WO2020207611A1

WO2020207611A1 - Phenazines as inhibitors of discoidin domain receptors 2 (ddr2)

Info

Publication number: WO2020207611A1
Application number: PCT/EP2019/080133
Authority: WO
Inventors: Lucio Claudio Rovati; Michael Pilkington-Miksa; Clara BOVINO; Stefano Mandelli; Tiziano ZANELLI
Original assignee: Rottapharm Biotech S.R.L.
Priority date: 2019-04-09
Filing date: 2019-11-04
Publication date: 2020-10-15
Also published as: WO2020207570A1

Abstract

The invention concerns a compound of Formula (I) or a pharmaceutically acceptable salt or derivative thereof, wherein R₁ and R₂ are independently H or a group -O(CHR₅)nR₆, where n is an integer value from 1 to 3, R₅ is independently H or CH₃ for each integer value of n, R₆ is H, an optionally substituted saturated heterocyclic ring or NR₇R₈, where R₇ and R₈ are independently H or (C₁-C₃)alkyl, R₄ is H or CH₃, R₃ is a group selected from the group consisting of an optionally substituted aryl, (C₃-C₆)alkyl, benzyl, an optionally substituted saturated (C₃-C₆)cycloalkyl and an optionally substituted unsaturated heterocyclic ring for use as a medicament. The compounds as medicament are used in the treatment (DDR2)-mediated diseases and disorders such as a cancer, acute and chronic pain, osteoarthritis, inflammation-associated disorder as arthritis, rheumatoid arthritis, atherosclerosis, various fibrotic disorders, fibrosis or kidney injury.

Description

"PHENAZINES AS INHIBITORS OF DISCOIDIN DOMAIN RECEPTORS 2 (DDR2)”

_{********************}

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to novel phenazine compounds, processes for their preparation, pharmaceutical compositions containing them and their use as medicaments for the treatment or alleviation of Discoidin Domain Receptor 2 (DDR2)- mediated diseases particularly cancer, acute and chronic pain, osteoarthritis, inflammation-associated disorder as arthritis, rheumatoid arthritis, atherosclerosis, fibrosis, kidney injury.

BACKGROUND OF THE INVENTION

Discoidin domain receptors, DDR1 and DDR2, lie at the intersection of two large receptor families, the extracellular matrix and tyrosine kinase receptors (RTKs). As such, DDRs are uniquely positioned to function as sensors for extracellular matrix and to regulate a wide range of cell functions from migration and proliferation to cytokine secretion and extracellular matrix homeostasis/remodeling. While activation of DDRs by extracellular matrix collagens is required for normal development and tissue homeostasis, aberrant activation of these receptors following injury or in disease is detrimental.

DDRs bind a number of different collagen types through their ability to recognize specific amino acid sequence motifs. The major binding site for DDRs is on fibrillar type (Xu et al Matrix Biol, 2011 ).

DDR1 and DDR2 are composed of an extracellular Discoidin (DS) homology domain which encompasses the collagen binding site, a DS-like domain which contributes to collagen-induced receptor activation, an extracellular juxtamembrane region and matrix metallo- proteinase cleavage sites. There is a transmembrane domain which mediates collagen-independent receptor dimerization, a large intracellular juxtamembrane region which contains phosphorylatable tyrosines that may serve as docking sites for DDR binding proteins and an intracellular tyrosine kinase domain with homology to Trk and insulin receptor family members. (Fu et al J Biol Chem, 2013)

The DDRs are widely expressed in different tissues: DDR1 mRNA is found with high levels in brain, lung, kidney and spleen, whereas DDR2 is mainly expressed in skeletal and heart muscle, kidney and lung (Leitinger Europe PMC Founders Group, 2014) While DDR2 is expressed as a single protein, the DDR1 sub-family may be expressed as five different isoforms that are created by alternative splicing. Unlike classical receptor tyrosine kinases in which ligand binding precedes receptor dimerization, DDRs appear to exist in dimeric complexes prior to ligand activation. DDRs undergo auto phosphorylation after binding to collagen. However, unlike the classical RTKs, the phosphorylation of DDRs after ligand binding occurs minutes to hours later. Accordingly, collagen binding to DDRs is associated with sustained, slow responses instead of the acute, rapid responses that are typically associated with signaling through receptor tyrosine kinases.

DDRs promotes inflammation in atherosclerosis, various fibrotic disorders, kidney injury, osteoarthritis and cancer progression (Villoutreix et al. Trends Pharmacol Sci 2016). In the rapidly evolving field of cancer immunotheraphy, it is reported that targeting DDR2 elicits a significantly enhanced response to immune checkpoint blockade with PD-1 inhibitors (Tu et al Sci Adv, 2019). Moreover it was recently published that the loss of E-cadherin, an adherent junction protein, besides increasing cancer cell migration, reduces checkpoint blockade (anti-CTLA-4/PD-1 ) responsiveness in melanoma (Shields BD et al Cancer Res, 2019). E-cadherin expression is known to be downregulated by DDR2 activation. These data further support, that an inhibition of DDR2 enhance the response to immune checkpoint inhibitors therapy.

Most drugs targeting RTKs are of two classes. The first is receptor antibodies that block ligand binding, receptor dimerization or receptor internalization. The second is small molecule tyrosine kinase inhibitors (TKIs) that interact with the intracellular kinase domain. While TKIs inhibiting DDR2 have been identified, these compounds exhibit only a preference for DDR2 inhibition. Effective and lasting use of traditional TKI strategies have been hampered by the emergence of drug resistance and acquired gatekeeper mutations in DDR2 treated with TKIs have already been reported. Therefore, development of inhibitors of DDR2 with alternative mechanisms of action could be highly advantageous.

A small molecule allosteric regulator that targets the extracellular domain (ECD) of an RTK has been described. Allosteric or non classical small molecule inhibitors of RTKs offer significant therapeutic advantages. An extracellularly acting small molecule allosteric inhibitor of DDR2 that functions to disrupt DDR2 receptor collagen ligand interaction has also been described (Grither et al PNAS, 2018).

Alternative compounds and methods for inhibiting DDR2 activity and treating cancers and/or inflammatory disorders are still needed.

SUMMARY OF THE INVENTION

The objects above indicated have been achieved by a compound of Formula (I):

where n is an integer value from 1 to 3,

R5 is independently H or Chb for each integer value of n,

R6 is H, an optionally substituted saturated heterocyclic ring or NR Rs, where R7 and Re are independently H or (Ci-C3)alkyl,

R4 is H or CH3,

R3 is a group selected from the group consisting of an optionally substituted aryl, (Ci- C3)alkyl, benzyl, an optionally substituted saturated (C3-C6)cycloalkyl and an optionally substituted unsaturated heterocyclic ring,

with the proviso that

a) when Ri, R2 and R4 are H, R3 is not methyl, ethyl, n-propyl and phenyl;

b) when Ri and R2 are H and R4 is methyl, R3 is not methyl; and

c) 2,7-diethoxyfenazine is excluded.

The present invention is further directed to a compound or a salt, particularly a pharmaceutically acceptable salt thereof for the use in the inhibition of Discoid in Domain Receptor 2 (DDR2).

In another aspect the invention relates to a compound of Formula (I):

or a pharmaceutically acceptable salt or derivative thereof,

wherein:

Ri and R2 are independently H or

where n is an integer value from 1 to 3,

R5 is independently H or Chb for each integer value of n,

R6 is H, an optionally substituted saturated heterocyclic ring or NR7R8, where R7 and Re are independently H or (Ci-C3)alkyl,

R4 is H or CH3,

R3 is a group selected from the group consisting of an optionally substituted aryl, (Ci- C3)alkyl, benzyl, an optionally substituted saturated (C3-C6)cycloalkyl and an optionally substituted unsaturated heterocyclic ring for use as a medicament.

The compounds of the invention are inhibitors of DDR2 and may be useful for the treatment of DDR2-mediated diseases and disorders. Therefore in another aspect the invention relates to the compound of the invention as a medicament for use in the treatment of (DDR2)-mediated diseases and disorders, particularly such as cancer, acute and chronic pain, osteoarthritis, inflammation-associated disorder as arthritis, rheumatoid arthritis, atherosclerosis, various fibrotic disorders, fibrosis or kidney injury. Accordingly, the invention is further directed to a method of treating a (DDR2)-mediated disease or condition in a patient which comprises administering to the patient a therapeutically effective amount of a compound according to Formula (I), or a pharmaceutically acceptable salt thereof.

"Treating" or "treatment" is intended to mean at least the mitigation of a disease condition in a patient. The methods of treatment for mitigation of a disease condition include the use of the compounds in this invention in any conventionally acceptable manner, for example for prevention, retardation, prophylaxis, therapy or cure of a mediated disease.

The present invention is also directed to a pharmaceutical composition comprising a compound as a medicament of the invention or its salt and a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable excipient" means a material, composition or vehicle involved in giving form or consistency to the composition.

DESCRIPTION OF THE FIGURES

Figure 1 reports spheroids shrinkage in 3D cell model of breast cancer cells of example 4.

Figure 2 reports Vitality in 3D cell model of breast cancer cells of example 4.

Figure 3 reports gene expression inhibition of collagen type I, a key marker in the fibrosis processes in human lung fibroblasts after stimulation of TGF i .

Figure 4 reports gene expression inhibition of MMP13, one of the proteases involved in the fibrosis processes in human lung fibroblasts after stimulation of TGF i

DETAILED DESCRIPTION OF THE INVENTION

Therefore the invention relates to a compound of Formula (I):

or a pharmaceutically acceptable salt or derivative thereof,

wherein:

RI and R2 are independently H or

where n is an integer value from 1 to 3,

R5 is independently H or Chb for each integer value of n,

R4 is H or CH3,

with the proviso that

a) when Ri, R2 and R4 are H, R3 is not methyl, ethyl, n-propyl and phenyl;

b) when Ri and R2 are H and R4 is methyl, R3 is not methyl; and

c) 2,7-diethoxyfenazine is excluded.

