 3,4-difluoro-2-(4-methyl-3- quinolylamino)benzoic acid of preparation 11 (800 mg, 2.5 mmol). N-cyclopropylmethoxy-2-(4-methyl-3-quinolylamino)-3,4-difluo robenzamide was obtained as a yellow solid (100 mg, yield: 10.4%), which was characterized by 1 H-MNR and LC-MS.

¹ HNMR (300MHz, CDCI ₃ ):δ 10.63 (s, 1 H), 8.82 (s, 1 H), 8.15 (3, 1 H), 8.05 (m, 2 H), 7.62 (m, 2 H), 7.38 (m, 1 H), 6.70 (m, 1 H), 3.82 (d, J = 7.2 Hz, 2 H), 2.68 (s, 3 H), 1.15 (m, 1 H). 0.56 (m, 2 H), 0.29 (m, 2 H).

Example 10: N-cyclopropylmethoxy-2-(7-chloro-3-quinolylamino)-3,4- difluorobenzamide

The compound was prepared and purified following the procedure of

Example 4 using as starting material 3,4-difluoro-2-(7-chloro-3- quinolylamino)benzoic acid of Preparation 1 m (575 mg, 1.72 mmol). N-cyclopropylmethoxy-2-(7-chloro-3-quinolylamino)-3,4-difluo robenzamide was obtained as an orange powder (80 mg, yield: 11.5%), which was characterized by 1 H-MNR and LC-MS.

¹ HNMR (300MHz, DMSO):δ 11.62 (s, 1 H), 8.86 (s, 1 H), 8.73 (d, J = 2.4Hz, 1 H), 7.90 (d, J = 2.4 Hz, 1 H), 7.77 (d, J = 8.7 Hz, 1 H), 7.50 (dd, J = 9, 2.7 Hz, 1 H), 7.35 (m, 3 H), 3.37 (d, J = 7.5 Hz, 2 H), 0.90 (m, 1 H), 0.41 (m, 2 H), 0.08 (m, 2 H). Example 11 : N-cyclopropylmethoxy-2-(5-bromo-2-pyridylamiπo)-3,4- difluorobenzamide

A solution of methyl 3,4-difluoro-2-(5-bromo-2-pyridylamino)benzoate of Preparation 2 (250 mg, 0.65 mmol) in toluene (3 ml) was placed into a sealed tube and added with cyclopropylmethoxylamine hydrochloride (240 mg, 1.94 mmol), triethylamino (0.27 ml, 1.9 mmol) and dropwise AI(CH3)3

(1.25 ml, 2N solution in hexane, 2.5 mmol). The reaction mixture heated at 100 ⁰ C for 30 min using a microwave apparatus. The mixture was cooled at 0 ⁰ C, added with NaOH 1.5 N (3 ml) and stirred for further 10 m. The heterogeneous solution was diluted with CHaCb.and the organic phase washed with NaHCO3 5%, dried over anhydrous Na2SO4, filtered and evaporated from the solvent under reduced pressure. The crude material was purified by chromatography using a Mega Varian Bond Elut cartridge and petrol ether/ethyl acetate mixture. The expected product was obtained as yellow solid (28 mg, 10% yield). Analysis:

¹ H-NMR (d6-DMSO) 11.51 (s,1H,CONH); 8.95 (s,1 HPh-NH-Ph); 8.06 (d,1 H,JHH=2.5Hz); 7.78-7.68 (m,1 H); 7.32-7.22 (m,2H); 6.86 (d,1 H,J=8.6Hz); 3.52 (d,2H,J=7.1 Hz,*CH2-Cyclopr); 1.09-0.9 (m,1 H, *CH cycl); 0.52-0,40 m,2H); 0,23-0.11 (m,2H). HPLC-UV: Rt = 6.81 m, 100% purity by UV 220 nm

MS [M-1] = 399

Example 12: N-cycIopropylmethoxy^-Cδ-methyl-i -oxy-2-pyridylamino)- 3,4-difluorobenzamide

