EP4619099A1 - Pyrido oxazolidinone derivatives as alk5 inhibitors - Google Patents

Abstract

The present invention generally relates to compounds of formula (I) inhibiting the transforming growth factor beta (TGF beta) type I receptor (ALK5) (hereinafter ALK5 inhibitors), methods of preparing such compounds, pharmaceutical compositions containing them and therapeutic use thereof; the compounds of the invention may be useful for instance in the treatment of many diseases, disorder, or condition associated with ALK5 signaling pathway.

Description

PYRIDO OXAZOLIDINONE DERIVATIVES AS ALK5 INHIBITORS

FIELD OF THE INVENTION

The present invention generally relates to compounds inhibiting the transforming growth factor P (TGF P) type I receptor (ALK5) (hereinafter ALK5 inhibitors), methods of preparing such compounds, pharmaceutical compositions containing them and therapeutic use thereof; the compounds of the invention may be useful for instance in the treatment of many diseases, disorder, or condition associated with ALK5 signaling pathway.

BACKGROUND OF THE INVENTION

The Transforming Growth Factor P (TGF P) is a protein belonging to the TGF P superfamily. It is involved in several processes, both cellular, such as proliferation, migration and differentiation, and biological, including wound healing, immunosuppression, cancerogenesis and extracellular matrix production.

The TGF P superfamily also includes, among others, other members known as activins (Acts) (see e.g., Hinck AP, FEBS Letters 586 (2012); 1860-1870). The binding of the peptide initiates the TGF P signalling cascade through the formation of a heterotetrameric complex composed of two different serine/threonine kinases receptors: type 1 (TGFPR1/ALK5) and type 2 (TGFPR2). TGFPR1/ALK5 is recruited and activated through the phosphorylation of its intracellular domain by TGFPR2, leading in turn to the phosphorylation of the receptor-activated (R)-Smad family, resulting in the activation of target gene transcription (see e.g., Sheppard D., Proc Am Thorac Soc. (2006) ;(3):413- 417). Similarly, to the TGF P signalling, the type I receptor for activin, ALKA, leads to the activation of target gene transcription (see e.g., Heldin CH et al., Cold Spring Harb Perspect Biol. (2016) Aug 1;8(8)). Several studies have linked an excessive and/or dysregulated TGF P activity with many diseases including cancer and fibrosis (see e.g., Syed V, J Cell Biochem. (2016) Jun; 117(6): 1279-87; Jakowlew SB. Cancer Metastasis Rev. (2006) Sep;25(3):435-57). Among fibrotic disorders, a crucial role of TGFP has been shown in organs such as lung, heart, liver, and kidney (see e.g., Alhamad EH, J Thorac Dis. (2015);7(3):386- 93). In particular, TGFP expression is increased in fibrotic lung diseases, such as idiopathic pulmonary fibrosis (IPF), and in chronic inflammatory conditions, such as chronic obstructive pulmonary disease and asthma (see e.g., Thomas BJ et al., Am J Respir Cell Mol Biol. (2016); (55):759-766). In lung, TGFP is expressed in several cell types, like epithelial cells, endothelial cells, connective tissue cells, macrophages and fibroblasts. These cell populations may produce excess of TGFP in IPF human lung tissue. Moreover, high levels of TGFP have been detected in lung tissue and BAL of IPF patients (see e.g., Bergeron A et al., Eur Respir J (2003);22:69-76). TGFp gene expression and TGFp protein production have been observed to increase in a variety of animal models of pulmonary fibrosis caused by bleomycin, silica, asbestos, and radiation (see e.g., Wei F et al., Int Immunopharmacol. (2017) Jul;48:67-75; Choe JY et al., Inflamm Res. (2010) Mar;59(3): 177-88; Wang X et al., Respir Res (2009); 10, 36) and it has also been reported how the TGFp expression is sufficient to induce progressive fibrosis in rodents (see e.g. Sime PJ et al., J Clin Invest (1997);100:768-776; Kim KK et al.). Contrarily, TGFP signalling inhibition obtained by employing knockout (KO) animals can inhibit fibrosis development through TGFP-linked mechanisms (see e.g., Bonniaud P et al., Am J Respir Crit Care Med (2005); 171 :889-898; 34). Similar results have been achieved with inhibition of TGFpRl in mouse bleomycin disease model (see e.g., Wei Y et al., J Clin Invest. (2017);127(10):3675-3688). Activin signalling dysregulation, similarly to TGFP, is associated to fibroblasts proliferation, myofibroblasts differentiation and accumulation of extracellular matrix (ECM) (see e.g., Yamashita et al., J. Am. Soc. Nephrol. (2004) 15, 91-101). Moreover, overexpression of activin has been linked to pathological conditions and fibrosis development in different organs, such as liver (see e.g., Patella et al., Am. J. Physiol. Gastrointest. Liver Physiol. (2006) 290, G137-G144), kidney (see e.g., Agapova et al., Kidney Int. (2016) 89, 1231-1243), heart (see e.g., Yndestad et al., Circulation (2004) 109,1379- 1385), and lung (see e.g., de Kretser et al., Crit.Care (2013) 17: R263). Taken together these data suggest the importance of targeting ALK5 to treat pharmacologically the aforementioned diseases, linked to the dysregulated TGF signalling pathway. The TGFP signalling is strongly involved in the cardiovascular homeostasis (see e.g., van Meeteren LA et al., Springer (2013)). Several studies in humans and mice have shown the main role of TGFP in angiogenesis and vascular morphogenesis. Moreover, TGFP plays a key role in the development and functionality of cardiac valves. It is therefore clear the importance of a selective regulation of TGFp pathway to target the pathological effects avoiding the suppression of the signalling needed for a correct homeostasis.

The answer to this crucial point could be addressed by using the inhalation route to deliver an antiTGFp drug. The inhalatory route would allow the treatment of the affected lung compartment bypassing the issue of the heart exposure.

Various compounds have been described in the literature as ALK5 and/or ALK4 receptor inhibitors.

Of note, inhibition of ALK5 receptor may be useful for the treatment of fibrosis and disease, disorder and conditions that result from fibrosis.

Several efforts have been done in the past years to develop novel ALK5 receptor inhibitors useful for the treatment of several diseases and some of those compounds have shown efficacy also in humans. However, there remains a potential for developing inhibitors of receptors ALK5 characterized by good potency, useful for the treatment of diseases or conditions associated with a dysregulation of ALK5 signaling pathway, in particular fibrosis.

In particular, there remains a potential for developing inhibitors of receptors ALK5 useful for the treatment of diseases or conditions associated with a dysregulation of ALK5 signaling in the respiratory field, in particular idiopathic pulmonary fibrosis (IPF), to be administered by the inhalation route and characterized by a good inhalatory profile, that corresponds to a good activity on the lung, a good lung retention and to a low metabolic stability in order to minimize the systemic exposure and correlated safety issues.

In this direction, we have surprisingly found a new series of compounds of general formula (I) that solves the problem of providing potent inhibitors of ALK5 receptor for administration by inhalation, that shows, at the same time, a good inhalatory profile, low metabolic stability, low systemic exposure and a good selectivity across the kinome.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to compounds of formula (I) wherein Ri is selected from the group consisting of Al and A2

Al A2

I<2 is selected from the group consisting of phenyl optionally substituted by one or more halogen atoms; pyridyl optionally substituted by one or more -(Ci-Ce)alkyl; and thiazolyl optionally substituted by one or more -(Ci-Ce)alkyl;

Ri is H or is selected from the group consisting of -NH2, -NHC(O)-(Ci-Ce)alkyl and -NHC(O)- (Ci-Ce)alkylene-NRARB; RA is -(Ci-Ce)alkyl;

RB is -(Ci-Ce)alkyl; and pharmaceutically acceptable salts thereof.

In a second aspect, the invention refers to a pharmaceutical composition comprising a compound of formula (I) and pharmaceutically acceptable salts thereof in admixture with one or more pharmaceutically acceptable carrier or excipient.

In a third aspect, the invention refers to a compound of formula (I) and pharmaceutically acceptable salts, or to a pharmaceutical composition comprising a compound of formula (I) and pharmaceutically acceptable salts thereof, for use as a medicament.

In a further aspect, the invention refers to a compound of formula (I) and pharmaceutically acceptable salts thereof, or to a pharmaceutical composition comprising a compound of formula (I) and pharmaceutically acceptable salts thereof, for use in preventing and/or treating a disease, disorder or condition mediated by ALK5 receptor in a mammal.

In a further aspect, the invention refers to a compound of formula (I) and pharmaceutically acceptable salts thereof, or to a pharmaceutical composition comprising a compound of formula (I) and pharmaceutically acceptable salts thereof, for use in the prevention and/or treatment of fibrosis and/or diseases, disorders, or conditions that involve fibrosis.

In a further aspect, the invention refers to a compound of formula (I) and pharmaceutically acceptable salts thereof, or to a pharmaceutical composition comprising a compound of formula (I) and pharmaceutically acceptable salts thereof, for use in the prevention and/or treatment idiopathic pulmonary fibrosis (IPF).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise specified, the compound of formula (I) of the present invention is intended to include also pharmaceutically acceptable salts thereof.