The alternative definitions for the various groups and substituent groups of compounds of Formula (I) provided throughout the specification are intended to particularly describe each compound species disclosed herein, individually, as well as groups of one or more compound species. The scope of this invention includes any combination of these group and substituent group definitions.

The compounds according to Formula (I) may contain one or more asymmetric centre (also referred to as a chiral centre) and may, therefore, exist as individual enantiomers, diastereomers, or other stereoisomeric forms, or as mixtures thereof. Chiral centres, such as a chiral carbon may also be present in the compounds of this invention. Where the stereochemistry of a chiral centre present in a compound of this invention, or in any chemical structure illustrated herein, is not specified the structure is intended to encompass all individual stereoisomers and all mixtures thereof. Thus, compounds according to Formula (I) containing one or more chiral centre may be used as racemic mixtures, enantiomerically enriched mixtures, or as enantiomerically pure individual stereoisomers.

It is to be understood that a solid form of a compound of the invention may exist in crystalline forms, non-crystalline forms or a mixture thereof. Such crystalline forms may also exhibit polymorphism (i.e. the capacity to occur in different crystalline forms). These different crystalline forms are typically known as "polymorphs." Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which may be used for identification. One of ordinary skill in the art will appreciate that different polymorphs may be produced, for example, by changing or adjusting the conditions used in crystallizing/re-crystallizing the compound.

In addition, the compounds of this invention, depending on further substitution, may exist in other tautomeric forms. All tautomeric forms of the compounds described herein are intended to be encompassed within the scope of the present invention. It is to be understood that any reference to a named compound of this invention is intended to encompass all tautomers of the named compound and any mixtures of tautomers of the named compound.

In the present invention, when the following terms are used:

- "compound(s) of the invention" or "compound(s) of this invention" mean a compound of Formula (I), as defined above, in any form, i.e., any salt or non-salt form (e.g., as a free acid or base form, or as a salt, particularly a pharmaceutically acceptable salt thereof) and any physical form thereof (e.g., including non-solid forms (e.g., liquid or semi-solid forms), and solid forms (e.g., amorphous or crystalline forms, specific polymorphic forms, solvate forms, including hydrate forms (e.g., mono-, di- and hemi- hydrates)), and mixtures of various forms;

- "optionally substituted" means unsubstituted groups or rings or groups or rings substituted with one or more specified substituents; among the substituents one or more of the following can be cited: (Ci-C3)alkyl, preferably methyl or ethyl, (Ci-C3)alkoxy, preferably methoxy or ethoxy, halogen preferably fluoro or chloro, sulphonyl, (2-hydroxyethyl)-1 -piperazinyl-sulfonyl, hydroxyl, hydroxy(Ci- C3)alkyl, preferably hydroxymethyl or 2-hydroxy-ethyl and hydroxy(Ci- C3)alkoxy, preferably hydroxymethoxy or 2-hydroxy-ethyloxy;

- “saturated heterocyclic ring” means a saturated carbocyclic ring wherein one or two atoms of carbon are replaced by heteroatoms such as nitrogen, oxygen or sulphur; non-limiting examples are tetrahydropyrane, pyrrolidine, imidazolidine, pyrazolidine, thiazolidine, tetrahydrofuran, 1 ,3-dioxolane, piperidine, piperazine and morpholine;

- “(Ci-C3)alkyl” means a linear or branched saturated hydrocarbon group containing froml to 3 carbon atoms;

- “aryl ring” means mono-, bi- or poly-carbocyclic hydrocarbon with from 1 to 4 rings, optionally further fused or linked to each other by single bonds, wherein at least one of the carbocyclic ring is“aromatic”, wherein“aromatic” refers to completely conjugated tt-electron bond system; non-limiting examples are phenyl, a- or b- naphthyl, a- or b-tetrahydronaphtalenyl, biphenyl and indanyl groups;

- “saturated (C3-C6)cycloalkyl” means saturated 3- to 6-membered all-carbon monocyclic rings; non-limiting examples are cyclopropane, cyclobutane, cyclopentane, cyclohexane;

- “unsaturated heterocyclic ring” means an unsaturated carbocyclic ring wherein one to three carbon atoms are replaced by heteroatoms such as nitrogen, oxygen or sulphur; non-limiting examples are pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, pyrrolyl, furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, thiadiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, indazolyl. Accordingly, a compound of the invention includes a compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof.

In a preferred and advantageous embodiment, one of Ri and R2 is hydrogen.

When Ri or R2 is

R5 is preferably hydrogen and/or n is preferably 1 or 2, more preferably 2.

R6 can be H, an optionally substituted saturated heterocyclic ring or NR Rs, where R7 and Re are independently H or (Ci-C3)alkyl.

For R6, when optionally substituted saturated heterocyclic ring is indicated, it is intended a saturated heterocyclic ring either unsubstituted or substituted preferably with one or more substituent selected from the group consisting of (Ci-C3)alkyl, (Ci-C3)alkoxy, halogen, sulphonyl, (2-hydroxyethyl)-1 -piperazinyl-sulfonyl, hydroxyl, hydroxy(Ci- C3)alkyl and hydroxy(Ci-C3)alkoxy, preferably (Ci-C3)alkyl, hydroxy(Ci-C3)alkyl. More preferably when R6 is a heterocyclic ring substituted with one or more substituents, it is substituted with methyl or 2-hydroxy-ethyl.

Preferably R6 is an optionally substituted saturated heterocyclic ring, more preferably R6 is an optionally substituted saturated heterocyclic ring selected from the group consisting of morpholinyl, pyrrolidinyl and piperazinyl, still more preferably piperazinyl. R4 is H or CHs.

R3 is a group selected from the group consisting of an optionally substituted aryl, (Ci- C3)alkyl, benzyl, an optionally substituted saturated (C3-C6)cycloalkyl and an optionally substituted unsaturated heterocyclic ring. For R3, when optionally substituted aryl is indicated, it is intended a ring either unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of (Ci-C3)alkyl, (Ci-C3)alkoxy, halogen, sulphonyl, (2-hydroxyethyl)-1 - piperazinyl-sulfonyl, hydroxyl, hydroxy(Ci-C3)alkyl and hydroxy(Ci-C3)alkoxy, more preferably (2-hydroxyethyl)-1 -piperazinyl-sulfonyl.

For R3, when optionally substituted saturated (C3-C6)cycloalkyl is indicated, it is intended a saturated (C3-C6)cycloalkyl either unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of (Ci-C3)alkyl, (Ci- C3)alkoxy, halogen, sulphonyl, (2-hydroxyethyl)-1 -piperazinyl-sulfonyl, hydroxyl, hydroxy(Ci-C3)alkyl and hydroxy(Ci-C3)alkoxy.

For R3, when optionally substituted unsaturated heterocyclic ring is indicated, it is intended an unsaturated heterocyclic ring either unsubstituted or substituted, preferably with one or more substituent selected from the group consisting of (Ci-C3)alkyl, (Ci- C3)alkoxy, halogen, sulphonyl, (2-hydroxyethyl)-1 -piperazinyl-sulfonyl, hydroxyl, hydroxy(Ci-C3)alkyl and hydroxy(Ci-C3)alkoxy.

Preferably R3 is an optionally substituted aryl, more preferably it is an optionally substituted phenyl. In a more preferred embodiment R3 is an optionally substituted piperazinyl-sulfonyl phenyl, still more preferably R3 is [N-(N-(2- hydroxyethyl)piperazinyl)sulfonyl]phenyl.

Preferably R3 is an optionally substituted (Ci-Cs)alkyl, more preferably it is propyl, ethyl, isopropyl or methyl, still more preferably it is isopropyl or ethyl.

Preferably R3 is an optionally substituted saturated (C3-C6)cycloalkyl, more preferably, cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl, still more preferably cyclopropyl. Preferably R3 is an optionally substituted unsaturated heterocyclic ring, more preferably pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, pyrrolyl, furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, thiadiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl or indazolyl, still more preferably thienyl.

According to the various and preferred aspect indicated above, the present invention provides a compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof, selected from the group consisting of:

(R)-2-methoxy-7-((1 -phenylpropan-2-yl)oxy)phenazine (compound 1 )

2-(cyclopropylmethoxy)-7-(2-(4-methylpiperazin-1 -yl)ethoxy)phenazine (compound 2) 4-(2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 3) 2-(cyclopropylmethoxy)-7-(2-(pyrrolidin-1 -yl)ethoxy)phenazine (compound 4)

2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)-N,N-dimethylethan-1 -amine (compound

5)

(R)-1 -((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)propan-2-amine (compound 6) 2-(4-(2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)ethyl)piperazin-1 -yl)ethan-1 -ol (compound 7)

4-(3-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)propyl)morpholine (compound 8) 2-(benzyloxy)-7-(2-(piperazin-1 -yl)ethoxy)phenazine (compound 9)

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10)

4-(2-((8-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 11 )

4-(2-((8-(sec-butoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 12)

4-(3-((8-(cyclopropylmethoxy)phenazin-2-yl)oxy)propyl)morpholine (compound 13) 4-(2-((8-(thiophen-2-ylmethoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 14) 4-(2-((8-(thiophen-3-ylmethoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 15) 4-(2-((8-(benzyloxy)phenazin-2-yl)oxy)ethyl)morpholine(compound 16)

4-(3-((8-isobutoxyphenazin-2-yl)oxy)propyl)morpholine (compound 17)

(R)-2-((1 -phenylpropan-2-yl)oxy)phenazine (compound 18)

2-(4-((4-((phenazin-2-yloxy)methyl)phenyl)sulfonyl)piperazin-1 -yl)ethan-1 -ol

(compound 19).

Preferred compounds are selected from the group consisting of:

2-(cyclopropylmethoxy)-7-(2-(4-methylpiperazin-1 -yl)ethoxy)phenazine (compound 2) 2-(cyclopropylmethoxy)-7-(2-(pyrrolidin-1 -yl)ethoxy)phenazine (compound 4)

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10)

4-(2-((8-(benzyloxy)phenazin-2-yl)oxy)ethyl)morpholine(compound 16).

Accordingly, a compound of the invention includes a compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof.