A solution of methyl 3,4-difluoro-2-(5-methyl-1-oxy-2- pyridylamino)benzoate of Preparation 6 (145 mg, 0.49 mmol) in toluene (3 ml) was added with cyclopropylmethoxylamine hydrochloride (182 mg, 1.47 mmol), triethylamine (0.24 ml, 1.72 mmol) and dropwise AI(CH3)3 (1.47 ml, 2N solution in hexane, 2.95 mmol). The reaction mixture was stirred at 65 °C

for 18 h, cooled at 0 ⁰ C and added with NaOH 1.5 N (3 ml) and stirred for further 10 m. The heterogeneous solution was filtered through a Chem Elut Carteridge which adsorb the aqueous phase washing it with CH2CI2. After evaporating the solvent at reduced pressure, the crude material was purified by chromatography using a Bond Elut cartridge and ChkCfe/MeOH 100/5 solvent. The expected product was obtained as pink solid (83 mg, 48% yield).

¹ H-NMR (d6-DMSO) 11.72 (s,1 H,CO ^* NH); 9.48 (s,1 H,Ph-*NH-Ph); 8.15 (m,1 H,Py); 7.37 (m,2H,H ⁶ -Ph+Py); 7.08 (m,1H,Py); 6.68 (m,1 H, H^-Ph); 3.36 (d,2H,J=7.2Hz,*CH2-CH); 2.17 1.0 (m,1 H,CH ₂ -*CH); 0.47 (m,2H); 0.20 (m,2H).

HPLC-UV: Rt = 4.17 m, 100% purity by UV 220 nm

MS [M-1] = 350

Example 13; N-cyclopropylmethoxy-2-(3-methyl-1 -oxy-2-pyridylamino)-

3,4-difluorobenzamide

The compound was prepared and purified using the same procedure described for N-cyclopropylmethoxy-2-(5-methyl-1 -oxy-2-pyridylamino)-3,4- difluorobenzamide of Example 12 starting from methyl 3,4-difluoro-2-(3- methyl-1-oxy-2-pyridylamino)benzoate of Preparation 7 (160 mg, 0.545 mmol) as starting material. The product was obtained as a white solid (95 mg, 50% yield).

1H-NMR (d6-DMSO) 11.73 (s,1H,CO*NH); 9.14 (s,1H,Ph-*NH-Ph);

8.12 (m,1H,Py); 7.37 (m,1 H, H^-Ph); 7.30 (m,1H,Py); 7.18 (m,1 H,Py); 6.95 (m,1 H, H ⁵ -Ph); 3.62 (d,2H,J=7.2Hz,*CH ₂ -CH); 1.98 0.80 (m,1 H,CH ₂ -*CH);

0.46 (m,2H); 0.22 (m,2H).

HPLC-UV:Rt = 4.15 m; 99% purity by UV 220 nm.

MS [M+1] = 350

Example 14: N"Cyclopropylmethoxy-2-(5-bromo-1 -oxy-2-pyridylamino)- 3,4-difluorobenzamide

The compound was prepared and purified using the same procedure described for N-cycIopropylmethoxy-2-(5-methyl-1-oxy-2-pyridylamino)-3,4- difluorobenzamide of Example 12 starting from methyl 3,4-difluoro-2-(5- bromo-1-oxy-2-pyridylamino)benzoate pf Preparation 5 (105 mg, 0.29 mmol) as starting material. The product was obtained as a white solid (70 mg, 58% yield).

¹ H-NMR (d6-DMSO) 10.0 (s,1 H,CO*NH); 9.29 (s,1H,Ph-*NH-Ph); 8.30 (m,1H,Py); 7.57 (m,1H, H ⁶ -Ph) 7.23 (m,1H,Py); 7.20 (m,1 H, Py); 6.49 (m,1 H, H ⁵ -Ph); 3.69 (d,2H,J=7.2Hz,*CH2-CH); 1.06 (m,1 H,CH2-*CH); 0.50 (m,2H); 0.22 (m,2H).

