EP4637757A1 - Phosphoglicerate dehydrogenase inhibitors for the treatment of fibrosis - Google Patents

Abstract

The present invention relates to a new therapeutic use of compounds of PHGDH inhibitors of general formula (I), for the prevention and/or treatment of fibrosis, in particular idiopathic pulmonary fibrosis (IPF). The invention is related also the use of the pharmaceutical compositions and combinations comprising said compounds for the prevention and/or treatment of fibrosis and in particular IPF.

Description

PHOSPHOGLICERATE DEHYDROGENASE INHIBITORS FOR THE TREATMENT OF FIBROSIS

FIELD OF INVENTION

The present invention relates to the use of compounds of general formula (I) as sole agent or in combinations with other active ingredients, as well as to the use of pharmaceutical compositions and combinations comprising said compounds for the prevention and/or treatment of fibrosis, in particular idiopathic pulmonary fibrosis (IPF).

BACKGROUND OF THE INVENTION

Fibrosis is a pathological wound healing in which connective tissue replaces normal parenchymal tissue to the extent that it goes unchecked, leading to considerable tissue remodelling and the formation of permanent scar tissue (see Wynn, Thomas A., 2004 Nature Reviews 4 (8): 583-594)

Fibrosis can occur in many tissues within the body, typically as a result of inflammation or damage, and examples include liver, lungs, kidney, brain and heart.

Idiopathic pulmonary fibrosis (IPF) is a rare, progressive illness of the respiratory system, characterized by the thickening and stiffening of lung tissue, associated with the formation of scar tissue. It is a type of chronic scarring lung disease characterized by a progressive and irreversible decline in lung function, (see HR, Egan JJ, et al. American Journal of Respiratory and Critical Care Medicine, 2011, 183 (6): 788-824.)

About 3 million people are affected globally and seems to become more common over the year, the incidence is expected to double by 2030 (see Fernandex Perez ER, et al, Chest, 2020; 137:129-137). Mortality is comparable to lung cancer, the median survival is 2-3 years after diagnosis, and respiratory failure is the most common cause of death (see Strongman H, et a., Adv Ther. 2018;35: 724-736)

The currently two FDA-approved drugs on the market, Nintedanib and Pirfenidone, slow the progression of the disease and prolong the life expectancy of IPF patients; however, the treated patients continue to experience lung function loss and premature death and the adverse events of both Nintedanib and Pirfenidone critically affect the patient’s quality of life.

The IPF is therefore a high unmet medical need and many efforts have been done in the recent past years to develop alternative treatments.

3 -phosphoglycerate dehydrogenase (PHGDH) is the enzyme that catalyzes the first ratelimiting step in the de novo biosynthesis of serine starting from glucose, and it is considered as a major enzyme in the diversion of glycolysis towards serine synthesis. Serine, a critical amino acid for protein and nucleic acid biosynthesis, in turn is metabolized and incorporated into a variety of biomolecules including glycine (see Yang M., Vousden, K H. Nature Reviews Cancer 2016,16, 650 662)

PHGDH is required to promote collagen protein synthesis. Indeed, glycine accounts for one- third of all amino acids within the collagen molecule, and the high content of glycine is critical for the stabilization of collagen helix.

Collagen is the main structural protein in the extracellular space, and it is produced in excess in IPF patients during fibroblasts into myofibroblasts differentiation that accumulates within the fibrotic tissue and leads to loss of organ architecture and function.

Moreover, PHGDH and the serine/glycine synthesis pathway is part of a wider network which links glycolysis with one-carbon metabolism and nucleotide synthesis contributing to cell proliferation in pathologies such as inflammation and fibrosis.

Therefore, the PHGDH inhibition has the potential to reduce the aberrant production and release of collagen such as inhibit cell proliferation (see Selvarajah et al., Science Signaling, 2019;12(582): eaav3048).

Various compounds have been described in the literature as PHGDH inhibitors.

WO 2017156179 (Raze Therapeutics) discloses the compound of general formula (I) effective as PHGDH inhibitors, and their use in the treatment of many PHGDH-mediated disorder, in particular melanoma, breast, or lung cancer.