Because the compounds of the invention contain basic moieties, a desired salt form may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, selected from the group consisting of hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid selected from the group consisting of maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, salicylic acid, citric acid, tartaric acid, p- toluensulfonic acid.

Because of their potential use in medicine, the salts of the compounds of Formula (I) are preferably pharmaceutically acceptable salts. Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention.

The present invention is also directed to a pharmaceutical composition comprising a compound of the invention or its salt and a pharmaceutically acceptable carrier.

The invention relates also to a compound of Formula (I):

or a pharmaceutically acceptable salt or derivative thereof,

wherein:

Ri and R2 are independently H or

where n is an integer value from 1 to 3,

R5 is independently H or Chb for each integer value of n,

R4 is H or CH3, R3 is a group selected from the group consisting of an optionally substituted aryl, (Ci- C3)alkyl, benzyl, an optionally substituted saturated (C3-C6)cycloalkyl and an optionally substituted unsaturated heterocyclic ring for use as a medicament.

In a preferred and advantageous embodiment, one of Ri and R2 is hydrogen.

When Ri or R2 is

where n is an integer value from 1 to 3

R5 is preferably hydrogen and/or n is preferably 1 or 2, more preferably 2.

R6 can be H, an optionally substituted saturated heterocyclic ring or NR7R8, where R7 and Re are independently H or (Ci-C3)alkyl.

R3 is a group selected from the group consisting of an optionally substituted aryl, (Ci- C3)alkyl, benzyl, an optionally substituted saturated (C3-C6)cycloalkyl and an optionally substituted unsaturated heterocyclic ring.

According to the various and preferred aspect indicated above, the present invention provides a compound of Formula (I), or a salt thereof, for use as a medicament particularly in a pharmaceutically acceptable salt form thereof, selected from the group consisting of:

(R)-2-methoxy-7-((1 -phenylpropan-2-yl)oxy)phenazine (compound 1 )

5)

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10)

4-(2-((8-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 11 )

4-(2-((8-(sec-butoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 12)

4-(3-((8-isobutoxyphenazin-2-yl)oxy)propyl)morpholine (compound 17)

(R)-2-((1 -phenylpropan-2-yl)oxy)phenazine (compound 18)

2-(4-((4-((phenazin-2-yloxy)methyl)phenyl)sulfonyl)piperazin-1 -yl)ethan-1 -ol

(compound 19).

Preferred compounds as a medicament are selected from the group consisting of: 2-(cyclopropylmethoxy)-7-(2-(4-methylpiperazin-1 -yl)ethoxy)phenazine (compound 2) 2-(cyclopropylmethoxy)-7-(2-(pyrrolidin-1 -yl)ethoxy)phenazine (compound 4)

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10)

4-(2-((8-(benzyloxy)phenazin-2-yl)oxy)ethyl)morpholine(compound 16)

In another aspect the invention relates a pharmaceutical composition comprising a compound of Formula (I) or a salt thereof for use as a medicament and a pharmaceutically acceptable carrier.

The compounds of the invention may be administered by any suitable route of administration, including both systemic administration and topical administration.

Systemic administration includes oral administration, parenteral administration, trans- dermal administration, rectal administration, and administration by inhalation.

The compounds of the invention may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for a compound of the invention depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the skilled artisan. In addition, suitable dosing regimens, including the duration of such regimens, are administered, for a compound of the invention depending on the condition being treated, the severity of the condition being treated, the age and the physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan.

The compounds of the invention will be normally, but not necessarily, formulated into a pharmaceutical composition prior to administration to a patient. The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art.

The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein an effective amount of a compound of the invention can be extracted and then given to the patient such as with powders, syrups, and solutions for injection. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form. A dose of the pharmaceutical composition contains at least a therapeutically effective amount of a compound of this invention (i.e., a compound of Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof). When prepared in unit dosage form, the pharmaceutical compositions may contain from 1 mg to 1000 mg of a compound of this invention.

The compounds of the invention and the pharmaceutically acceptable excipient or excipients will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration.

Conventional dosage forms include those adapted for (1 ) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) trans-dermal administration such as trans-dermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols and solutions; and (6) topical administration such as creams, ointments, lotions, pastes, sprays and gels.

Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, granulating agents, coating agents, wetting agents, suspending agents, emulsifiers, sweeteners, , flavour masking agents, colouring agents, anti-caking agents, humectants, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch, cellulose, calcium sulphate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch, gelatin, sodium alginate, alginic acid, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc. Suitable carriers for oral dosage forms include but are not limited to magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, methylcellulose, sodium carboxymethyl cellulose, and the like. Techniques used to prepare oral formulations are the conventional mixing, granulation and compression or capsules filling.

The compounds of the present invention may be also formulated for parenteral administration with suitable carriers including aqueous vehicles solutions (i.e.: saline, dextrose) or and/or oily emulsions.

In a still further aspect, the invention relates to a compound or a salt, particularly a pharmaceutically acceptable salt for use as a medicament in the inhibition of a DDR2 receptor.

The compounds of the invention are inhibitors of DDR2 and may be useful for the treatment of (DDR2)-mediated diseases and disorders.

Therefore, in another aspect the invention relates to the compound of the invention for use in the treatment of (DDR2) kinase-mediated diseases and disorders, particularly cancer (preferably cancer of colon, prostate, breast and lung cancer) acute and chronic pain, osteoarthritis, inflammation-associated disorder as arthritis, rheumatoid arthritis, atherosclerosis, fibrosis, various fibrotic disorders, kidney injury.

Accordingly, the invention is further directed to a method of treating a DDR2-mediated disease or condition in a patient, which comprises administering to the patient a therapeutically effective amount of a compound according to Formula (I), or a pharmaceutically acceptable salt thereof.

A therapeutically "effective amount" is intended to mean the amount of a compound that, when administered to a patient in need of such treatment, is sufficient to carry out treatment, as defined herein. Thus, e.g., a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is a quantity of an inventive agent that, when administered to a human in need thereof, is sufficient to modulate or inhibit the activity of DDR2 such that a disease condition which is mediated by that activity is reduced, alleviated or prevented. The amount of a given compound that will correspond to such an amount will vary depending upon factors such as the particular compound (e.g., the potency (IC50), efficacy (ECso), and the biological half- life of the particular compound), disease condition and its severity, the identity (e.g., age, size and weight) of the patient in need of treatment, but can nevertheless be routinely determined by one skilled in the art. Likewise, the duration of treatment and the time period of administration (time period between dosages and the timing of the dosages, e.g., before/with/after meals) of the compound will vary according to the identity of the human in need of treatment (e.g., weight), the particular compound and its properties (e.g., pharmaceutical characteristics), disease or condition and its severity and the specific composition and method being used, but can nevertheless be determined by one of skill in the art.

As above stated the compounds of the present invention may be administered orally or parenterally, in a pharmacological effective amount. For all methods of treatment herein discussed for the compounds of formula (I), the daily oral dosage regimen will preferably be from about 0.05 to about 30 mg/Kg of total body weight. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of formula (I) will be determined by the nature and extent of the condition being treated.

Accordingly, appropriate pharmaceutical composition of compounds of formula (I) or their pharmaceutically acceptable salts, optionally together with pharmaceutically acceptable carriers can be used for the treatment of diseases involving destruction of articular cartilage such as traumatic joint injuries, arthritis including osteoarthritis, rheumatoid arthritis and psoriatic arthritis. Furthermore, appropriate pharmaceutical composition of compounds of formula (I) and their pharmaceutically acceptable salts can be used for treatment of different forms of cancer, in particular colon, prostate, breast and lung cancer.

In addition, appropriate pharmaceutical composition of compounds of formula (I) and their pharmaceutically acceptable salts can be used for treatment of acute and chronic pain, including but not limited to inflammatory pain and associated hyperalgesia and allodynia, osteoarthritis pain, postoperative pain, visceral pain, pain associated with cancer, trigeminal neuralgia, acute herpetic and post herpetic neuralgia, neuropathic pain, diabetic neuropathy.

Furthermore, appropriate pharmaceutical composition of compounds of formula (I) and their pharmaceutically acceptable salts can be used for treatment of atherosclerosis, various fibrotic disorders, fibrosis or kidney injury.

The compounds of Formula (I) may be obtained by using synthetic procedures.

In a further aspect the invention relates to a process for preparing a compound of Formula (I).

General syntheses used for preparing compounds of Formula (I) are described in the following schemes 1 , 2 and 3

Scheme 1

a) Conversion of the starting material (ST) into compounds of formula (II), using an alkyl alcohol and a suitable base as sodium hydride;

b) Formation of the nitroso aniline derivatives (III) using potassium propanolate and fluoro aniline in TFIF;

c) Formation of the phenazine ring using trimethylsilyl (E)-N-(trimethylsilyl) acetimidate in acetonitrile;

d) Removal of the protecting group in non-aqueous acidic media as TFA in DCM e) Ether formation using a suitable alkyl bromide and a base or a suitable alcohol under Mitsunobu conditions to obtain intermediates (VI); and

f) Formation of compounds (VII) using a suitable alcohol and a base as sodium hydride in THF.

Two synthetic pathways are described from the starting (ST) nitro phenol

Pathway 1

a) Conversion of the starting material (ST) into compounds of formula (VIII), using di-tert-butyl dicarbonate and magnesium perchlorate in DCM; b) Formation of the nitroso aniline derivatives (IX) using potassium propanolate and fluoro aniline in THF;

d) Formation of compounds (XI) using a suitable alcohol and a base as sodium hydride in THF;

e) Removal of the protecting group in non-aqueous acidic media as TFA in DCM; and

f) Formation of compounds (XV) using a suitable alcohol and a base as sodium hydride in THF.

Pathway 2 g) Removal of the protecting group in acidic media as TFA in DCM to obtain phenazine (XIII);

h) Ether formation using a suitable alcohol under Mitsunobu conditions to obtain intermediates (XIV); and

i) Formation of compounds (XV) using a suitable alcohol and a base as sodium hydride in THF.