HPLC-UV:Rt = 4.67 m, 99% purity by UV 220 nm.

MS [M+1] = 415 Examples 15-222

The compounds reported below in table 2 are prepared following the procedures of Examples 1-3 and using the heteroarylaminobenzoic acids of preparations 1-67 and suitable hydroxylamines as starting products.

Table 2

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Example 223: N-((R)-2,3-Dihydroxypropoxy)-2-(3,4,5- trichlorothiophene-2-carbonyl)benzamide

This compound is prepared following the procedure of example 1 , using 2-(3,4,5-trichlorothiophene-2-carbonyl)benzoic acid (commercially available from Aurora Fine Chemicals) as starting material.

Example 224: N-((R)-2,3-Dihydroxypropoxy)-2-(thiopheπe-2- carbonyl)benzamide

The product is prepared following the procedure of example 1 , using 2-(thiophene-2-carbonyl)benzoic acid, which is commercially available, as starting material.

Example 225: N-((R)-2,3-Dihydroxypropoxy)-2-(thiophene-2- methyQbenzamide

This compound is prepared following the procedure of example 1 , using 2-(thiophene-2-methyl)benzoic acid (commercially available from Asinex) as starting material.

PHARMACOLOGICAL SECTION MEK1 kinase assay

MEK1 kinase activity is monitored in the presence of test compounds by an enzyme-linked immunosorbent assay (ELISA) method essentially as described in R. Mallon et al. Anal. Biochem. 294, 48 (2001). In this assay system, activated MEK1 phosphorylates ERK2 which is specifically detected (vs nonphosphorylated ERK2) by antibodies making possible the ELISA development. A DELFIA (dissociation-enhanced lanthanide fluorescence immunoassay) format is used as readout to determine inhibition of MEK1 enzyme activity.

First stage: kinase reaction Assay reactions were run in 100-μl volumes in assay dilution buffer (20 rtiM MOPS, pH 7.2, 25 mM β-glygerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol) in 96-well plates.

Assay reactions contain:

• Activated MEK1 (30 nM, Upstate) • Nonactivated GST-ERK2 (60 nM, Upstate)

• ATP (100 μM)

• MgCI ₂ (5 mM)

• Solvent (DMSO, 1 % final cone.) or test compounds (various dilutions in solvent). Reactions were incubated for 30 min at room temp, under agitation and then stopped with EDTA (70 mM final cone).

Second stage: DELFIA

Reactions are transferred to an anti-GST Ab-coated 96-well plate (goat anti-GST from GE Healthcare; Plate for fluorescence from Nunc) and

incubated for 60 min at room temperature under agitation.

Wells are washed 3 times with TBST (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 0.05% Tween-20).

Anti-phospho-ERK (Monoclonal SIGMA, 1 :3000 in 100 μl TBST) is added and incubated for 60 min at room temp, under agitation.

Wells are washed 3 times with TBST.

Secondary anti-mouse Ab/europium conjugate (PerkinElmer, 1 :500 in 100 μl TBST) is added and incubated for 60 min at room temp, under agitation. Wells are washed 3 times with TBST.

DELFIA enhancement solution is added and incubated for 10 min at room temp, under agitation.

Europium counts are measured on a TECAN Plate Reader and analyzed in Excel for single point percentage inhibition and IC50 determinations.

In vivo evaluation of antitumour activity

Materials

The MEK inhibitors used for in vivo antitumour activity studies can be formulated in a suitable medium for endovenous (ev) or oral administration (os). For example, for endovenous administration the compounds can be administered in 0.9% NaCI or in 0.9%NaCI, solutol HS15 and dimethylsulphoxide mixtures, for example in 87:10:3 (v:v:v) ratio respectively.