Unless otherwise specified, the compound of formula (I) of the present invention is intended to include also the compounds of formula (la) and (lb).

The term “pharmaceutically acceptable salts”, as used herein, refers to derivatives of compounds of formula (I) wherein the parent compound is suitably modified by converting any of the free acid or basic group, if present, into the corresponding addition salt with any base or acid conventionally intended as being pharmaceutically acceptable.

Suitable examples of said salts may thus include mineral or organic acid addition salts of basic residues such as amino groups, as well as mineral or organic basic addition salts of acid residues such as carboxylic groups.

Cations of inorganic bases which can be suitably used to prepare salts comprise ions of alkali or alkaline earth metals such as potassium, sodium, calcium or magnesium.

Those obtained by reacting the main compound, functioning as a base, with an inorganic or organic acid to form a salt comprise, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methane sulfonic acid, camphor sulfonic acid, acetic acid, oxalic acid, maleic acid, fumaric acid, succinic acid and citric acid.

The term “halogen” or “halogen atoms” or “halo” as used herein includes fluorine, chlorine, bromine, and iodine atom.

The term "(C_x-C_y)alkyl" wherein x and y are integers, refers to a straight or branched chain alkyl group having from x to y carbon atoms. Thus, when x is 1 and y is 6, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n- hexyl.

Throughout the specification the use of an asterisk in the definition of a structural formula, indicates the point of attachment for the radical group to the rest of the molecule.

A dash that is not between two letters or symbols is meant to represent the point of attachment for a substituent.

The compounds of the invention are active as inhibitors of ALK5 receptor, they are potent and show improved properties such as a low metabolic stability, a low systemic exposure, a good inhalatory profile and a good selectivity across the kinome. In this respect, the state of the art does not describe or suggest pyrido oxazolidinone derivatives of general formula (I) of the present invention having an inhibitory activity on receptor ALK5 which represents a solution to the aforementioned need.

In more details, the present invention refers to a series of compounds represented by the general formula (I) as herein below described in details, which are endowed with an inhhibitory activity on receptor ALK5 receptor. Advantageously, the inhibitory action on receptor ALK5 can be effective in the treatment of those diseases where these receptors play a relevant role in the pathogenesis such as fibrosis and disease, disorder, and condition from fibrosis.

Differently from similar compounds of the prior art, the compounds of formula (I) of the present invention are able to act as antagonists of ALK5 receptor, particularly appreciated by the skilled person when looking at a suitable and efficacious compounds useful for the treatment of fibrosis, in particular idiopathic pulmonary fibrosis. As indicated in the experimental part, in particular in Table 3, the compounds of formula (I) of the present invention show a notable potency with respect to their inhibitory activity on receptor ALK5, with a pKi greater than 7.5, confirming that they are able to inhibit the ALK5 receptor involved in fibrosis and diseases that result from fibrosis. Advantageously, the compounds of the present invention are endowed with a high potency, they could be administered in human at a lower dosage respect to the compounds of the prior art, thus reducing the adverse events that typically occur administering higher dosages of a drug. In addition to being notably potent with respect to their inhibitory activity on receptor ALK5, the compounds of the present invention are also characterized by a good inhalatory profile, that permits to act effectively on the lung compartment and have, at the same time, a low metabolic stability, that allows to minimize the drawbacks associated with the systemic exposure, such as safety and tolerability issues.

Moreover, as indicated in the experimental part, comparative examples, in particular in Table 4, it is shown that, conversely to the compounds Cl and C2 characterized by lacking a pyridinyl or a pyridinyl condensed group linked to the amino group bearing the pyridazine ring, the presence of a pyridinyl or a pyridinyl condensed group linked to the amino group bearing the pyridazine ring in the present invention compounds unexpectedly and remarkably determines a relevant increase in the inhibitory activity on the ALK5 receptor.

Therefore, the compounds of the present invention are particularly appreciated by the skilled person when looking at a suitable and efficacious compounds useful for the treatment of fibrosis, in particular idiopathic pulmonary fibrosis, administered by the inhalation route and characterized by a good inhalatory profile, that corresponds to a good activity on the lung, a good lung retention and to a low metabolic stability, that minimizes the systemic exposure and correlated safety issues.

Thus, in one aspect the present invention relates to a compound of general formula (I) wherein Ri is selected from the group consisting of Al and A2

Al A2 I<2 is selected from the group consisting of phenyl optionally substituted by one or more halogen atoms; pyridyl optionally substituted by one or more -(Ci-Ce)alkyl; and thiazolyl optionally substituted by one or more -(Ci-Ce)alkyl;

R; is H or is selected from the group consisting of -NH2, -NHC(O)-(Ci-Ce)alkyl and - NHC(O)-(Ci-C₆)alkylene-NR_ARB;

RA is -(Ci-Ce)alkyl;

RB is -(Ci-Ce)alkyl; and pharmaceutically acceptable salts thereof.

In another preferred embodiment the present invention refers to a compound of formula (I), wherein Ri is Al

Al represented by the formula (la) wherein Rz is selected from the group consisting of phenyl optionally substituted by one or more halogen atoms; pyridyl optionally substituted by one or more -(Ci-Ce)alkyl; and thiazolyl optionally substituted by one or more -(Ci-Ce)alkyl;

Rs is H or is selected from the group consisting of -NH2, -NHC(O)-(Ci-Ce)alkyl and -NHC(O)- (Ci-Ce)alkylene-NRARB;

RA is -(Ci-Ce)alkyl;

RB is -(Ci-Ce)alkyl; and pharmaceutically acceptable salts thereof.

According to a preferred embodiment, the invention refers to at least one of the compounds of Formula (la) listed in the Table 1 below and pharmaceutically acceptable salts thereof. These compounds are particularly active on receptor ALK5, as shown in Table 3. Table 1: List of preferred compounds of Formula (la) In another preferred embodiment the present invention refers to a compound of formula (I), wherein Ri is A2

A2 represented by the formula (lb) wherein Rz is selected from the group consisting of phenyl optionally substituted by one or more halogen atoms; pyridyl optionally substituted by one or more -(Ci-Ce)alkyl; and thiazolyl optionally substituted by one or more -(Ci-Ce)alkyl; and pharmaceutically acceptable salts thereof.

According to a preferred embodiment, the invention refers to at least one of the compounds of Formula (lb) listed in the Table 2 below and pharmaceutically acceptable salts thereof. These compounds are particularly active on receptor ALK5, as shown in Table 3.

Table 2: List of preferred compounds of Formula (lb)

In a more preferred embodiment, the present invention refers to a compound of formula (I), wherein Rz is phenyl substituted by a chlorine and a fluorine atom.

In an even more preferred embodiment, the present invention refers to a compound of formula (I), wherein Rz is -(5-chloro-2-fluorophenyl).

In a further preferred embodiment, the present invention refers to a compound of formula (I), wherein Rz is pyridyl substituted by a methyl.

In an even more preferred embodiment, the present invention refers to a compound of formula (I), wherein Rz is -(6-methylpyridin-2-yl). In a more preferred embodiment, the present invention refers to a compound of formula (I), wherein Rz is thiazolyl substituted by a methyl.

In a further preferred embodiment, the present invention refers to a compound of formula (I), wherein Rz is -(4-methylthiazol-2-yl).

The compounds of the invention, including all the compounds here above listed, can be prepared from readily available starting materials using the following general methods and procedures outlined in detail in the Schemes shown below, or by using slightly modified processes readily available to those of ordinary skill in the art. Although a particular embodiment of the present invention may be shown or described herein, those skilled in the art will recognize that all embodiments or aspects of the present invention can be obtained using the methods described herein or by using other known methods, reagents and starting materials. When typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. While the optimum reaction conditions may vary depending on the particular reactants or solvent used, such conditions can be readily determined by those skilled in the art by routine optimization procedures. Thus, processes described below should not be viewed as limiting the scope of the synthetic methods available for the preparation of the compounds of the invention.

In some cases, a step is needed in order to mask or protect sensitive or reactive moieties, generally known protective groups (PG) could be employed, in accordance to general principles of chemistry (Protective group in organic syntheses, 3rd ed. T. W. Greene, P. G. M. Wuts).

The compounds of formula (I) of the present invention have surprisingly been found to effectively inhibit the receptor ALK5. Advantageously, the inhibition of ALK5 may result in efficacious treatment of the diseases or condition wherein the ALK5 receptor is involved. In this respect, it has now been found that the compounds of formula (I) of the present invention have an inhibitory drug potency, expressed as pICso (negative logarithm of IC50, half maximal inhibitory concentration) and subsequently converted to pKi (negative logarithm of dissociate function Ki), equal or higher than 7.5 on ALK5, as shown in the experimental part. Preferably, the compounds of the present invention have a pKi on ALK5 between 7.5 and 8.9, more preferably higher or equal than 9.0.

In one aspect, the present invention refers to a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use as a medicament. Thus, the invention refers to a compound of formula (I) in the preparation of a medicament, preferably for use in the prevention and/or treatment of a disease, disorder or condition associated with ALK5 signaling pathway.