Scheme 3

a) Removal of the protecting group using sodium ethanethiolate to obtain intermediate (XVI); and

b) Ether formation to obtain compounds (XVII) using triphenyl phosphine in suitable solvents as DMF or DME.

Experimental part

Reagents used in the following examples were commercially available from various suppliers and used without further purifications. Solvents were used in dry form. Reactions in anhydrous environment were run under a positive pressure of dry N2. Proton Nuclear Magnetic Resonance (¹FI NMR) spectra were recorded on Bruker Avance 400 MFIz instrument. Chemical shifts are reported in ppm (d) using the residual solvent line as internal standard. Splitting patterns are designated as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad signal.

Mass spectra (MS) were run on a Ion Trap Thermo LCQ classic spectrometer, operating in positive ES(+) and negative ES(-) ionization mode.

UPLC spectra were performed on a Waters Acquity UPLC-SQD instrument using an Acquity UPLC-BEFI C18 column (1 7mM, 50x2.1 mm).

Chiral-FIPLC spectra were performed using Agilent 1200 apparatus equipped UV-Vis detector.

Preparative FIPLC was performed on Waters GX-281 FIPLC system equipped with a UV-detector.

Flash silica gel chromatography was performed on Biotage automatic flash chromatography systems (Sp1 and Isolera systems) using Biotage SNAP HP silica cartridges or Biotage SNAP KP-NH cartridges.

Reverse phase chromatography was performed on Biotage automatic flash chromatography systems (Isolera systems) using RediSep Gold C-18Aq cartridges. Purifications of some basic compounds were performed using Phenomenex Strata SCX cartridges (55pm, 70A).

Thin layer chromatography was carried out using Merck TLC plates Kieselgel 60F-254, visualized with UV light, aqueous permanganate solution, iodine vapours.

The following abbreviations are used herein: AcOH: acetic acid; DIAD: diisopropyl (E)- diazene-1 ,2-dicarboxylate; Boc: terbutyloxycarbonyl; DCM: dichloromethane; DCE: 1 ,2-dichloroethane; TFA: trifluoroacetic acid; DMF: dimethylformamide; THF: tetrahydrofuran; RT: room temperature; DMAP: dimethylamino pyridine; AcOEt: ethyl acetate; NaOH: sodium hydroxyde; KOH: potassium hydroxyde; DIPEA: N,N- diisopropylethylamine; TEA: triethyl amine; NaHCOs: sodium bicarbonate; Na2S04: sodium sulphate; PdCl2(PPh3)2: bis(triphenylphosphine)palladium(ll)chloride;

CS2CO3: cesium carbonate

Preparation of Intermediates (II) of scheme 1

Intermediate 1 1 -(tert-butoxy)-4-nitrobenzene

To 1 -fluoro-4-nitrobenzene (5 g, 35.4 mmol) in DMF (50 ml) under nitrogen at RT, was added at once potassium 2-methylpropan-2-olate (4.97 g, 44.3 mmol). The resulting mixture was left to stir 24 hours, then the reaction was stripped of volatiles under vacuum. To the remaining residue was added cyclohexane (100 ml_) followed by 1 M aqueous sodium hydroxide (100 ml_). After brief mixing, the aqueous layer was separated and the organic layer was washed twice more with 1 M aqueous sodium hydroxide. The organic layer was finally washed with water (100 ml_), then dried over anhydrous sodium sulphate and then stripped of volatiles to give 6.26 g (Yield 90%) of the title compound as a light brown oil. 1 H NMR (400 MHz, DMSO-d6) d ppm 8.11 - 8.22 (2 H, m), 7.14 - 7.24 (2 H, m), 1.44 (9 H, s). ESI+ m/z 140 [M-tBu+2H+]

Intermediate 2 1-(benzyloxy)-4-nitrobenzene

To 4-nitrophenol (5 g, 35.9 mmol) and potassium carbonate (7.45 g, 53.9 mmol) under nitrogen and in dry Acetonitrile (50 ml) was added dropwise (bromomethyl)benzene (4.28 ml, 35.9 mmol). The resulting mixture was then stirred overnight at 60°C, cooled to RT and then filtered through Celite. The filtrate was the stripped of volatiles to give a solid. To this solid was added water (150 ml_) and the resulting suspension was left to stir overnight. The next day the solid was recovered by filtration and then dried under vacuum overnight to give 8.11 g of title compound as a cream-colored solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 8.16 - 8.27 (2 H, m), 7.33 - 7.53 (5 H, m), 7.19 - 7.28 (2 H, m), 5.28 (2 H, s). ESI+ m/z 230 [M+H]

Intermediate 3 1 -isobutoxy-4-nitrobenzene

To 2-methylpropan-1 -ol (26.3 g, 354 mmol) under nitrogen was added sodium hydride (1.772 g, 44.3 mmol). The resulting mixture was left to stir at RT until gas evolution had finished. Solid 1 -fluoro-4-nitrobenzene (3.76 ml, 35.4 mmol) was then added at once and the resulting mixture was left to stir at RT under nitrogen overnight, then the reaction was stripped of volatiles under vacuum.

The residue was dissolved in DCM (100 ml_), washed with water (50 ml_) and sat. aq. sodium bicarbonate solution (100 ml_); the organic layer was separated, dried over anhydrous sodium sulphate and then evaporated to give a brown oil.

After column chromatography (Biotage HP-Sil 100g silica column; cHex/DCM 3:1 ) a light yellow oil was obtained. (6.05g; 76%).

1 H NMR (400 MHz, DMSO-d6) d ppm 8.14 - 8.24 (2 H, m), 7.09 - 7.19 (2 H, m), 3.90 (2 H, d), 2.06 (1 H, dt), 0.93 - 1.06 (6 H, m). ESI+ m/z 196 [M+H]

Preparation of Intermediates fill) of scheme 1

Intermediate 4 5-(tert-butoxy)-N-(4-fluorophenyl)-2-nitrosoaniline

To potassium 2-methylpropan-2-olate (8.62 g, 77 mmol) in dry THF (100 ml) at -78°C was added first 4-fluoroaniline (2.426 ml, 25.6 mmol) and then intermediate 1 (5 g, 25.6 mmol). After 2 hours at -78°C, the reaction mixture was quickly poured into a sat. aq. ammonium chloride solution (100 ml_). After brief mixing, EtOAc (50 ml_) was added and the two layers were separated. The organic layer was dried over anhydrous sodium sulphate and evaporated to give a brown oil which was used directly for the next reaction (Intermediate 7)

ESI+ m/z 290 [M+H]

Intermediate 5 5-(benzyloxy)-N-(4-fluorophenyl)-2-nitrosoaniline

To potassium 2-methylpropan-2-olate (1.469 g, 13.09 mmol) in dry THF (30 ml) at - 78°C nitrogen was added first 4-fluoroaniline (0.413 ml, 4.36 mmol) and then a solution of intermediate 2 (1 g, 4.36 mmol) in dry THF (10 ml). After 2 hours at -78°C, the reaction mixture was quickly poured into a sat. aq. ammonium chloride solution (100 ml_). After brief mixing, EtOAc (50 ml_) was added and the two layers were separated. The organic layer was dried over anhydrous sodium sulphate and evaporated to give a brown oil which was used directly for the next reaction (Intermediate 8).

ESI+ m/z 323 [M+H]

Intermediate 6 N-(2-fluorophenyl)-5-isobutoxy-2-nitrosoaniline

To potassium 2-methylpropan-2-olate (2.59 g, 23.05 mmol) in dry THF (50 ml) at -78°C nitrogen was added first 4-fluoroaniline (0.764 ml, 8.07 mmol) and then intermediate 3 (1.5 g, 7.68 mmol). After 2 hours at -78°C, the reaction mixture was quickly poured into a sat. aq. ammonium chloride solution (150 ml_). After brief mixing, EtOAc (50 ml_) was added and the two layers were separated. The organic layer was dried over anhydrous sodium sulphate and evaporated to give a brown oil which was used directly for the next reaction (Intermediate 9)

ESI+ m/z 289 [M+H]

Preparation of Intermediates (IV) of scheme 1

Intermediate 7 2-(tert-butoxy)-7-fluorophenazine

To intermediate 4 (7.21 g, 25 mmol) in Acetonitrile (250ml) under nitrogen was added trimethylsilyl (E)-N-(trimethylsilyl)acetimidate (24.45ml, l OOmmol). After 24 hours at reflux, the reaction was cooled to RT and the solvent evaporated. The resulting solid was loaded onto a Biotage HP-Sil 340g column eluting with DCM. 3.45 g (51 %) of title compound were recovered as an orange-yellow solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 8.23 - 8.33 (1 H, m), 8.08 - 8.18 (1 H, m), 7.85 - 8.02 (2 H, m), 7.57 - 7.67 (2 H, m), 1.55 (9 H, s). ESI+ m/z 271 [M+H]

Intermediate 8 2-(benzyloxy)-7-fluorophenazine

To intermediate 5 (1.4g, 4.34mmol) in acetonitrile (100ml) was added trimethylsilyl (E)- N-(trimethylsilyl) acetimidate (7.43 ml, 30.4 mmol). The resulting mixture was heated to 70°C for 24 hours, then the reaction was cooled to RT and the solvent evaporated. The resulting solid was loaded onto a Biotage HP-Sil 100g column eluting with DCM. 903 mg (68%) of title compound were recovered as a yellow solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 8.27 (1 H, dd), 8.15 (1 H, d), 7.85 - 8.01 (2 H, m), 7.71 - 7.79 (1 H, m), 7.54 - 7.66 (3 H, m), 7.34 - 7.49 (3 H, m), 5.40 (2 H, s).

ESI+ m/z 305 [M+H]

Intermediate 9 2-fluoro-7-isobutoxyDhenazine

To intermediate 6 (2.2g, 7.7 mmol) in acetonitrile (100ml) was added trimethylsilyl (E)- N-(trimethylsilyl) acetimidate (9.4 ml, 38.4 mmol). The resulting mixture was heated to 70°C for 24 hours, then the reaction was cooled to RT and the solvent evaporated. The resulting solid was loaded onto a Biotage HP-Sil 100g column eluting with DCM. 1.45 g (69%) of title compound were recovered as a yellow solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 8.24 (1 H, dd), 8.11 (1 H, d), 7.83 - 7.99 (2 H, m), 7.66 (1 H, dd), 7.48 (1 H, d), 4.03 (2 H, d), 2.16 (1 H, dt), 1.07 (6 H, d).