Alternative PHGDH inhibitors promising for the prevention and/or treatment of fibrosis, are described as noncompetitive inhibitors or allosteric inhibitors of PHGDH enzyme (see Pacold et al, Nat Chem Biol. 2016; 12: 452-458).

Examples of said PHGDH inhibitors useful for treating fibrotic diseases are disclosed in WO2016115463 (Whitehead Institute For Biomedical Research; Dana-Farber Cancer Institute, Inc).

In particular one compound, identified as NCT-503, has been tested in a bleomycin-induced Pulmonary Fibrosis model (see Hamanaka et al. , AmJRespir Cell Mol Biol. 2018 May; 58(5): 585- 593) showing a preliminary efficacy in attenuation of lung fibrosis in treated mice 7 days after intratracheal instillation of bleomycin.

Despite the above prior art, there remains a potential for developing medicaments comprising a PHGDH inhibitor useful for the prevention and/or treatment of fibrosis, in particular IPF, with an improved efficacy profile.

The underlying problem of the present invention therefore lies in the provision of a medicament comprising a compound of formula (I) for the prevention and/or treatment of fibrosis, in particular IPF. SUMMARY OF THE INVENTION

In a first aspect the invention refers to a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of fibrosis, wherein

R¹ is hydrogen or C1-4 alkyl;

R² is selected from the group consisting of F, Cl, -CF3, -OCF3, -

OCHF2, -OCH2PI1, -OCH3, -CN, -CH₃, ,. ;

R³ is halogen, -OR, -CN, C1-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R⁸; or R² and R³ are optionally taken together with the carbon atoms to which they are attached and any intervening atoms to form a 5-8 membered partially unsaturated ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R is independently hydrogen or an optionally substituted group selected from Cn 6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each L is independently a C1-6 bivalent straight or branched hydrocarbon chain wherein 1-4 methylene units of the chain are independently and optionally replaced with -O-, -C(O)- , -C(O)O-, -OC(O)-, -OC(O)N(R)-, -(R)NC(O)O-, -C(O)N(R)-, -(R)NC(O)-, -N(R)-, - N(R)C(O)N(R)-, -S-, -SO-, or -SO2-;

R⁸ is hydrogen, C1-6 aliphatic, or an optionally substituted 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R⁴ is hydrogen, halogen, -OR⁵, -CN, Ci-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R⁸;

R⁵ is hydrogen, -(CH2)_n-phenyl, or Ci-6 alkyl optionally substituted with 1, 2, or 3 halogens;

R⁶ is hydrogen or Ci-4 alkyl;

R⁷ is hydrogen, -CO2R, C1-6 optionally substituted aliphatic group, or a bivalent 3-7 membered ring;

L¹ is a C1-8 bivalent straight or branched hydrocarbon chain wherein 1-5 methylene units of the chain are independently and optionally replaced with -O-,-C(O)-, -C(O)O-, - OC(O)-, -N(R)-, -C(O)N(R)-, -(R)NC(O)-, - OC(O)N(R)-, -(R)NC(O)O-, -N(R)C(O)N(R)-, halogen, cycloalkyl, -S-, -SO-, -SO2-, -SO₂N(R)-, -(R)NSO₂-, -C(S)-, -C(S)O-, -OC(S)-, - C(S)N(R)-, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy-;

L² is a covalent bond, or a C1-8 bivalent straight or branched hydrocarbon chain wherein 1-5 methylene units of the chain are independently and optionally replaced with -O-, -C(O)- , -C(O)O-, -OC(O)-, -N(R)-, -C(O)N(R)-, -(R)NC(O)-, -OC(O)N(R)-, -(R)NC(O)O-, - N(R)C(O)N(R)-, halogen, cycloalkyl, halogen, cycloalkyl, -S-, -SO-, -SO2-, -SO2N(R)-, - (R)NSO₂-, -C(S)-, -C(S)O-, -OC(S)-, -C(S)N(R)-, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy-; each -Cy- is independently a bivalent 6-membered arylene ring containing 0-2 nitrogen atoms, or a bivalent 5-membered heteroarylene ring with 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bivalent partially unsaturated 8-10 membered bicyclic heterocyclene ring with 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein -Cy- is optionally substituted with 1 or 2 substituents independently selected from C1-4 alkyl or -OR; and n is independently 0, 1, 2, 3, 4, or 5; wherein said compound is other than compound 4-[(lS)-l-({5-chloro-6-[(2-oxo-l,3- oxazolidin-5-yl)methoxy]-lH-indol-2-yl}formamido)-2 hydroxyethyl]benzoic acid and 4- [(1 S)-l -[[5-Chl oro-6-[2-(2 -hydroxy ethylamino)-2-oxo-ethoxy]-lH-indole-2-carbonyl] amino]-2-hydroxy-ethyl]benzoic acid.