In a preferred embodiment, the invention refers to a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of a disease, disorder or condition associated with ALK5 signaling pathway.

In one embodiment, the present invention refers to a compound of formula (I) useful for the prevention and/or treatment of fibrosis and/or diseases, disorders, or conditions that involve fibrosis.

The terms "fibrosis" or "fibrosing disorder," as used herein, refers to conditions that are associated with the abnormal accumulation of cells and/or fibronectin and/or collagen and/or increased fibroblast recruitment and include but are not limited to fibrosis of individual organs or tissues such as the heart, kidney, liver, joints, lung, pleural tissue, peritoneal tissue, skin, cornea, retina, musculoskeletal and digestive tract.

Preferably, the compounds of formula (I) of the present invention, or a pharmaceutical composition comprising a compound of formula (I), are useful for the treatment and/or prevention of fibrosis such as pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), hepatic fibrosis, renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis and systemic sclerosis.

More preferably, the compounds of formula (I) of the present invention, or a pharmaceutical composition comprising a compound of formula (I), are useful for the treatment of idiopathic pulmonary fibrosis (IPF).

As used herein, "safe and effective amount" in reference to a compound of formula (I) or a pharmaceutically acceptable salt thereof or other pharmaceutically active agent means an amount of the compound sufficient to treat the patient's condition but low enough to avoid serious side effects and it can nevertheless be routinely determined by the skilled artisan.

The compounds of formula (I) may be administered once or according to a dosing regimen wherein several doses are administered at varying intervals of time for a given period of time. Typical daily dosages may vary depending upon the route of administration chosen.

The present invention also refers to a pharmaceutical composition comprising a compound of formula (I) in admixture with at least one or more pharmaceutically acceptable carrier or excipient.

In one embodiment, the invention refers to a pharmaceutical composition of compounds of formula (I) in admixture with one or more pharmaceutically acceptable carrier or excipient, for example those described in Remington’s Pharmaceutical Sciences Handbook, XVII Ed., Mack Pub., N.Y., U.S.A.

Administration of the compounds of the invention and their pharmaceutical compositions may be accomplished according to patient needs, for example, orally, nasally, parenterally (subcutaneously, intravenously, intramuscularly, intrastemally and by infusion) and by inhalation. Preferably, the compounds of the present invention are administered orally or by inhalation. More preferably, the compounds of the present invention are administered by inhalation.

In one preferred embodiment, the pharmaceutical composition comprising the compound of formula (I) is a solid oral dosage form such as tablets, gelcaps, capsules, caplets, granules, lozenges and bulk powders.

In one embodiment, the pharmaceutical composition comprising the compound of formula (I) is a tablet. The compounds of the invention can be administered alone or combined with various pharmaceutically acceptable carriers, diluents (such as sucrose, mannitol, lactose, starches) and known excipients, including suspending agents, solubilizers, buffering agents, binders, disintegrants, preservatives, colorants, flavorants, lubricants and the like.

In a further embodiment, the pharmaceutical composition comprising a compound of formula (I) is a liquid oral dosage forms such as aqueous and non-aqueous solutions, emulsions and suspensions. Such liquid dosage forms can also contain suitable known inert diluents such as water and suitable known excipients such as preservatives, wetting agents, sweeteners, flavorants, as well as agents for emulsifying and/or suspending the compounds of the invention.

In a further embodiment, the pharmaceutical composition comprising the compound of formula (I) is an inhalable preparation such as inhalable powders, propellant-containing metering aerosols or propellant-free inhalable formulations.

For administration as a dry powder, single- or multi-dose inhalers known from the prior art may be utilized. In that case the powder may be filled in gelatine, plastic or other capsules, cartridges or blister packs or in a reservoir.

A diluent or carrier chemically inert to the compounds of the invention, e.g., lactose or any other additive suitable for improving the respirable fraction may be added to the powdered compounds of the invention.

Inhalation aerosols containing propellant gas such as hydrofluoroalkanes may contain the compounds of the invention either in solution or in dispersed form. The propellant-driven formulations may also contain other ingredients such as co-solvents, stabilizers and optionally other excipients.

The propellant-free inhalable formulations comprising the compounds of the invention may be in form of solutions or suspensions in an aqueous, alcoholic or hydroalcoholic medium and they may be delivered by j et or ultrasonic nebulizers known from the prior art or by soft-mist nebulizers.

The compounds of the invention can be administered as the sole active agent or in combination with other pharmaceutical active ingredients.

The dosages of the compounds of the invention depend upon a variety of factors including among others the particular disease to be treated, the severity of the symptoms, the route of administration and the like.

The invention is also directed to a device comprising a pharmaceutical composition comprising a compound of formula (I) according to the invention, in form of a single- or multidose dry powder inhaler or a metered dose inhaler.

All preferred groups or embodiments described above for compounds of formula (I) may be combined among each other and apply mutatis mutandis as well.

In a first embodiment of the present invention, compounds of formula (I) can be prepared according to the following synthetic routes described in Scheme 1.

Scheme 1

Route A demethylation amine protection

Br

PG= tert butyloxy carbonyl

Buchwald- Hartwig ring formation amination

Route B

Buchwald- cross-coupling reaction Br

(X) ring formation

Compounds of formula (III) may be obtained by oxidation of commercially available pyridines (II) suitably substituted for further elaboration. Oxidation conditions may comprise the use of 3 -chloroperbenzoic acid in a suitable solvent, such as di chloroethane and at an appropriate temperature, as for example 60 °C. Compounds of formula (III) treated under nitration conditions may afford compounds (IV). Typical conditions include heating compounds (III) in the presence of sulfuric acid and nitric acid at high temperature, such as 60 to 90 °C. Reduction of nitro group of compounds (IV) may lead to compounds of formula (V). Such reaction may be carried out in the presence of reductive agents, as for example iron powder, in a suitable polar protic solvent, such as ethanol and at the proper temperature, as 70 °C. According to Route A, a compound of formula (V) may first undergo demethylation followed by proper protection of the free amino group to afford compounds (VII). Demethylation can be carried out under standard conditions represented using boron tribromide in a suitable apolar and aprotic solvent, such dichloromethane, as very well known to the person skilled in the art.

The resulting compounds of formula (VI) may be suitably protected at the amine group, as described in “Protective group in organic syntheses, 3rd ed. T. W. Greene, P. G. M. Wuts”, using for example, di-tert butyl dicarbonate in the presence of a base, such as sodium hydroxide and in an apolar solvent, as for example 1,4-di oxane, at room temperature. Compounds (VII) can participate to metal -catalyzed cross coupling reaction to introduce the proper R2 group leading to compounds (VIII). Cross-coupling reactions may be Suzuki coupling, as described in “Transition Metals for 15 Organic Synthesis", 2nd Ed, 1, 2004. Representative Suzuki reaction conditions include reacting compound (VII) with a suitable boronic acid, in the presence of base, such as K2CO3, KF and the like, and Pd catalyst, as PdChfPPhs^ DCM or PdChfPhsP in a mixture of solvents, such as 1,4 dioxane, MeCN and water, at an appropriate temperature, such as, for example, 115 °C under conventional heating or microwave irradiation.

Alternatively, as described in Route B of Scheme 1, a compound of formula (V) can first partecipate in a metal -catalyzed cross coupling reactions to afford compounds (X). Such reactions may comprise Suzuki cross-coupling as described above or Stille coupling to introduce appropriate R2 groups. Typical Stille coupling conditions involve the use of the proper organostannane, in the presence of a suitable catalyst such as PdChfPhsP^ and a copper salt, like copper iodide, in an appropriate solvent such as DMF or toluene and at an appropriate temperature, such as, for example, 100 °C. Next, compounds (X) can undergo demethylation according to the conditions described above to lead to compounds (VIII).

As depicted in Route A and Route B of Scheme 1, compounds of formula (VIII) can react with a suitable halide under standard Buchwald-Hartwig amination conditions to afford compounds (IX). Typical reaction conditions comprise the use of a base, such as CS2CO3 or sodium /c/V-butoxide, a suitable ligand reagent, as Xantphos, and a suitable catalyst like Pd(OAc)2 or Pd2(dba)3, in an appropriate solvent as, for example, 1,4-di oxane and at an appropriate temperature, such as, for example, 100 °C or 140 °C under conventional heating or microwave irradiation. Finally, compounds of formula (I) can be obtained by carbamate ring formation enabled by treating compounds (IX) with 1,1’ -carbonyldiimidazole. Representative reaction conditions include the use of an organic base, such as diisopropylethylamine, triethylamine and the like, in a polar solvent as for instance DMF and at an appropriate temperature, such as 55 °C.

The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way. PREPARATIONS OF INTERMEDIATES AND EXAMPLES

Chemical Names of the compounds were generated with Structure To Name Marvin Sketch Iodine.1 version 21.15.1.

All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or are known compounds or may be formed from known compounds by known methods by a person skilled in the art.