ESI+ m/z 271 [M+H]

Preparation of Intermediates (V) of scheme 1

Intermediate 10 7-fluorophenazin-2-ol

To intermediate 7 (1 g, 3.70 mmol) was added TFA (30 ml) and the resulting mixture was left to stir at RT for 3 hours. After 3 hours the reaction was evaporated. To the remaining residue was added ethanol (50ml_) and the resulting solution was evaporated once more. This operation was then repeated other 4 times. The resulting yellow solid was dried under high vacuum for 48 hours. Yield 0.79g (91 %). ESI+ m/z 215 [M+H]

Preparation of Intermediates (VI) of scheme 1

Intermediate 11 2-(cvcloorooylmethoxy)-7-fluoroohenazine

To intermediate 10 (600 mg, 2.80 mmol), cesium carbonate (2738 mg, 8.40 mmol) and 4A molecular sieves in dry DMF (20 ml) under nitrogen at RT, was added (bromomethyl)cyclopropane (0.340 ml, 3.50 mmol). After 18 hours at RT, the reaction was filtered and evaporated and to the remaining solid was added DCM (20 ml_). The resulting mixture was then filtered again and evaporated. The brown solid was purified by normal phase column chromatography on a Biotage HP-Sil 50g column eluting with DCM only. Yield 634 mg (83%) of the title compound as a yellow solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 8.24 (1 H, dd), 8.10 (1 H, d), 7.82 - 7.99 (2 H, m), 7.67 (1 H, dd), 7.45 (1 H, d), 4.10 (2 H, s), 1.28 - 1.42 (1 H, m), 0.59 - 0.72 (2 H, m), 0.37 - 0.50 (2 H, m). ESI+ m/z 269 [M+H]

Intermediate 12 (R)-2-fluoro-7-((1-ohenylorooan-2-yl)oxy)ohenazine

To intermediate 10 (250 mg, 1.16 mmol), and triphenylphosphine (459 mg, 1.751 mmol) under nitrogen was added dry THF (45 ml). Once complete dissolution was obtained, (S)-1 -phenylpropan-2-ol (0.208 ml, 1.517 mmol) was added and the resulting solution was then cooled to 0°C. Once at 0°C, diisopropyl (E)-diazene-1 ,2- dicarboxylate (0.345 ml, 1.751 mmol) was added dropwise. After stirring at 0°C for 6 hours and 18 holurs at RT, the reaction was stripped of volatiles under vacuum and the resulting residue was then loaded onto a Biotage KP-NH 28g column attached in series to a Biotage HP-Sil 50g column all primed with cHex/DCM 1 :1. The columns were eluted with cHex/DCM 1 :1 for 1 CV and the the eluent was changed to DCM only over 6CV and then eluted with DCM only. After drying at 80°C, 357 mg (92%) of the title compound were obtained.

1 H NMR (400 MHz, DMSO-d6) d ppm 8.24 (1 H, dd), 8.10 (1 H, d), 7.83 - 7.99 (2 H, m), 7.52 - 7.65 (2 H, m), 7.26 - 7.39 (4 H, m), 7.16 - 7.25 (1 H, m), 5.02 - 5.16 (1 H, m), 2.96 - 3.17 (2 H, m), 1.39 (3 H, d). ESI+ m/z 333 [M+H]

Preparation of Intermediate (VIII) of scheme 2 Intermediate 13 1 -(tert-butoxy)-3-nitrobenzene

To 3-nitrophenol (5 g, 35.9 mmol) and di-tert-butyl dicarbonate (18.99 ml, 83 mmol) in dry DCM (40 ml) was added magnesium perchlorate (0.802 g, 3.59 mmol). The resulting mixture was left to stir for 48 hours at RT under nitrogen. After 48 hours sat. aq. sodium bicarbonate solution was added (60 ml_) and after brief mixing the organic layer was separated, dried over anhydrous sodium sulphate and then stripped of volatiles. The desired product was then recovered from the resulting residue by normal phase column chromatography on a Biotage HP-Sil 100g cartridge eluting initially with cHex only and then gradually changing to DCM only. Yield 4.8 g (65%) of title compound were recovered.

1 H NMR (400 MHz, DMSO-d6) d ppm 7.94 (1 H, ddd), 7.72 (1 H, t), 7.59 (1 H, t), 7.43 - 7.51 (1 H, m), 1.36 (9 H, s).

Preparation of Intermediates (IX) of scheme 2

Intermediate 14 4-itert-butoxy)-N-i4-fluorophenyl)-2-nitrosoaniline

To potassium 2-methylpropan-2-olate (6.90 g, 61.5 mmol) in dry THF (100 ml) at -78°C, was added first 4-fluoroaniline (2.135 ml, 22.54 mmol) and then intermediate 12 (4 g, 20.49 mmol). The resulting mixture was left to stir at -78°C for 2 hours, then was quickly poured into a sat. aq. ammonium chloride solution (100 ml_). After brief mixing, EtOAc (50 ml_) was added and the two layers were separated. The organic layer was dried over anhydrous sodium sulphate and then stripped of volatiles to give a brown-black oil which was used directly for the next reaction (intermediate 16)

ESI+ m/z 289 [M+H]

Intermediate 15 5-(benzyloxy)-N-(3-fluorophenyl)-2-nitrosoaniline

To potassium 2-methylpropan-2-olate (2.94 g, 26.2 mmol) in dry THF (30 ml) at -78°C (dry ice/acetone) and under nitrogen was added first 3-fluoroaniline (0.839 ml, 8.72 mmol) and then a solution of intermediate 2 (2 g, 8.72 mmol) in dry THF (10 ml). The resulting mixture was left to stir at -78°C for 2 hours.

After this time, the reaction mixture was quickly poured into a sat. aq. ammonium chloride solution (100 ml_). After brief mixing, EtOAc (50 ml_) was added and the two layers were separated. The organic layer was dried over anhydrous sodium sulphate and then stripped of volatiles to give a brown-black oil which was used directly for the next experiment (intermediate 17)

ESI+ m/z 323 [M+H]

Preparation of Intermediates ( X ) of scheme 2

Intermediate 16 2-(tert-butoxy)-8-fluoroohenazine

To intermediate 14 (5.88 g, 20.4 mmol) in Acetonitrile (250 ml) was added trimethylsilyl (E)-N-(trimethylsilyl)acetimidate (19.95 ml, 82 mmol). The resulting mixture was heated to reflux for 24 hours. The reaction was cooled to RT and the stripped of solvent.

The resulting solid was loaded onto a Biotage HP-Sil 340g column primed with DCM. The column was then eluted with DCM only. 3.94 g (71 %) of a yellow solid was recovered. Analysis of the solid showed it to be a mixture of two regioisomers. The solid was used as such in subsequent reactions (intermediate 18 and 19).

1 H NMR (400 MHz, DMSO-d6) d ppm 8.33 (1 H, ddd), 7.48 - 8.19 (5 H, m), 1.43 - 1.62 (9 H, m). ESI+ m/z 271 [M+H]

Intermediate 17 2-(benzyloxy)-8-fluorophenazine

To intermediate 15 (2.80 g, 8.7 mmol) in Acetonitrile (100 ml) under nitrogen was added trimethylsilyl (E)-N-(trimethylsilyl)acetimidate (12.76 ml, 52.2 mmol). The resulting mixture was heated to reflux for 24 hours, then cooled to RT and evaporated. The resulting solid was loaded onto a Biotage HP-Sil 3400g column primed with cHex/DCM 3:7; the column was then eluted for 1 CV with cHex/DCM 3:7 and then the eluent was gradually changed to DCM only over 3CV and then the column was eluted with DCM only Yield 1.51 g (57%) of title compound.

Preparation of Intermediates (XI) of scheme 2

Intermediate 18 4-(3-((8-(tert-butoxy)ohenazin-2-yl)oxy)orooyl) morpholine

To intermediate 16 (mixture of isomers) (1.25 g, 2.312 mmol) in dry Dioxane (40 ml) 3-morpholinopropan-1 -ol (4.80 ml, 34.7 mmol) followed by 60% sodium hydride in mineral oil (0.647 g, 16.19 mmol) were added. The reaction mixture was left to stir at 60°C overnight under nitrogen, then was cooled to RT and then stripped of volatiles. To the remaining residue was added DCM (100 ml_), water (50 ml_) and sat. aq. sodium bicarbonate solution (50 ml_). After brief mixing, the organic layer was dried over anhydrous sodium sulphate and then stripped of volatiles to give an orange residue that was purified by normal phase column chromatography on a Biotage HP-Sil 100g column eluting initially with ethyl acetate only for 2CV and then gradually changing to EtOAc/MeOH 85:15 over 8CV. Yield 1g yellow solid (partially pure)

ESI+ m/z 396 [M+H]

Intermediate 19 4-(2-((8-(tert-butoxy)Dhenazin-2-yl)oxy)ethyl)morDholine

To intermediate 16 (mixture of isomers) (1.25 g, 2.312 mmol) in dry Dioxane (40 ml) 3-morpholinopropan-1 -ol (4.80 ml, 34.7 mmol) followed by 60% sodium hydride in mineral oil (0.647 g, 16.19 mmol) were added. The reaction mixture was left to stir at 60°C overnight under nitrogen, then was cooled to RT and then stripped of volatiles. To the remaining residue was added DCM (100 ml_), water (50 ml_) and sat. aq. sodium bicarbonate solution (50 ml_). After brief mixing, the organic layer was dried over anhydrous sodium sulphate and then stripped of volatiles to give an orange residue that was purified by normal phase column chromatography on a Biotage HP-Sil 100g column eluting initially with ethyl acetate only for 2CV and then gradually changing to EtOAc/MeOH 85:15 over 8CV. Yield 0.9g yellow solid (partially pure)