In a second aspect, the invention refers to a compound of formula (I) or a pharmaceutically acceptable salt for use in the prevention and/or treatment of IPF.

In a third aspect, the invention refers to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in admixture with one or more pharmaceutically acceptable carrier or excipient, for use in the prevention and/or treatment of fibrosis. In a fourth aspect, the invention refers to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in admixture with one or more pharmaceutically acceptable carrier or excipient, for use in the prevention and/or treatment ofIPF.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Collagen protein expression

Figure 2: Inhibition of a-SMA/GAPDH

Figure 3: Inhibition of cell proliferation

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "carbocycle," “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “lower alkyl” refers to a C1-4 straight or branched alkyl group. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C1-4 straight or branched alkyl group that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2//-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N- substituted pyrrolidinyl)).

The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

As used herein, the term “bivalent Cns (or Ci-e) saturated or unsaturated, straight or branched, hydrocarbon chain”, refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylene chain” is a polymethylene group, i.e., -(CH2)_n-, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalent cyclopropyl group of the following structure: .

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic or bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “hetero ar alkoxy,” refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 % electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, TH-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

As used herein, the terms “heterocycle”, “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2J/-pyrrolyl), NH (as in pyrrolidinyl), or ⁺NR (as in TV-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3/7-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation but is not intended to include aryl or heteroaryl moieties, as herein defined.

As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.

Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.

Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.

The term “pharmaceutically acceptable salts”, as used herein, refers to derivatives of compounds 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.

The term “fibrotic disease” or “fibrosis” of “fibrotic conditions” 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 pulmonary fibrosis, familiar pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or cirrhosis.

The term “PHGDH” or “phosphoglycerate dehydrogenase”, is alternatively referred to as 3- PGDH, 3PGDHm HEL-S-113, NLS, PDG, PGAD, PGD, PGDH, PHGDHD, or SERA. The term “PHGDH” encompasses mutants, variants, homologs, fragments, and synthetically modified phosphoglycerate dehydrogenases.

The term “treating” or “treatment” of a disease state includes: (i) inhibiting the disease state, i.e. arresting the development of the disease state or its clinical symptoms, or (ii) relieving the disease state, i.e. causing temporary or permanent regression of the disease state or its clinical symptoms.

The term “preventing” or “prevention” of a disease state includes causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state. For example, treating or preventing a respiratory disease or disorder includes treating or preventing the symptoms the disorder such as cough and/ or urge to cough associated with a respiratory disease.

The term “therapeutically effective amount” means an amount of a compound that, when administered to a subject for treating a disease state, is sufficient to affect such treatment for the disease state. The "therapeutically effective amount" will vary depending on the compound, disease state being treated, the severity or the disease treated, the age and relative health of the subject, the route, and form of administration.

The present invention relates to the compounds of general formula (I) as herein defined for the use in prevention and/or treatment of fibrosis.

Differently from the other PHGDH inhibitors of the prior art, such as NCT-503, the compounds of formula (I) are surprisingly efficacious in the treatment of fibrosis, in particular IPF, in a substantive and effective way, particularly appreciated by the skilled person when looking at improved treatment for fibrosis.

As indicated in the Experimental part, the compounds of formula (I) have shown an improved in vitro profile and demonstrated an antifibrotic activity in in vitro and in vivo model.