ABBREVIATION - MEANING

BBrs= boron tribromide; Boc2O= di-tert-butyl dicarbonate; cHex= cyclohexane; Cs2CO3= cesium carbonate; DCE= 1,2-di chloroethane; DCM= dichloromethane; DIPEA= N,N- diisopropylethylamine; DMF= dimethylformamide; DMSO= dimethylsulfoxide; Et2O= diethyl ether; EtOH= ethanol; EtOAc= ethyl acetate; HCOOH= formic acid; h= hour; H2= hydrogen; HNO3= nitric acid; H2O= water; H2SO4= sulfuric acid; KF= potassium fluoride; LC-MS= liquid chromatography/mass spectrometry; MeCN= acetonitrile; MeOH= methanol; min= minutes; MW= microwaves; N2= nitrogen; NaCl= sodium chloride; NaH= sodium hydride; Na2SO4= sodium sulfate; NaHCCh = sodium bicarbonate; NH3= ammonia; NH4C1= ammonium chloride; NH4HCO3= ammonium bicarbonate; NMR= nuclear magnetic resonance; Pd/C= palladium on carbon; PdC12(dppf)= [1,1 '-bis(diphenylphosphino)ferrocene]dichloropalladium(II); PdCl₂(PPh3)₂= bis(triphenylphosphine) palladium(II) dichloride; Pd2(dba)3= tris(dibenzylideneacetone)dipalladium(0); RT= room temperature; r.t.= retention time; SCX= strong cation exchange; /-BuONa= sodium /c/V-butoxide; UPLC-MS= ultra-performance liquid chromatography mass spectrometry; Xantphos= 4,5-bis(diphenylphosphino)-9,9- dimethylxanthene.

General Experimental Details and methods

Analytical method

Instruments, materials and methods employed for analyses.

¹ H-NMR spectra were performed on a Varian MR-400 spectrometer operating at 400 MHz (proton frequency), equipped with: a self-shielded Z-gradient coil 5 mm IH/nX broadband probe head for reverse detection, deuterium digital lock channel unit, quadrature digital detection unit with transmitter offset frequency shift, or on Agilent VNMRS-500, or on a Bruker Avance 400, or on a Bruker Avance III HD 400 MHz or on a Bruker Fourier 300 MHz; or on a Agilent Inova 600 operating at 600 MHz equipped with 5mm PFG PENTA Probe spectrometers. Chemical shifts are reported as 5 values in ppm relative to trimethylsilane (TMS) as an internal standard. Coupling constants (J values) are given in hertz (Hz) and multiplicities are reported using the following abbreviation (s= singlet, d= doublet, t= triplet, q= quartet, m= multiplet, br. s= broad singlet, dd= double-doublet, ddd=double-double-doublet, dt= doublet of triplets).

LC/UV/MS Analytical Methods

UPLC-MS measurements were performed on:

Method A'. Waters ACQUITY UPLC I-Class PLUS System with Waters SQ Detector 2 (ESIMS, capillary voltage: 3000 V, cone voltage: 40 V, de-solvation gas: 1000 L/h, de-solvation temp.: 500 °C), equipped with Acquity UPLC BEH C18 1.7 pm (2.1 x 100 mm), column no. 186002352, using 20 - 100% MeCN in water gradient with 0.1% formic acid (flow: 0.5 mL/min); analysis time: 3.0 min.

Method B: Shimadzu LCMS-2020 Single Quadrupole Liquid Chromatograph Mass Spectrometer, equipped with Acquity UPLC BEH - Waters, 1.7 pm C18 (2.1 x 100 mm), 130 A, column no. 186002352, using 20 - 100% ACN in water gradient with 0.1% formic acid (flow: 0.5 mL/min); analysis time: 6.0 min.

LC-MS retention times are estimated to be affected by an experimental error of +0.5 min.

LC-MS may be recorded under the following conditions: diode array DAD chromatographic traces, mass chromatograms and mass spectra may be taken on UPLC/PDA/MS Acquity TM system coupled with Micromass ZQTM or Waters SQD single quadrupole mass spectrometer operated in positive and/or negative electron spray ES ionization mode and/or Fractionlynx system used in analytical mode coupled with ZQTM single quadrupole operated in positive and/or negative ES ionisation mode. Quality Control methods used operated under low pH conditions or under high pH conditions:

Method 7, low pH conditions column: Acquity CSH C18 2.1x50mm 1.7um, the column temperature was 40 °C; mobile phase solvent A was milliQ water+0.1% HCOOH, mobile phase solvent B MeCN+0.1% HCOOH. The flow rate was 1 mL/min.

The gradient table was t=0 min 97% A 3% B, t=1.5 min 0.1% A 99.9% B, t=1.9 min 0.1% A 99.9% B and t=2 min 97% A 3% B. The UV detection range was 210-350 nm and ES+ZES- range was 100 to 1500 AMU.

Method 2, high pH conditions: column: Acquity Kinetex 1.7 um EVO C18 100A, 2.1x50mm, the column temperature was 40 °C; mobile phase solvent A was 10 mM aqueous solution of NH4HCO3 adjusted to pH=10 with ammonia, mobile phase solvent B MeCN. The flow rate was 1 mL/min. The gradient table was t=0 min 97% A 3% B, t=1.5 min 0.1% A 99.9% B, t=1.9 min 0.1% A 99.9% B and t=2 min 97% A 3% B. The UV detection range was 210-350 nm and ES+/ES- range was 100 to 1500 AMU. LC-MS measurements may be performed on Dionex UHPLC Ultimate 3000 with DAD detector/Thermo Scientific MSQ Plus system, equipped with Kinetex® 2.6 pm XB-C18 (4.6 x 50mm), 110A, column no. 00B-4496-E0. Detection range: 190 - 350 nm ± 4 nm. Flow: 1.0 mL/min. Column temperature: 25 °C. Autosampler temperature: 20 °C.

Method 3, analysis time: 6 min. Mobile phase A was milliQ water, mobile phase B was MeCN. The gradient table was t= 0.00 - 3.35 min 80% A, 20% B, t=3.35 - 3.75 min 20% A, 80% B, t=3.90 - 4.75 min 5% A, 95% B, t= 5.00 - 6.00 min 80% A, 20% B.

Method 4, analysis time: 6 min. Mobile phase A was milliQ water, mobile phase B was MeCN. The gradient table was t=0.00 - 3.35 min 70% A, 30% B, t=3.35 - 3.75 min 20% A, 80% B, t=3.90 - 4.75 min 5% A, 95% B, t=5.00 - 6.00 min 70% A, 30% B.

Method 5, analysis time: 7 min. Mobile phase A was millQ water+0.1% HCOOH, mobile phase B was MeCN+0.1% HCOOH. The gradient was t=0.00 - 2.00 min 80% A, 20% B, t=2.00 - 2.35 min 20% A, 80% B, t=2.45 - 4.25 min 5% A, 95% B, t=5.00 - 7.00 min 80% A, 20% B.

PREPARATIONS OF INTERMEDIATES

Intermediate 1: 2-bromo-5-methoxypyridine

To a stirred solution of 2-bromo-5-hydroxypyridine (4.0 g, 23 mmol) in DMF (40 mL), at RT and under a N2 atmosphere, NaH 60% dispersion in oil (1.29 g, 32.25 mmol) was cautiously added portionwise (vigorous gas development), then the mixture was stirred at RT for 10 min. lodomethane (2.8 mL, 45 mmol) was added dropwise and the resulting reaction mixture was stirred at RT for Ih. The reaction mixture was diluted with Et2O and washed twice with H2O. The organic phase was washed with saturated NaCl aqueous solution, dried over Na2SO4, and evaporated under reduced pressure to afford title compound (4.3 g, 23 mmol, recovery assumed quantitative). LC- MS (ESI): m/z (M+l): 189.9 (Method 1).

Intermediate 2: 2-bromo-5-methoxypyridin-l-ium-l-olate

XX . T X

To a stirred solution of Intermediate 1 (5.4 g, 28.72 mmol) in DCE (70 mL), at RT, 3- chloroperbenzoic acid (8.42 g, 48.83 mmol) was added portionwise, then the resulting reaction mixture was stirred at 60 °C for 12h. The reaction mixture was allowed to reach RT, quenched by portion-wise addition of diethylamine (2.5 mL, 24.15 mmol), then volatiles were removed under reduced pressure. The residue was purified by flash chromatography on Biotage NH silica cartridge (gradient of elution from 0 to 55% of EtOAc in cHex) to afford title compound (5.2 g, 25.49 mmol, 89% yield). LC-MS (ESI): mlz (M+l): 203.9 (Method 1).

Intermediate 3: 2-bromo-5-methoxy-4-nitropyridin-l-ium-l-olate

95-98% H2SO4 (15 mL, 281.4 mmol) was cautiously added to Intermediate 2 (5.2 g, 25.49 mmol) followed by dropwise addition of 70% HNO3 (3 mL, 67.13 mmol) at RT. The stirred mixture was heated to 90 °C then it was allowed to reach 60 °C and additional 70% HNO3 (12 mL, 268.53 mmol) was added dropwise. The resulting mixture was stirred at 60 °C for 15 min, then it was cooled and poured into ice/EEO mixture (~ 250 mL). The mixture was filtered, the solid was washed with H2O, EtOAc and dried under reduced pressure. The solid was collected, suspended in DCM, then filtered washing with DCM and dried under reduced pressure to afford a first crop (1.62 g). Mother liquor was concentrated under reduced pressure and the crude material was purified by flash chromatography on Biotage NH silica cartridge (gradient of elution from 0 to 50% of EtOAc in cHex). Proper fractions were collected and mixed with the first crop to afford title compound (2.11 g, 8.47 mmol, 33% yield). LC-MS (ESI): mlz (M+l): 248.9 (Method 1).