ESI+ m/z 382 [M+H]

Preparation of Intermediates (XII) of scheme 2

Intermediate 20 8-(3-morDholinoproDoxy)Dhenazin-2-ol

To intermediate 18 (1 g, 2.52 mmol) was added TFA (30 ml). After 3 hours at RT, the reaction was stripped of volatiles under vacuum. To the remaining residue was then added ethanol (50 ml_) and the resulting solution was then stripped of volatiles under vacuum once more. This was repeated a further two times. The remaining solid was then loaded onto a Biotage HP-Sil 100g column primed with DCM only. The column was then eluted for 2CV with DCM only and then the eluent was gradually changed over 8CV to DCM/MeOH 9:1. Yield 0.78 g (95%) yellow solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 10.70 - 10.97 (1 H, m), 9.62 - 10.08 (1 H, m), 8.05 (2 H, s), 7.38 - 7.56 (3 H, m), 7.21 - 7.34 (1 H, m), 4.24 - 4.42 (2 H, m), 3.89 - 4.15 (2 H, m), 3.61 - 3.79 (2 H, m), 3.45 - 3.61 (2 H, m), 3.32 (2H, m), 2.96 - 3.23 (2 H, m), 2.17 - 2.35 (2 H, m). ESI+ m/z 340 [M+H]

Intermediate 21 8-(2-moroholinoethoxy)ohenazin-2-ol

To intermediate 19 (0.9 g, 2.36 mmol) was added TFA (30 ml). After 3 hours at RT, the reaction was stripped of volatiles under vacuum. To the remaining residue was then added ethanol (50 ml_) and the resulting solution was then stripped of volatiles under vacuum once more. This was repeated a further two times. The remaining solid was then loaded onto a Biotage HP-Sil 100g column primed with DCM only. The column was then eluted for 2CV with DCM only and then the eluent was gradually changed over 8CV to DCM/MeOH 9:1. Yield 0.75 g (97%) yellow solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 10.86 (1 H, bs), 8.06 - 8.12 (2 H, m), 7.50-7.55 (3 H, m), 7.30 (1 H, d), 4.61 (2 H, m), 3.85 - 4.05 (2 H, m), 3.62 - 3.80 (2 H, m), 3.15 - 3.55 (6 H, m). ESI+ m/z 326 [M+H]

Preparation of Intermediate (XIII) of scheme 2

Intermediate 22 8-fluorophenazin-2-ol

To intermediate 16 (4.5 g, 16.65 mmol) was added TFA (50 ml). The resulting mixture was left to stir at RT for 3 hours, then the reaction was stripped of volatiles under vacuum.

To the remaining residue was added ethanol (100 ml_) and the solvent was stripped under vacuum. This was repeated twice. Acetonitrile (100 ml_) was then added and then the solvent was stripped under vacuum. This was also repeated twice. The resulting solid was then dried under high vacuum to give 3.56 g (88%) of the title compound as a yellow solid.

ESI+ m/z 215 [M+H]

Preparation of Intermediates (XIV) of scheme 2

Intermediate 23 2-fluoro-8-(thioohen-2-ylmethoxy)ohenazine

To intermediate 22 (200 mg, 0.934 mmol), triphenylphosphine (367 mg, 1.401 mmol) and thiophen-2-ylmethanol (0.106 ml, 1.120 mmol) in dry THF under nitrogen and at 0°C was added dropwise diethyl (E)-diazene-l ,2-dicarboxylate (0.238 ml, 1.401 mmol). After 18 hours at RT solvent was evaporated, to the remaining residue was then added DCM (25 ml_) and water (50 ml_). After prolonged mixing, the organic layer was separated, dried over anhydrous sodium sulphate and evaporated. The residue was purified by normal phase column chromatography on a Biotage HP-Sil 50g column eluting initially for 2CV with cHex/DCM 1 :1 and then changing to DCM only over 8CV and then eluting with DCM only. Yield 115mg (39%), yellow solid.

ESI+ m/z 311 [M+H]

Intermediate 24 2-fluoro-8-(thioohen-3-ylmethoxy)ohenazine

To intermediate 22 (200 mg, 0.934 mmol), triphenylphosphine (367 mg, 1.401 mmol) and thiophen-3-ylmethanol (0.106 ml, 1.120 mmol) in dry THF under nitrogen and at 0°C was added dropwise diethyl (E)-diazene-l ,2-dicarboxylate (0.238 ml, 1.401 mmol). After 18 hours at RT solvent was evaporated, to the remaining residue was then added DCM (25 ml_) and water (50 ml_). After prolonged mixing, the organic layer was separated, dried over anhydrous sodium sulphate and evaporated. The residue was purified by normal phase column chromatography on a Biotage HP-Sil 50g column eluting initially for 2CV with cHex/DCM 1 :1 and then changing to DCM only over 8CV and then eluting with DCM only. Yield 120mg (43%), yellow solid.

ESI+ m/z 311 [M+H]

Preparation of Intermediate (XVI) of scheme 3

Intermediate 25 phenazin-2-ol , Sodium salt

To 2-ethoxyphenazine (750 mg, 3.34 mmol) in dry DMF (20 ml) was added sodium ethanethiolate (844 mg, 10.03 mmol). The resulting mixture was then heated in a microwave in a sealed vial to 150°C for 40 minutes, then the reaction was cooled to RT and then stripped of DMF under vacuum. To the remaining residue was added water (40 ml_) and diisopropyl ether (40 ml_). The resulting mixture was then cooled to 0°C and was then slowly adjusted to pH 2 with cone aqueous hydrochloric acid. Once at pH 2 the mixture was filtered to recover the precipitate that had formed.

This precipitate was then dissolved in 0.1 M aqueous sodium hydroxide solution and the resulting solution was then loaded onto a RediSep C18Aq 150g column primed with water only. The column was run for 1 CV with water only and then the eluent was rapidly changed over 5CV to acetonitrile only. Yield 310 mg of title compound as a red solid.

1 H NMR (400 MHz, DMSO-d6) d ppm 7.78 - 7.88 (1 H, m), 7.69 (1 H, d), 7.45 - 7.59 (2 H, m), 7.26 - 7.36 (1 H, m), 7.01 (1 H, dd), 6.27 (1 H, d)

ESI+ m/z 197 [M+H]

Intermediate 26 2-(4-( (4-(bromometh vDohen vDsulfon vDoioerazin- 1-yl)ethan-1-ol

To 4-(bromomethyl)benzenesulfonyl chloride (1 g, 3.71 mmol) in dry Et2<D (50 ml) at - 5°C was slowly added 2-(piperazin-1 -yl)ethan-1 -ol (0.483 g, 3.71 mmol) over a period of 1 hour. Once addition was complete, the mixture was left to stir at -5°C for another 2 hours. After this time, the reaction mixture was stripped of solvent under vacuum at a low water bath temperature (30°C maximum). The remaining solid was then dissolved in DCM (50 ml_) and this solution was then washed with 1 M aqueous HCI (50 ml_) and then water (15 ml_) with sat. aq. sodium bicarbonate solution (25 ml_). The aqueous solutions were then back extracted with DCM (20 ml_). The organic solutions were combined, dried over anhydrous sodium sulphate and then stripped of solvent under vacuum to give, on drying under high vacuum, a white solid (1.25g, 93%).

ESI+ m/z 364 [M+H]

General procedures used to prepare compounds of the invention (formula I):

General procedure 1 :

To sodium hydride (26.8 mg, 0.671 mmol) was added dry THF (6 ml) followed by the corresponding alcohols (all commercially available) (1.32mmol). Once gas evolution had subsided, solid 2-(cyclopropylmethoxy)-7-fluorophenazine (60 mg, 0.224 mmol) was added then heated to 65°C for 18 hours. The reaction was evaporated, to the residue was then added acetic acid (2 ml_) and DMSO (3 ml_). The resulting solution was loaded onto a RediSep C18Aq 50g column primed with water + 0.1 % AcOH only. The column was then eluted with water + 0.1 % AcOH only for 3CV and then the eluent was gradually changed to acetonitrile + 0.1 % AcOH only over 10CV. General procedure 2:

To 2-(benzyloxy)-7-fluorophenazine (100 mg, 0.329 mmol) in dry Diethylene glycol dimethyl ether (3 ml) was added 2-(piperazin-1 -yl)ethan-1 -ol (1070 mg, 8.21 mmol) followed by sodium hydride (158 mg, 3.94 mmol). After 2 hours at 50°C the reaction was evaporated.

To the remaining residue was added DMSO (5 ml_) and acetic acid (1 ml_) and the resulting solution was then loaded onto a RediSep C18Aq 50g column primed with water + 0.1 % AcOH only. The column was eluted with water + 0.1 % AcOH only for 3CV and then the eluent was gradually changed to acetonitrile + 0.1 % AcOH only over 10CV.

General procedure 3:

To sodium hydride in mineral oil 60% (31.1 mg, 0.777 mmol) in dry THF (10 ml) under nitrogen was added 2-morpholinoethan-1 -ol (0.188 ml, 1.554 mmol). Once gas evolution had subsided, solid 2-fluoro-7-isobutoxyphenazine (70 mg, 0.259 mmol) was added at once. The resulting mixture was stirred at 65°C for 24 hours, then evaporated. To the remaining residue was added DCM (40 ml_) and sat. aq. sodium bicarbonate solution (50 ml_). After brief mixing, the organic layer was separated and dried over anhydrous sodium sulphate, then evaporated. To the residue was then added acetic acid (2 ml_) and DMSO (3 ml_). The resulting solution was then loaded onto a RediSep C18Aq 50g column primed with water + 0.1 % AcOH only. The column was then eluted with water + 0.1 % AcOH only for 3CV and then the eluent was gradually changed to acetonitrile + 0.1 % AcOH only over 10CV.