Cell lines

The following murine and human tumour cells from different tissue hystotypes can be used to evaluate the antitumour activity of the compounds of the invention: A375 (human, melanoma), H460 (human, lung), A2780

(human, ovary), PC-3 (human, prostate), LoVo (human, colon), HCT116

(human, colon), BXPC3 (human, pancreas), PANC-1 (human, pancreas),

MX-1 (human, breast), MOLT (human, leukemia), multiple myeloma (human, myeloma), YC8 (murine, lymphoma), L1210 (murine, leukemia), 3LL (murine, lung).

Animal species Immunocompetent or immunosuppressed mice of 5-6 weeks are commercially available. CD1 nu/nu mice are maintained under sterile conditions.

Implant and growth of tumour cells

Solid tumours from different hystotypes (lung, ovary, breast, prostate, pancreas, colon) can be transplanted subcutaneously (sc) in the axillary region of immunocompetent mice (murine models) or in immunodeprived

(human models). Human tumours cell lines, originally obtained from ATCC, can be adapted to grow in vivo as solid tumours from "in vitro" cultures.

Murine or human haematological tumours models can be transplanted in different sites (ev, ip, ic o sc) of immunocompetent mice (murine tumours) or in immunodeprived mice (leukemia, lymphoma and human myeloma tumours).

Pharmacological treatment

Mice bearing solid or haematological tumours are randomized in experimental groups (10 mice/group). As far as solid tumours are concerned, a tumour mean weight of 80-100 mg for each group is required to start the treatment; mice with larger or smaller tumours are excluded.

The experimental groups are randomly allocated into the treatment group with the test substance or into the control group. The animals can be treated with the compounds through the intravenous, oral or intraperitoneal route, according to the bioavailability of the products and following different treatment protocols, for example iv once or twice a week or daily per os.

In solid tumour models, treatment with the compound can start when

tumour weight is comprised between 80 and 100 mg after tumour transplant (day 0).

The compounds can be administered in a volume of 10 mL/Kg body weight /mouse in a suitable solvent. Antitumour activity parameters

The following parameters can be determined for the evaluation of the antitumour activity:

• solid primary tumour growth, controlled twice a week in each mouse with a caliper; . survival time of treated mice with respect to control mice;

. evaluation of the body weight of each mouse twice a week. Inhibition of tumour growth, TWI% (percentage of inhibition of tumour growth compared to a control group treated with vehicle only), or relative inhibition of tumour growth RTWI% in case of advanced tumours is evaluated one week after treatment with the compound and tumour weight (TW) can be calculated as follows:

TW = 1/2 ab ² where a and b are the short and long diameters of the tumour mass expressed in millimiters. Antitumour activity can be determined as inhibition of tumour growth

(TWI %), calculated according to the following formula: mean TW (treated)

TWI% = 100 - x 100 mean TW (controls) The relative percentage of inhibition of tumour growth compared to the control group treated with administration vehicle only (RTWI%) is evaluated one week after the last treatment with the product, according to the following formula:

mean RV of treated mice

RTWI% = 100 - x 100 mean RV of control mice Vt (tumour weight at t day) where RV=

Vo (initial weight of the tumour at the beginning of the treatment)

The tumour regression percentage can be calculated as weight regressions of the tumour, determined as tumour weight at a certain day divided by the initial weight of the tumour at the beginning of the treatment.

In models of haematological tumours the antitumour activity can be determined as percentage increase of the mice mean survival time expressed as (T/C%) ratio between the mean survival time of the treated group (T) and the control group (C). Animals without tumours at the end of the experiment (60 days after tumour transplant) are excluded from calculation and are considered as long-term survivors (LTS). Toxicity evaluation in tumour-bearing mice

Toxicity can be evaluated daily both on the basis of approximate autoptic results and of loss of body weight. When mice die before control animals treated with the vehicle only, or when a meaningful weight loss

(>20%) and/or a reduction of spleen and liver dimensions is observed, it is assumed that death is due to toxicity.

The percentage change of body weight (BWC%) is calculated as follows: 100- (mean body weight of the mice at a given day / mean body weight of the mice at the beginning of the treatment) x 100. This value is calculated one week after treatment with the test compound.