Intermediate 4: 2-bromo-5-methoxypyridin-4-amine

To a stirred solution Intermediate 3 (1.62 g, 6.51 mmol) in EtOH (16 mL) at RT, iron powder (3.6 g, 64.46 mmol) was added followed by a solution of NH4CI (340 mg, 6.36 mmol) in H2O (4 mL). The resulting reaction mixture was heated to 70 °C and stirred for Ih. The mixture was allowed to reach RT then it was filtered over Celite® and the filtrate was concentrated under reduced pressure. The residue was taken up with EtOAc and H2O, the organic phase was washed with saturated NaCl aqueous solution, dried over Na2SO4 and the solvent removed under reduced pressure to afford title compound (0.96 g, 4.73 mmol, 73% yield). LC-MS (ESI): mlz (M+l): 202.9 (Method 2). Intermediate 5: 2-(5-chloro-2-fluorophenyl)-5-methoxypyridin-4-amine

Intermediate 4 (300 mg, 1.48 mmol), (5-chloro-2-fluorophenyl)boronic acid (305 mg, 1.75 mmol) and KF (210 mg, 3.61 mmol) were mixed in MeCN (2.4 mL) and H2O (2.4 mL). N2 was bubbled for 5 min before adding PdC12(PPh3)2 (102 mg, 0.14 mmol), then the vial was sealed and irradiated with MW apparatus at 115 °C for 30 min. The mixture was diluted with EtOAc and H2O, the two phases were separated and the organic layer was washed with H2O, dried over Na2SO4 and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography on Biotage NH silica cartridge (gradient of elution from 0 to 20% of EtOAc in cHex) to afford title compound (254 mg, 1.0 mmol, 67% yield). LC-MS (ESI): m/z (M+l): 253.0 (Method 1).

Intermediate 6: 2-bromo-5-hydroxypyridin-4-aminium bromide

To a suspension of Intermediate 4 (7.7 g, 34.7 mmol) in dry DCM (45 mL), cooled to - 30 °C under argon atmosphere, BB (IM in DCM, 45 mL, 45.0 mmol) was added dropwise over 30 min. The mixture was vigorously stirred for 24h. After that time, the suspension was warmed to RT and the precipitate was filtered and washed with DCM. The solid was dried under reduced pressure to give title compound as HBr salt (7.1 g, 26.5 mmol, 76% yield). ^JH NMR (300 MHz, Deuterium Oxide) 5 7.54 (s, 1H), 6.96 (s, 1H).

Intermediate 7: tert-butyl N-(2-bromo-5-hydroxypyridin-4-yl)carbamate

To a solution of Intermediate 6 (8.1 g, 30.2 mmol) in 1,4-dioxane (90 mL), NaOH aqueous solution (1.0M, 70 mL, 70 mmol) was added and the mixture was cooled to 0 °C before addition of BOC2O (16.5 g, 75.5 mmol) portionwise. The reaction was stirred at RT for 1.5h, then a second portion of BOC2O (3.0 g, 13.6 mmol) was added and the mixture was allowed to stir for 1.5h. Volatiles were removed under reduced pressure, the residue diluted with H2O (35 mL) and extracted with EtOAc (3 * 200 mL). Combined organics were dried over Na2SO4 and the solvent removed under reduced pressure. The residue was triturated with hexanes, filtered, washed with additional portion of hexanes to afford title compound (6.6 g, 22.9 mmol, 76% yield). ^!H NMR (300 MHz, DMSO-O 5 7.77 (s, 1H), 6.80 (s, 1H), 6.49 (s, 2H), 1.49 (s, 9H).

Intermediate 8: 4-amino-6-(5-chloro-2-fluorophenyl)pyridin-3-ol

Method A

In a suitable vial, to a mixture of Intermediate 7 (210 mg, 0.73 mmol), (5-chloro-2- fluorophenyl)boronic acid (152 mg, 0.87 mmol) and PdChfPPhs)? (55 mg, 0.08 mmol), MeCN (1.9 mL) was added followed by a solution of KF (109 mg, 1.88 mmol) in H2O (1.9 mL). N2 was bubbled for 2 min then the vial was sealed and irradiate with MW apparatus at 115 °C for 30 min. The mixture was concentrated under reduced pressure, the residue was taken up with MeOH and the solution charged on SCX cartridge, washing with MeOH, and eluting with IN NH3 in MeOH. Basic fractions were collected and concentrated to afford title compound (0.73 mmol, recovery assumed quantitative).

Method B

To a stirred solution of Intermediate 5 (40 mg, 0.16 mmol) in DCM (2 mL), cooled at -10 °C and under N2 atmosphere, BBrs (1.0M in DCM, 0.19 mL, 0.19 mmol) was added dropwise. After 2 min the cooling-bath was removed and the resulting reaction mixture was stirred at RT for 2h. The reaction was quenched by addition of MeOH, then the mixture was concentrated under reduced pressure. The residue was dissolved in MeOH, and the solution was charged on SCX cartridge, washing with MeOH, and eluting with IN NH3 in MeOH to afford title compound (36 mg, 0.15 mmol, 95% yield). LC-MS (ESI): m/z (M+1): 239.0 (Method 2).

Intermediate 9: 5-methoxy-6'-methyl-[2,2'-bipyridin]-4-amine In avial, to a suspension of Intermediate 4 (100 mg, 0.49 mmol) in toluene (2.2 mL), tributyl- (6-methyl-2-pyridinyl)stannane (284 mg, 0.74 mmol) was added then N2 was bubbled in for several minutes. Copper (I) iodide (19 mg, 0.10 mmol) and PdChfPPhs)? (35 mg, 0.05 mmol) were added, the vial was sealed and heated at 105 °C for 12h. Additional PdCh(PPh3)2 (17 mg), copper(I) iodide (10 mg) and tributyl-(6-methyl-2-pyridinyl)stannane (140 mg) were added, followed by DMF (1 mL). The vial was sealed and heated overnight at 105 °C. The reaction mixture was concentrated under reduce pressure, the residue was charged on SCX cartridge, washing with MeOH, and eluting with IN NH3 in MeOH. Basic fractions were collected, the residue was purified by flash chromatography on Biotage NH silica cartridge (gradient of elution from 0 to 50% of EtOAc in cHex) to afford title compound (56 mg, 0.26 mmol, 53% yield). LC- MS (ESI): mlz (M+l): 216.0 (Method 2).

Intermediate 10: 4-amino-6'-methyl-[2,2'-bipyridin]-5-ol

Intermediate 10 was prepared following the procedure used for the synthesis of Intermediate 8 Method B starting from Intermediate 9 (142 mg, 0.66 mmol) to afford title compound (95 mg, 0.47 mmol, 71% yield). LC-MS (ESI): mlz (M+l): 202.0 (Method 2).

Intermediate 11: 6'-methyl-4-[(pyridin-4-yl)amino]-[2,2'-bipyridin]-5-ol

In a suitable vial, to a mixture Intermediate 10 (36 mg, 0.18 mmol), 4-bromopyridine hydrochloride (34 mg, 0.17 mmol), Z-BuONa (32 mg, 0.33 mmol), Pd2(dba)3 (15 mg, 0.02 mmol) and Xantphos (15 mg, 0.03 mmol), 1,4-dioxane (2.6 mL) was added. The vial was sealed and irradiate with MW apparatus at 140 °C for 20 min. The mixture was diluted with MeOH, filtered, and charged on SCX cartridge, washing with MeOH, and eluting with IN NH3 in MeOH. Basic fractions were collected and evaporated to afford title compound (0.17 mmol, recovery assumed quantitative). LC-MS (ESI): mlz (M+l): 279.1 (Method 2). Intermediate 12: 6'-methyl-4-[(quinolin-4-yl)amino]-[2,2'-bipyridin]-5-ol

Intermediate 12 was prepared following the procedure used for the synthesis of Intermediate 11 starting from Intermediate 10 (22 mg, 0.11 mmol) and using 4-bromoquinoline (28 mg, 0.13 mmol) to afford title compound (35 mg, 0.10 mmol, 98% yield). LC-MS (ESI): m/z (M+l): 329.1 (Method 2).

Intermediate 13: N-(4-bromopyridin-2-yl)acetamide

Pyridine (3.7 mL, 46 mmol) was added to a stirred solution of 4-bromo-2-pyridinamine (500 mg, 2.89 mmol) in DCM (3.8 mL) at RT, followed by acetic anhydride (0.41 mL, 4.34 mmol). The resulting reaction mixture was stirred at RT for 12h. The mixture was concentrated under reduced pressure and the residue was taken up with EtOAc and a saturated NaHCCh aqueous solution. The organic phase was washed with saturated NaCl aqueous solution, dried over Na2SC>4 and the solvent was removed under reduced pressure to afford title compound (2.89 mmol, recovery assumed quantitative). LC-MS (ESI): m/z (M+l): 214.9 (Method 1).