General procedure 4:

To (R)-2-fluoro-7-((1 -phenylpropan-2-yl)oxy)phenazine (60 mg, 0.181 mmol) in dry THF (4 ml) under nitrogen was added sodium methanolate 25% in methanol (780 mg, 3.61 mmol). The resulting mixture was stirred at 65°C for 24 hours, then evaporated. To the remaining residue was added DCM (5 ml_) and sat. aq. sodium bicarbonate solution (5 ml_). After brief mixing, the organic layer was separated and dried over anhydrous sodium sulphate, then evaporated. To this residue was added DMSO (3 ml_) and acetic acid (0.5 ml_) and the resulting solution was then loaded onto a RediSep C18Aq 50g column primed with water + 0.1 % AcOH only. The column was eluted with water + 0.1 % AcOH only for 3CV and then the eluent was gradually changed to acetonitrile + 0.1 % AcOH only over 10CV.

General procedure 5:

To alcooxy-phenazin-2-ol (0.246 mmol), cesium carbonate (401 mg, 1.229 mmol) and 4A molecular sieves in dry DMF (10 ml) under nitrogen at RT was added the bromo alkane (1 mmol) and the reaction was left to stir overnight. The reaction was stripped of volatiles under vacuum and to the remaining solid was added DCM (20 ml_). The resulting mixture was then filtered and stripped of volatiles to give a solid.

The desired product was recovered from this solid by normal phase column chromatography on a Biotage HP-Sil 25g column eluting initially with DCM only and then changing to EtOAc and eluting with EtOAc only

General procedure 6:

To intermediate 23 or 24 (0.93 mmol) was added dry THF (20 ml) followed by 2- morpholinoethan-1 -ol (612 mg, 4.67 mmol) and then 60% sodium hydride in mineral oil (74.7 mg, 1.867 mmol). The resulting mixture was then heated to 50°C under nitrogen and left at this temperature overnight. The next day the reaction was cooled to RT and then stripped of volatiles. To the remaining residue was added DCM (20 ml_), water (20 ml_) and sat. aq. sodium bicarbonate (20 ml_). After brief mixing, the organic layer was separated, dried over anhydrous sodium sulphate and stripped of volatiles under vacuum to give an orange residue.

To this residue was then added acetic acid (1 ml_) and DMSO (4 ml_). The resulting solution was then loaded onto a RediSep C18Aq 50g column primed with water + 0.1 % AcOH only. The column was then eluted with water + 0.1 % AcOH only for 3CV and then the eluent was gradually changed to acetonitrile + 0.1 % AcOH only over 10CV. General procedure 7:

To phenazin-2-ol (100 mg, 0.510 mmol) and triphenylphosphine polymer bound (408 mg, 0.561 mmol) in DME (15 ml) was added the corresponding alcohol (0.637 mmol) dropwise. The reactions was left on a shaker plate overnight at RT. The reactions were filtered to remove the resins and the resins were washed with DCM and MeOH. The filtrates were then stripped of volatiles under vacuum to give a residue that was then dissolved in DCM and loaded onto a Biotage KP-Sil 28g column attached in series to a Biotage HP-Sil 25g column primed with DCM only. The column was then eluted with DCM only.

General procedure 8:

To phenazin-2-ol (110 mg, 0.561 mmol), cesium carbonate (199 mg, 0.609 mmol), intermediate 26 (177 mg, 0.488 mmol) and 4A molecular sieves was added dry DMF (5 ml). The resulting mixture was left to stir at RT for 24 hours. The reaction was filtered and evaporated. The residue was purified by normal phase column chromatography on a Biotage KP-NH 55g column eluting initially for 3CV with DCM only and then gradually over 10CV changing to DCM/EtOAc 6:4.

The compounds obtained are indicated below with reference to the used procedure.

Pharmacological evaluation of the compounds of the invention

Compounds of Formula (I), according to the invention, have been studied in vitro in order to establish their potential DDR2 inhibition and their efficacy in cellular models for testing DDR2 inhibition.

Example 1

Alphascreen assay on HEK293 cells

HEK293 cells, transfected to express human DDR2 receptor, were plated 30.000 cells per well in a 96 wells plate coated with poly D Lysine (Corning) and maintained at 37°C in a 5% C02 incubator for 48 hours. Subsequently cells were stimulated for 6 hours with 75pg/ml Bovine Articular Cartilage Collagen II (DiscoverX) ± tested compounds in a concentration range from 0.1 mM to 10mM.

At the end of the incubation time total cell lysates were obtained by adding 50mI AlphaLISA lysis buffer (Perkin Elmer) per well, plates were maintained in 300rpm agitation on ice for 15’. Afterwards 5mI of cell lysates were transferred to a white bottom 384 wells plate and 10mI anti-p-Tyr 100 acceptor beads 50pg/ml were added and solution was incubated for 1 hour at room temperature in dark. Then 10mI anti-FLAG donor beads 50 pg/ml were added (both donor and acceptor beads were from Perkin Elmer). Finally plates were read after an hour incubation at room temperature using the fluorescence reader ENVISION (Perkin Elmer) with excitation at 680 nm and emission wavelength of 520-620 nm.

The DDR2 inhibitory activity of representative examples of compounds of Formula (I) is reported in the following table.

Table 1 : Alphascreen assay

As it is evident from Table 1 all the compounds showed a good inhibitory activity. Compounds 2, 4, 6, 10, 14, 15 and 16 showed the better inhibitory activity.

Example 2

In vitro binding assay

The compound affinities were evaluated using a solid phase collagen binding method Type II collagen, diluted at 20pg/ml_ in“collagen dilution buffer” (Chondrex), was coated onto Immulon 2 HB 96-well plates overnight at 4°C. Wells were blocked 1 h in assay buffer ( 1 mg/mL of bovine serum albumin in PBS + 0.05% Tween-20), then a mixed solution of recombinant DDR2-His (final assay concentration 50 nM) and compounds diluted in assay buffer was added for 3 h at room temperature. Compounds were dissolved in DMSO (1 mM master stock) and the final DMSO concentration in the assay was 1 %. Background values were determinated adding only assay buffer to the corresponding wells. Bound DDR2-His was detected with anti-His coupled horseradish peroxidase (HRP) antibody solution, incubated for 1 h at room temperature. After every step, wells were extensively washed with assay buffer using a microplate washer. Detection was achieved using the HRP substrate o-phenylenediamine dihydrochloride. The reaction was stopped with 3M sulfuric acid, and plates were read at Abs 492 nm using the plate reader Victor II (Perkin Elmer). The results were expressed as inhibition percentage compared to DMSO control, after background subtraction.

Table 2: DDR2 binding assay

As it is evident from Table 2 all the compounds showed a good inhibitory activity. Compounds 1 , 6, 8, 9, 10 17 and 18 showed inhibitory activity in the nanomolar concentration range.

Example 3:

MMP3 expression in rat chondrocytes

Rat articular chondrocytes were obtained from young adult Sprague Dawley male rats (7 weeks). All studies involving animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health. Articular cartilage from shoulders, femoral heads and knees was prepared according to Berenbaum (J Biol Chem, 2003). The cell suspension was then diluted to 5x104 cells/ml and seeded into 6-well culture plates (2 ml/well) in DMEM supplemented with 10% FBS and gentamicin (50pg/ml). Cells were maintained in an incubator at 37°C with 5% C02. The cells reached confluence in 10-12 days. Confluent cells were synchronized by 16h incubation in DMEM supplemented with 0.4% FBS. Cells were then co-treated in DMEM GlutaMAX I 0.4% with collagene type

II 80pg/ml and the different products for 32h.

At the end of the incubation, cells were lysed and total RNA was purified using the RNeasy® kit from Quiagen, following supplier instructions. Total RNA was subsequently retrotran scribed using the High Capacity cDNA Reverse Transcription Kit following supplier instructions. RT-PCR was finally performed in 7500 fast Real Time PCR system. The specific probes and primers are from Applied Biosystems as Assays- on-DemandTM while the probe and primers of the endogenous control 18S is a PDAR (Pre-Developed TaqMan®) Assay.

The data analysis, with the normalisation on the amplified values for the 18S was done following the specific instruction of Applied Biosystems for the relative quantification of gene expression. All single data are the result of three analyses for each sample. Table 3: Inhibition of DDR2-mediated MMP13 expression in rat chondrocytes

The tested compounds showed a good inhibitory activity in rat cells. Compounds 16 and 2 emerged as the better inhibitors.

Example 4:

3D cell model of breast cancer cells

MDA-MB-231 cells, a highly aggressive human breast cancer cell line was used to generate tumor spheroids. 1500 cell/well in DMEM 10% were plated in MW96 CELLCARRIER SPHEROID ULA-96 microplates (Perkin Elmer), which have an Ultra- Low Attachment (ULA)-coated surface that enables the formation of consistently round spheroids. After 24h incubation small spheroids were already formed. At this time point, the cells were treated directly in the well with 10mM of compound 2 for further 72h. 24h after treatment, pictures of the spheroids were made. The spheroids shrinkage was evaluated by measuring its dimension (Figure 1 , A and B). At the end of the incubation time cell vitality was analysed with CellTiter-Glo® 3D Reagent as described by Promega. The CellTiter-Glo® 3D Cell Viability Assay is a homogeneous method to determine the number of viable cells in 3D cell culture based on quantitation of the ATP present, which is a marker for the presence of metabolically active cells. All single data were the result of six replicates for each condition (Figure 2).

Compound 2 was able to reduce the 3D spheroids dimension and to knock down vitality in 3D model of breast cancer.

Example 5:

Determination of the pharmacokinetics of the invention compounds The pharmacokinetics of compound 2 were studied in rats treated intravenously and orally (n=3 for each route) with the compound in solution at the dose of 3 mg/Kg dissolved in 5% EtOH/saline, pH 7. The rats were fitted with a jugular cannula for serial sampling. A full pharmacokinetic profile was therefore obtained from each rat. Plasma extracts were quantitatively analyzed using a specific and sensitive LC-MS/MS bioanalytical method. Inter-individual variations between the three rats in each group were limited. After intravenous administration, the compound showed moderate volume of distribution and a low clearance. After oral administration, the compound was adsorbed at a moderate rate, with peak plasma concentration reached after 2 hours. The absolute oral bioavailability was very good (F 88%).