Intermediate 14: N-(4-{[2-(5-chloro-2-fluorophenyl)-5-hydroxypyridin-4-yl]amino} pyridin-2-yl)acetamide

Intermediate 14 was prepared following the procedure used for the synthesis of Intermediate 11 starting from Intermediate 8 (35 mg, 0.15 mmol) and using Intermediate 13 (33 mg, 0.15 mmol) to afford title compound (0.15 mmol, recovery assumed quantitative). LC-MS (ESI): m/z (M+l): 373.0 (Method 2). Intermediate 15: N-(4-bromopyridin-2-yl)-3-(dimethylamino)propanamide

To a solution of 3 -dimethylaminopropionic acid (100 mg, 0.85 mmol) in DCM (2 mL) at RT, oxalyl chloride (0.29 mL, 3.42 mmol) was added followed by a catalytic amount of DMF (2 drops). The reaction mixture was stirred at RT for Ih, then it was concentrated under reduced pressure. The residue was dissolved in DCM (3 mL) and the solution was added portionwise to a stirred solution of 4-bromo-2-pyridinamine (146 mg, 0.84 mmol) and pyridine (1.03 mL, 12.8 mmol) in DCM (2 mL) at RT. The resulting reaction mixture was stirred at RT for 12h. The reaction mixture was diluted with DCM and washed with H2O. The aqueous phase was extracted twice with DCM, the organic phases were combined, dried over Na2SO4, and concentrated under reduced pressure to afford title compound (86 mg, 0.32 mmol, 37% yield).

LC-MS (ESI): mlz (M+l): 272.0 (Method 2).

Intermediate 16: N-(4-{[2-(5-chloro-2-fluorophenyl)-5-hydroxypyridin-4-yl]amino} pyridin-2-yl)-3-(dimethylamino)propanamide

Intermediate 16 was prepared following the procedure used for the synthesis of Intermediate 11 starting from Intermediate 8 (35 mg, 0.14 mmol) and using Intermediate 15 (50 mg, 0.18 mmol) to afford title compound (62 mg, 0.14 mmol, 98% yield). LC-MS (ESI): mlz (M+l): 430.1 (Method 2).

Intermediate 17: 6-(5-chloro-2-fluorophenyl)-4-[(2-nitropyridin-4-yl)amino]pyridin-3- ol Intermediate 17 was prepared following the procedure used for the synthesis of Intermediate 11 starting from Intermediate 8 (60 mg, 0.26 mmol) and using CS2CO3 (182 mg, 0.56 mmol) as a base. The reaction was heated to 125 °C for 20 min under MW irradiation to afford title compound (94 mg, 0.26 mmol, recovery assumed quantitative). LC-MS (ESI): mlz (M+l): 360.9 (Method 2).

Intermediate 18: 6-(5-chloro-2-fluorophenyl)-l-(2-nitropyridin-4-yl)-lH,2H-

[l,3]oxazolo[5,4-c]pyridin-2-one

To a solution of Intermediate 17 (94 mg, 0.26 mmol) in DMF (1.84 mL) at RT, 1,1'- carbonyldiimidazole (66 mg, 0.41 mmol) and DIPEA (0.14 mL, 0.81 mmol) were subsequently added then the resulting reaction mixture was stirred at 55 °C for 3h. Additional 1,1'- carbonyldiimidazole (24 mg, 0.15 mmol) was added and the reaction mixture was stirred at 60 °C for 2h. The reaction mixture was diluted with EtOAc and washed with H2O and saturated NaCl aqueous solution. The organic phase was dried over Na2SC>4 and concentrated under reduced pressure. The residue was purified by flash chromatography on Biotage silica cartridge (gradient of elution from 0 to 35% of EtOAc in cHex) to afford title compound (28 mg, 0.07 mmol, 27% yield). LC-MS (ESI): mlz (M+l): 387.1 (Method 1).

Intermediate 19: 5-hydroxy-2-(4-methyl-l,3-thiazol-2-yl)pyridin-4-aminium chloride

Intermediate 19 was prepared following the procedure used for the synthesis of Intermediate 9, starting from Intermediate 7 (1.0 g, 3.46 mmol) and using 4-methyl-2-(tributylstannyl)-l,3- thiazole (1.45 mL, 5.19 mmol). The reaction mixture was sealed and stirred at 80 °C for 19h. After cooling to RT, volatiles were removed under reduced pressure. The residue was taken up with DCM, washed with aqueous ammonia solution and the product was extracted with 6N aqueous HC1. Combined acidic layers were dried under reduced pressure to afford title compound (1.0 g, crude) which was used in the next step without further purification. ^JH NMR (300 MHz, Methanol - tZ₄) 5 7.63 (s, 1H), 7.47 (d, J= 1.0 Hz, 1H), 7.34 (s, 1H), 2.55 (d, J= 1.0 Hz, 3H). Intermediate 20: sodium 6-(4-methyl-l,3-thiazol-2-yl)-4-[(pyridin-4-yl)amino]pyridin- 3-olate

Intermediate 20 was prepared following the procedure used for the synthesis of Intermediate 11 starting from Intermediate 19 (300 mg, 1.45 mmol), using 4-bromopyridine hydrochloride (240 mg, 1.23 mmol) and PdC12(dppf) (556 mg, 5.79 mmol) as catalyst. The reaction was irradiated with MW apparatus at 150 °C for 40 min. The mixture was diluted with MeOH and filtered through Celite®. Solvents were removed under reduced pressure and the residue was triturated with EtOAc. The solid was then dissolved in H2O and washed with DCM. Aqueous layer was dried under reduced pressure to afford title compound (980 mg, crude) which was used in the next step without further purification. 'H NMR (300 MHz, DMSO-t/₆) 8 11.27 (s, 1H), 10.65 (s, 1H), 8.39 (d, J = 4.9 Hz, 2H), 7.97 (s, 1H), 7.35 (d, J= 10.2 Hz, 1H), 7.21 (d, J= 6.9 Hz, 2H), 2.41 (s, 3H).

Intermediate 21: sodium 6-(4-methyl-l,3-thiazol-2-yl)-4-[(quinolin-4-yl)amino] pyridin-3-olate

Intermediate 21 was prepared following the procedure used for the synthesis of Intermediate 11, starting from Intermediate 19 (161 mg, 0.66 mmol) and using 4-bromoquinoline (124 mg, 0.6 mmol) to afford the title compound (307 mg, crude) which was used in the next step without further purification. UPLC-MS: m/z (M+l): 335.2, r.t. = 1.25 min (Method A).

Intermediate 22: 6-(5-chloro-2-fluorophenyl)-4-[(quinolin-4-yl)amino]pyridin-3-ol

Intermediate 22 was prepared following the procedure used for the synthesis of Intermediate

11, starting from Intermediate 8 (60 mg, 0.25 mmol) and using 4-bromoquinoline (47 mg, 0.23 mmol) to afford the title compound (53 mg, 0.15 mmol, 57% yield). 'H NMR (400 MHz, DMSO- tZ₆) 8 10.50 (s, 1H), 8.82 (d, J= 26.4 Hz, 1H), 8.57 (s, 1H), 8.33 (d, J= 11.3 Hz, 2H), 7.98 (dd, J = 6.9, 2.8 Hz, 1H), 7.91 (dd, J = 19.0, 7.6 Hz, 1H), 7.74 (ddd, J= 8.3, 6.8, 1.3 Hz, 1H), 7.68 (s, 1H), 7.57 (t, J= 7.6 Hz, 1H), 7.47 (ddd, J = 8.7, 4.1, 2.8 Hz, 1H), 7.39 - 7.30 (m, 1H), 6.83 (s, 1H).

Intermediate 23: 6-(5-chloro-2-fluorophenyl)-4-[(pyridin-4-yl)amino]pyridin-3-ol

Intermediate 23 was prepared following the procedure used for the synthesis of Intermediate 11, starting from Intermediate 8 (20 mg, 0.084 mmol) and using 4-bromopyridine hydrochloride (15 mg, 0.076 mmol) to afford title compound (30 mg, crude) which was used in the next step without further purification. UPLC-MS: m/z (M+l): 316.6, r.t. = 2.38 min (Method B).

Intermediate 24: 6-(5-chloro-2-fluorophenyl)-4-[(pyridin-3-yl)amino]pyridin-3-ol

Intermediate 24 was prepared following the procedure used for the synthesis of Intermediate 11, starting from Intermediate 8 (36 mg, 0.15 mmol) and using 3 -bromopyridine (25 mg, 0.16 mmol) to afford title compound (47 mg, 0.15 mmol, recovery assumed quantitative). LC-MS (ESI): m/z (M+l): 316.0 (Method 2).