Example 6

In vitro model of fibrosis

HFL-1 cells, human fetal lung fibroblasts, were obtained from ECACC General Cell Collection. They were grown in Ham F-12 GlutaMaxl medium supplemented with 10% FBS and 1 % Non Essential Amino Acids (NEAA) at 37°C with 5% C02. HFL-1 cells were used between passage 10 and 17. 3.5 x 10⁵ cells were plated in MW6 well for 24h, thereafter the cells were synchronized by 16h incubation in the growth medium supplemented with 0.4% FBS.

Synchronized cells were then stimulated with 10ng/ml human Transforming Growth Factor-bΐ (TGF i )(Sigma) and treated with compound 2 at 1 and 10mM for 24h.

At the end of the incubation, cells were lysed and total RNA was purified using the MagMAX™mirVana™ Total RNA Isolation Kit (ThemnoFisher Scientific), following supplier instructions. Total RNA was subsequently retrotran scribed using the High Capacity cDNA Reverse Transcription Kit following supplier instructions. RT-PCR was finally performed in 7500 fast Real Time PCR system. The specific probes and primers of genes modulated in key processes of fibrosis were from Applied Biosystems as Assays-on-Demand™ while the probe and primers of the endogenous control 18S is a PDAR (Pre-Developed TaqMan®) Assay.

The data analysis, with the normalisation on the amplified values for the 18S was done following the specific instruction of Applied Biosystems for the relative quantification of gene expression. All single data were the result of three analyses for each sample. Figure 3 illustrates the effects of compound 2 on collagen type I expression by lung fibroblasts stimulated by TGF b1 . A clear concentration-dependent inhibition was observed, reaching the level of unstimulated controls at the 10 mM concentration.

Figure 4 illustrates the effects of compound 2 on the expression of MMP13, a relevant protease in the fibrotic process, by lung fibroblasts stimulated by TGF b1 . Also, in this case a clear concentration-dependent effect was observed, with the lowest tested concentration able to reduce by more than 60% the expression of this marker.

CONCLUSION

TΰRb1 is the most potent stimulator of fibroblasts proliferation and collagen production and is a requisite for fibrosis of different aetiologies and tissues e.g. pulmonary fibrosis. Compound 2 potently inhibited the pro-fibrotic stimulation of TΰRb1 by reducing the gene expression of collagen type I and MMP13.

Claims

1. A compound of Formula (I):

Ri and R2 are independently H or

where n is an integer value from 1 to 3,

R5 is independently H or Chb for each integer value of n,

R4 is H or CH3,

with the proviso that

a) when Ri, R2 and R4 are H, R3 is not methyl, ethyl, n-propyl and phenyl;

b) when Ri and R2 are H and R4 is methyl, R3 is not methyl; and

c) 2,7-diethoxyfenazine is excluded.

2. The compound of claim 1 ,

wherein Ri or R2 is

3. The compound of claim 2 wherein Rs is preferably hydrogen.

4. The compound of claim 3 wherein n is 1 or 2, preferably 2.

5. The compound of anyone of claims 1 -4, wherein R6 is an optionally substituted saturated heterocyclic ring.

6. The compound of claim 5, wherein R6 is an optionally substituted saturated heterocyclic ring selected from the group consisting of morpholinyl, pyrrolidinyl and piperazinyl.

7. The compound of claim 6, wherein R6 is piperazinyl.

8. The compound of claim 1 , wherein R3 is an optionally substituted aryl, preferably an optionally substituted phenyl.

9. The compound of claim 8, wherein phenyl is substituted with an optionally substituted heterocyclyl sulfonyl.

10. The compound of claim 9, wherein R3 is [N-(N-(2- hydroxyethyl)piperazinyl)sulfonyl]phenyl.

11. The compound of anyone of claims 1 -7, wherein R3 is an optionally substituted saturated (C3-C6)cycloalkyl, preferably, cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl, more preferably cyclopropyl.

12. The compound of anyone of claims 1 -7, wherein R3 is an optionally substituted unsaturated heterocyclic ring, preferably pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, pyrrolyl, furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, thiadiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl or indazolyl ,more preferably thienyl.

13. The compound of anyone of claims 1 -12, wherein R4 is H or CH3.

14. The compound of claim 1 , wherein the compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof, is selected from the group consisting of:

(R)-2-methoxy-7-((1 -phenylpropan-2-yl)oxy)phenazine (compound 1 ),

2-(cyclopropylmethoxy)-7-(2-(4-methylpiperazin-1 -yl)ethoxy)phenazine (compound 2), 4-(2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 3), 2-(cyclopropylmethoxy)-7-(2-(pyrrolidin-1 -yl)ethoxy)phenazine (compound 4), 2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)-N,N-dimethylethan-1 -amine (compound

5),

(R)-1 -((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)propan-2-amine (compound 6), 2-(4-(2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)ethyl)piperazin-1 -yl)ethan-1 -ol (compound 7),

4-(3-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)propyl)morpholine (compound 8), 2-(benzyloxy)-7-(2-(piperazin-1 -yl)ethoxy)phenazine (compound 9),

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10),

4-(2-((8-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 1 1 ),

4-(2-((8-(sec-butoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 12),

4-(3-((8-(cyclopropylmethoxy)phenazin-2-yl)oxy)propyl)morpholine (compound 13), 4-(2-((8-(thiophen-2-ylmethoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 14), 4-(2-((8-(thiophen-3-ylmethoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 15), 4-(2-((8-(benzyloxy)phenazin-2-yl)oxy)ethyl)morpholine(compound 16),

4-(3-((8-isobutoxyphenazin-2-yl)oxy)propyl)morpholine (compound 17),

(R)-2-((1 -phenylpropan-2-yl)oxy)phenazine (compound 18), and

2-(4-((4-((phenazin-2-yloxy)methyl)phenyl)sulfonyl)piperazin-1 -yl)ethan-1 -ol

(compound 19).

15. The compound of claim 14, wherein the compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof, is selected from the group consisting of:

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10)

4-(2-((8-(benzyloxy)phenazin-2-yl)oxy)ethyl)morpholine(compound 16)

16. A compound of Formula (I):

or a pharmaceutically acceptable salt or derivative thereof,

wherein:

Ri and R2 are independently H or

where n is an integer value from 1 to 3,

R5 is independently H or Chb for each integer value of n,

R4 is H or CH3,

17. The compound for use of claim 16,

wherein Ri or R2 is

18. The compound for use of claim 17 wherein Rs is preferably hydrogen.

19. The compound for use of claim 18 wherein n is 1 or 2, preferably 2.

20. The compound for use of anyone of claims 16-19, wherein R6 is an optionally substituted saturated heterocyclic ring.

21. The compound for use of claim 20, wherein R6 is an optionally substituted saturated heterocyclic ring selected from the group consisting of morpholinyl, pyrrolidinyl and piperazinyl.

22. The compound for use of claim 21 , wherein R6 is piperazinyl.

23. The compound for use of claim 16, wherein R3 is an optionally substituted aryl, preferably an optionally substituted phenyl.

24. The compound for use of claim 23, wherein phenyl is substituted with an optionally substituted heterocyclyl sulfonyl.

25. The compound for use of claim 24, wherein R3 is [N-(N-(2- hydroxyethyl)piperazinyl)sulfonyl]phenyl.

26. The compound for use of anyone of claims 16-22, wherein R3 is an optionally substituted saturated (C3-C6)cycloalkyl, preferably, cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl, more preferably cyclopropyl.

27. The compound for use of anyone of claims 16-22, wherein R3 is an optionally substituted unsaturated heterocyclic ring, preferably pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, pyrrolyl, furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, thiadiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl or indazolyl, more preferably thienyl.

28. The compound for use of anyone of claims 16-27, wherein R4 is H or CH3.

29. The compound for use of claim 16, wherein the compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof, is selected from the group consisting of:

(R)-2-methoxy-7-((1 -phenylpropan-2-yl)oxy)phenazine (compound 1 ),

2-(cyclopropylmethoxy)-7-(2-(4-methylpiperazin-1 -yl)ethoxy)phenazine (compound 2), 4-(2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 3), 2-(cyclopropylmethoxy)-7-(2-(pyrrolidin-1 -yl)ethoxy)phenazine (compound 4),

2-((7-(cyclopropylmethoxy)phenazin-2-yl)oxy)-N,N-dimethylethan-1 -amine (compound 5),

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10), 4-(2-((8-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 11 ), 4-(2-((8-(sec-butoxy)phenazin-2-yl)oxy)ethyl)morpholine (compound 12),

4-(3-((8-isobutoxyphenazin-2-yl)oxy)propyl)morpholine (compound 17),

(R)-2-((1 -phenylpropan-2-yl)oxy)phenazine (compound 18), and

2-(4-((4-((phenazin-2-yloxy)methyl)phenyl)sulfonyl)piperazin-1 -yl)ethan-1 -ol

(compound 19).

30. The compound for use of claim 29, wherein the compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof, is selected from the group consisting of:

4-(2-((7-isobutoxyphenazin-2-yl)oxy)ethyl)morpholine (compound 10)

4-(2-((8-(benzyloxy)phenazin-2-yl)oxy)ethyl)morpholine(compound 16)

31. A pharmaceutical composition comprising a compound of Formula (I) of anyone of claims 16-30 and a pharmaceutically acceptable carrier.

32. A compound or a pharmaceutically acceptable salt thereof of anyone of claims 16-

30 for use in the inhibition of a DDR2 receptor in the treatment of (DDR2)-mediated diseases and disorders.

33. The compound for use according to claim 32, wherein the (DDR2)-mediated diseases and disorders is a cancer, preferably colon, prostate, breast and lung cancer.

34. The compound for use according to claim 32, wherein the (DDR2)-mediated diseases and disorders is acute and chronic pain, osteoarthritis, inflammation- associated disorder as arthritis, rheumatoid arthritis, atherosclerosis, fibrosis, various fibrotic disorders or kidney injury.