Intermediate 25: 6-(5-chloro-2-fluorophenyl)-4-[(5-nitropyridin-3-yl)amino]pyridin-3- ol

Intermediate 25 was prepared following the procedure used for the synthesis of Intermediate

9 starting from Intermediate 8 (100 mg, 0.41 mmol) and using 3 -bromo-5 -nitropyridine (100 mg, 0.49 mmol) to afford title compound (130 mg, 0.36 mmol, 88% yield). LC-MS (ESI): mlz (M+l): 361.0 (Method 1).

Intermediate 26: 6-(5-chloro-2-fluorophenyl)-l-(5-nitropyridin-3-yl)-lH,2H-[l,3] oxazolo[5,4-c]pyridin-2-one

Intermediate 26 was prepared following the procedure used for the synthesis of Intermediate 18 starting from Intermediate 25 (130 mg, 0.36 mmol) to afford title compound (42 mg, 0.11 mmol, 30% yield). LC-MS (ESI): mlz (M+l): 387.1 (Method 1).

PREPARATIONS OF EXAMPLES

Example 1: 6-(6-methylpyridin-2-yl)-l-(pyridin-4-yl)-lH,2H-[l,3]oxazolo[5,4- c]pyridin-2-one

Example 1 was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 11 (56 mg, 0.20 mmol) to afford title compound (11 mg, 0.04 mmol, 18% yield). LC-MS (ESI): mlz (M+l): 305.1 (Method 1). 'H NMR (400 MHz, Chloroform-d) 5 ppm 8.83 - 8.99 (m, 2H), 8.63 (s, 1H), 8.42 (s, 1H), 8.21 (d, J=7.9 Hz, 1H), 7.67 - 7.77 (m, 3H), 7.21 (d, .7=7,5 Hz, 1H), 2.62 (s, 3H).

Example 2: 6-(6-methylpyridin-2-yl)-l-(quinolin-4-yl)-lH,2H-[l,3]oxazolo[5,4- c]pyridin-2-one

Example 2 was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 12 (35 mg, 0.11 mmol) to afford title compound (6 mg, 0.02 mmol, 16% yield). LC-MS (ESI): mlz (M+l): 355.1 (Method 1). 'H NMR (400 MHz, Chloroform-d) 5 ppm 9.19 (d, 7=4.6 Hz, 1H), 8.70 (d, 7=0.7 Hz, 1H), 8.35 (d, 7=8.4 Hz, 1H), 8.19 (d, 7=7.9 Hz, 1H), 7.87-7.92 (m, 2H), 7.61-7.78 (m, 4H), 7.16 (d, 7=7.3 Hz, 1H), 2.50 (s, 3H).

Example 3: N-{4-[6-(5-chloro-2-fluorophenyl)-2-oxo-lH,2H-[l,3]oxazolo[5,4- c]pyridin-l-yl]pyridin-2-yl}acetamide

Example 3 was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 14 (56 mg, 0.15 mmol) to afford title compound (22 mg, 0.06 mmol, 66% yield). LC-MS (ESI): m/z (M+l): 399.9 (Method 1). 'H NMR (400 MHz, Chlor of orm-d) 5 ppm 8.71 (d, .7=0,9 Hz, 1H), 8.56 (d, 7=1.8 Hz, 1H), 8.50 (dd, 7=5.5, 0.7 Hz, 1H), 8.11 (s, 1H), 8.04 (dd, 7=6.7, 2.8 Hz, 1H), 7.84 (t, 7=1.0 Hz, 1H), 7.45 (dd, 7=5.5, 2.0 Hz, 1H), 7.34-7.39 (m, 1H), 7.15 (dd, 7=10.6, 8.8 Hz, 1H), 2.28 (s, 3H).

Example 4: N-{4-[6-(5-chloro-2-fluorophenyl)-2-oxo-lH,2H-[l,3]oxazolo[5,4- c]pyridin-l-yl]pyridin-2-yl}-3-(dimethylamino)propanamide

Example 4 was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 16 (62 mg, 0.14 mmol) and using THF as solvent to afford title compound (12 mg, 0.03 mmol, 18% yield). LC-MS (ESI): m/z (M+l): 456.0 (Method 2). 'HNMR (400 MHz, Chloroform-d) 5 ppm 11.58-11.75 (m, 1H), 8.69 (d, 7=0.9 Hz, 1H), 8.50-8.60 (m, 2H), 8.03 (dd, 7=6.8, 2.6 Hz, 1H), 7.83 (t, 7=0.9 Hz, 1H), 7.31-7.41 (m, 2H), 7.14 (dd, 7=10.7, 8.7 Hz, 1H), 2.70 (d, 7=6.4 Hz, 2H), 2.59 (d, 7=6.4 Hz, 2H), 2.44 (s, 6H).

Example 5: l-(2-aminopyridin-4-yl)-6-(5-chloro-2-fluorophenyl)-lH,2H-

[l,3]oxazolo[5,4-c]pyridin-2-one

To a solution of Intermediate 18 (27 mg, 0.07 mmol) in a mixture of EtOAc (5 mL)/THF (2 mL), 10% w/w Pd/C (22 mg, 0.21 mmol) was added and the resulting reaction was stirred under H2 atmosphere for 6h. Additional 10% w/w Pd/C (22 mg, 0.21 mmol) was added and the reaction stirred under H2 atmosphere for 12h. The mixture was filtered over Celite®, the filtrate was concentrated, and the residue was purified by reverse flash chromatography on Biotage Cl 8 cartridge (from 100% H₂O/MeCN 95:5 +0.1% HCOOH to 70% of MeCN/H₂O 95:5 + 0.1% HCOOH) to afford title compound (6 mg, 0.02 mmol, 24% yield). LC-MS (ESI): m/z (M+l): 357.1 (Method 2). *HNMR (400 MHz, Chlor of orm-d) 5 ppm 8.69 (s, 1H), 8.31 (d, J=5.7 Hz, 1H), 7.99- 8.07 (m, 1H), 7.72 (s, 1H), 7.34-7.42 (m, 1H), 7.14 (s, 1H), 6.90 (dd, J=5.6, 1.9 Hz, 1H), 6.80 (d, 7=1.5 Hz, 1H), 4.76 (br. s., 2H).

Example 6: 6-(4-methyl-l,3-thiazol-2-yl)-l-(pyridin-4-yl)-lH,2H-[l,3]oxazolo[5,4- c]pyridin-2-one

Example 6 was prepared followed the procedure used for the synthesis of Intermediate 18, starting from Intermediate 20 (200 mg, 0.70 mmol) to afford title compound (8.2 mg, 0.026 mmol, 4% yield). LC-MS (ESI): m/z (M+l): 352.2 (Method 3). ‘H NMR (400 MHz, DMSO-t/₆) 8 8.88 (d, J= 5.3 Hz, 2H), 8.75 (s, 1H), 7.87 (s, 1H), 7.79 (d, J= 5.2 Hz, 2H), 7.44 (s, 1H), 2.43 (s, 3H).

Example 7: 6-(4-methyl- 1 ,3-thiazol-2-yl)-l -(quinolin-4-yl)- 1H,2H- [1,3] oxazolo [5,4- c]pyridin-2-one

Example 7 was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 21 (35 mg, 0.105 mmol) to afford title compound (6.33 mg, 0.016 mmol, 17% yield). LC-MS (ESI): m!z (M+l): 361.2 (Method 4). 'HNMR (400 MHz, DMSO-t/₆) 5 9.20 (d, .7= 4.6 Hz, 1H), 8.80 (d, = 0.7 Hz, 1H), 8.27 (d, = 8.2 Hz, 1H), 8.09 (dd, J= 8.4, 1.3 Hz, 1H), 7.98 - 7.91 (m, 2H), 7.71 (ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 7.38 (s, 2H), 2.31 (d, J= 1.0 Hz, 3H).

Example 8: 6-(5-chloro-2-fluorophenyl)-l-(quinolin-4-yl)-lH,2H-[l,3]oxazolo[5,4- c]pyridin-2-one

Example 8 was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 22 (53 mg, 0.28 mmol) to afford title compound (25.5 mg, 0.064 mmol, 23% yield). LC-MS (ESI): m/z (M+l): 391.9 (Method 5). *H NMR (400 MHz, DMSO-t/₆) 5 9.17 (d, J = 4.6 Hz, 1H), 8.89 (s, 1H), 8.24 (d, J = 8.5 Hz, 1H), 8.08 (dd, J = 8.5, 1.4 Hz, 1H), 7.96 - 7.89 (m, 2H), 7.85 (dd, J= 6.7, 2.8 Hz, 1H), 7.70 (ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 7.51 (ddd, J = 8.8, 4.2, 2.8 Hz, 1H), 7.36 - 7.28 (m, 2H).

Example 9: 6-(5-chloro-2-fluorophenyl)-l-(pyridin-4-yl)-lH,2H-[l,3]oxazolo[5,4- c]pyridin-2-one

Example 9 was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 23 (28 mg, 0.089 mmol) to afford title compound (6.4 mg, 0.019 mmol, 21% yield). 'H NMR (400 MHz, DMSO-t/₆) 5 8.87-8.81 (m, 3H), 7.92 (dd, J= 6.7, 2.8 Hz, 1H), 7.81-7.75 (m, 3H), 7.58 (ddd, J= 8.8, 4.2, 2.8 Hz, 1H), 7.42 (dd, J= 10.7, 8.8 Hz, 1H).

Comparative newly synthesised compounds having a pyridinyl with the nitrogen atom in meta position with respect to the point of attachment with the rest of the molecule

Example Cl: 6-(5-chloro-2-fluorophenyl)-l-(pyridin-3-yl)-lH,2H-[l,3]oxazolo[5,4- c]pyridin-2-one

Example Cl was prepared following the procedure used for the synthesis of Intermediate 18, starting from Intermediate 24 (48 mg, 0.15 mmol). The residue was purified by flash chromatography on Biotage silica cartridge (gradient of elution from 0 to 30% of EtOAc in cHex) to afford title compound (22 mg, 0.06 mmol, 42% yield). LC-MS (ESI): m/z (M+l): 342.1 (Method 1). 'H NMR (500 MHz, Chloroform-d) 5 ppm 8.91 (d, .7=2,5 Hz, 1H), 8.78 (dd, J=4.9, 1.1 Hz, 1H), 8.69 (s, 1H), 8.01 (dd, J=6.9, 2.7 Hz, 1H), 7.98 (dt, J=8.2, 1.9 Hz, 1H), 7.54 - 7.66 (m, 2H), 7.32 - 7.39 (m, 1H), 7.11 (dd, J=10.7, 8.8 Hz, 1H).

Example C2: l-(5-aminopyridin-3-yl)-6-(5-chloro-2-fluorophenyl)-lH,2H-

[l,3]oxazolo[5,4-c]pyridin-2-one

Example C2 was prepared following the procedure used for the synthesis of Example 5, starting from Intermediate 26 (42 mg, 0.11 mmol) to afford title compound (13 mg, 0.04 mmol, 33% yield). LC-MS (ESI): m/z (M+l): 357.0 (Method 2). 'H NMR (400 MHz, Chloroform-d) 5 ppm 8.67-8.70 (m, 1H), 8.20-8.27 (m, 2H), 7.98-8.04 (m, 1H), 7.59-7.63 (m, 1H), 7.33-7.41 (m, 1H), 7.20-7.25 (m, 1H), 7.08-7.16 (m, 1H), 3.96-4.11 (m, 2H).

PHARMACOLOGICAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION

In vitro Assay

The enzymatic activity of compounds of the present invention was monitored measuring the formation of ADP using the ADP-GLO Kinases assay. Following the incubation of the purified enzyme, a substrate and ATP, the produced ADP was converted into ATP, which in turn was converted into light by Ultra-Gio Luciferase. The luminescent signal positively correlated with ADP amount and kinase activity. Briefly, the kinase reaction was performed by incubating 2.6nM of the purified, commercially available human ALK5 (recombinant TGF pi N-term GST-tagged, 80-end), a final concentration of TGFpi peptide 94.5pM (Promega, T36-58) and ultra-pure ATP (Promega V915B). The ATP concentration was set at the Km value (concentration of substrate which permits the enzyme to achieve half maximal velocity (Vmax)) of ALK5 (0.5pM). Compound and ALK5 kinase were mixed and incubated for 15 mins. Reactions were initiated by addition of ATP at a final concentration in the assay of 0.83 pM. After an incubation of 120 min, the reaction was stopped, and ADP production detected with ADP-Glo kit according to manufacturer’s indications. All reaction and incubation steps were performed at 25°C and the assays were performed in 384-well format and validated using a selection of reference compounds tested in 11 -point concentration-response curve. The results for individual compounds are provided below in Table 3 wherein the compounds are classified in term of potency with respect to their inhibitory activity on ALK5 receptor. Results were expressed as pICso (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociate function Ki) using the Cheng-Prusoff equation. The higher the value of pKi, the greater the inhibition of ALK5 activity. As it can be appreciated, all the compounds of Table 3 show pKi values greater than 7.5 when tested in the biochemical ALK5 assay.

Table 3

Comparative Examples

Compounds of the examples Cl and C2 were tested in the same in vitro assay described above.

Table 4

The compounds of the present invention, as shown in Table 3, have a pki higher than 7.5 whereas comparative examples Cl and C2 have a pki lower than 5.5.

These data demonstrate that, conversely to the compounds Cl and C2 characterized by having a pyridinyl with the nitrogen atom in meta position with respect to the point of attachment with the rest of the molecule, the presence of a pyridinyl with the nitrogen atom in para position with respect to the point of attachment with the rest of the molecule or a quinoline wherein the pyridinyl has the nitrogen atom in meta position with respect to the point of attachment with the rest of the molecule in the present invention compounds unexpectedly and remarkably determines a relevant increase in the inhibitory activity on the ALK5 receptor.

Claims

CLAIMS A compound of formula (I) wherein Ri is selected from the group consisting of Al and A2

Al A2

R2 is selected from the group consisting of phenyl optionally substituted by one or more halogen atoms; pyridyl optionally substituted by one or more -(Ci-Ce)alkyl; and thiazolyl optionally substituted by one or more -(Ci-Ce)alkyl; R; is H or is selected from the group consisting of -NH2, -NHC(O)-(Ci-Ce)alkyl and -

NHC(O)-(Ci-C₆)alkylene-NR_ARB;

RA is -(Ci-Ce)alkyl;

RB is -(Ci-Ce)alkyl; and pharmaceutically acceptable salts thereof. The compound of formula (I) according to claim 1, wherein Ri is Al represented by the formula (la) wherein Rz is selected from the group consisting of phenyl optionally substituted by one or more halogen atoms; pyridyl optionally substituted by one or more -(Ci-Ce)alkyl; and thiazolyl optionally substituted by one or more -(Ci-Ce)alkyl;

R; is H or is selected from the group consisting of -NH2, -NHC(O)-(Ci-Ce)alkyl and - NHC(O)-(Ci-C₆)alkylene-NR_ARB;

RA is -(Ci-Ce)alkyl;

RB is -(Ci-Ce)alkyl; and pharmaceutically acceptable salts thereof. The compound of formula (la) according to claim 2 selected from at least one of:

6-(6-methylpyridin-2-yl)-l-(pyridin-4-yl)-lH,2H-[l,3]oxazolo[5,4-c]pyridin-2-one; N-{4-[6-(5-chloro-2-fhrorophenyl)-2-oxo-lH,2H-[l,3]oxazolo[5,4-c]pyridin-l- yl]pyri din-2 -yl} acetamide;

N-{4-[6-(5-chloro-2-fhrorophenyl)-2-oxo-lH,2H-[l,3]oxazolo[5,4-c]pyridin-l- yl]pyridin-2-yl}-3-(dimethylamino)propanamide; l-(2-aminopyridin-4-yl)-6-(5-chloro-2-fluorophenyl)-lH,2H-[l,3]oxazolo[5,4- c]pyridin-2-one;

6-(4-methyl-l,3-thiazol-2-yl)-l-(pyridin-4-yl)-lH,2H-[l,3]oxazolo[5,4-c]pyri din-2- one;

6-(5-chloro-2-fluorophenyl)-l-(pyridin-4-yl)-lH,2H-[l,3]oxazolo[5,4-c]pyri din-2- one. The compound of formula (I) according to claim 1, wherein Ri is A2 represented by the formula (lb) wherein Rz is selected from the group consisting of phenyl optionally substituted by one or more halogen atoms; pyridyl optionally substituted by one or more -(Ci-Ce)alkyl; and thiazolyl optionally substituted by one or more -(Ci-Ce)alkyl; and pharmaceutically acceptable salts thereof. The compound of formula (lb) according to claim 4 selected from at least one of:

6-(6-methylpyridin-2-yl)-l-(quinolin-4-yl)-lH,2H-[l,3]oxazolo[5,4-c]pyridin-2-one; 6-(4-methyl-l,3-thiazol-2-yl)-l-(quinolin-4-yl)-lH,2H-[l,3]oxazolo[5,4-c]pyridin-2- one;

6-(5-chloro-2-fluorophenyl)-l-(quinolin-4-yl)-lH,2H-[l,3]oxazolo[5,4-c]pyri din-2- one. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 5, in admixture with one or more pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to claim 6 for administration by inhalation. A compound of formula (I) according to any one of claims 1 to 5 or a pharmaceutical composition according to claims 6 and 7 for use as a medicament. A compound of formula (I) or a pharmaceutical composition for use according to claim 8 in the prevention and/or treatment of a disease, disorder or condition mediated by ALK5 signaling pathway in mammals. A compound of formula (I) or a pharmaceutical composition for use according to claims 8 and 9 in the prevention and/or treatment of fibrosis and/or diseases, disorders or conditions that involve fibrosis. A compound of formula (I) or a pharmaceutical composition for use according to claim 10 in the prevention and/or treatment of fibrosis including pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), hepatic fibrosis, renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis and systemic sclerosis. A compound of formula (I) or a pharmaceutical composition for use according to claim 11 in the prevention and/or treatment idiopathic pulmonary fibrosis (IPF).