The antifibrotic activity of the more preferred compound of formula (I), Compound 15, has been monitored measuring the expression of fibrotic mediators, collagen I (Col-I) and alpha smooth muscle actin (a-SMA).

As can be appreciated in Table 2, the Compound 15 is able to reduce the fibroblast to myofibroblast transition (FMT) induced by TGF-P and to inhibit the release of and a-sma in NHLF treated with TGF-P (lOng/ml) for 48h and differently from the compound NCT-503, the Compound 15 is also able to reduce the cell proliferation induced by serum treatment in NHLFs (72h).

As reported in the Table 3 in Example 2, all other tested compounds, dose-dependently, reduce the expression of aSMA, after 72 hours of incubation, differently from the NCT-503 which partially reduces the expression of aSMA. Furthermore, tested compounds dose- dependently inhibit the collagen I deposition, after 72h of treatment.

The antifibrotic activity of compounds of formula (I) has been demonstrated also measuring the expression collagen I (Col-I), known fibrotic mediator, in Fibroblast to myofibroblast transition (FMT)in in vitro assay. Furthermore, collagen is a downstream effector of the PHGDH pathway, its inhibition underlines the key role of this marker in fibrosis processes. These findings suggest that de novo synthesis of serine and glycine is required for pulmonary fibrosis; the inhibition of myofibroblast differentiation and collagen deposition is strongly correlated and necessary for the antifibrotic activity of the compounds.

As further advantage, the compounds of the present invention are able to be efficacious also in the most applied in vivo model, the Bleomycin-induced pulmonary fibrosis in mice. Bleomycin- induced pulmonary fibrosis in mice is the most commonly applied in vivo experimental model to induce lung fibrosis. Instillation of Bleomycin in the trachea induce a multiphasic response that starts with an acute and severe inflammation followed by a diffuse matrix and collagen deposition that led to histological changes, with evident fibrosis accumulation and loss of functional parenchymal tissue, replicating certain pathological features consistent with those observed in the lungs of IPF patients. Indeed, the bleomycin animal model is widely used in the assessment of potential antifibrotic agents.

In one embodiment, the invention relates to the compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of fibrosis wherein

R¹ is hydrogen or Ci-4 alkyl;

R² is selected from the group consisting of F, Cl, -CF3, -OCF3, - o . o £

0CHF₂, -OCH2PI1, -OCH3, -CN, -CH₃, ,.and ^H ;

R³ is halogen, -OR, -CN, C1-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R⁸; or R² and R³ are optionally taken together with the carbon atoms to which they are attached and any intervening atoms to form a 5-8 membered partially unsaturated ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R is independently hydrogen or an optionally substituted group selected from Cn 6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each L is independently a C1-6 bivalent straight or branched hydrocarbon chain wherein 1-4 methylene units of the chain are independently and optionally replaced with -O-, -C(O)- , -C(O)O-, -OC(O)-, -OC(O)N(R)-, -(R)NC(O)O-, -C(O)N(R)-, -(R)NC(O)-, -N(R)-, - N(R)C(0)N(R)-, -S-, -SO-, or -SO2-;

R⁸ is hydrogen, C1-6 aliphatic, or an optionally substituted 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

R⁴ is hydrogen, halogen, -OR⁵, -CN, C1-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R⁸;

R⁵ is hydrogen, -(CH2)_n-phenyl, or C1-6 alkyl optionally substituted with 1, 2, or 3 halogens;

R⁶ is hydrogen or C1-4 alkyl;

R⁷ is hydrogen, -CO2R, C1-6 optionally substituted aliphatic group, or a bivalent 3-7 membered ring;

L¹ is a C1-8 bivalent straight or branched hydrocarbon chain wherein 1-5 methylene units of the chain are independently and optionally replaced with -O-,-C(O)-, -C(O)O-, - OC(O)-, -N(R)-, -C(O)N(R)-, -(R)NC(O)-, - OC(O)N(R)-, -(R)NC(O)O-, -

N(R)C(0)N(R)-, halogen, cycloalkyl, -S-, -SO-, -SO2-, -SO₂N(R)-, -(R)NSO₂-, -C(S)-, - C(S)O-, -OC(S)-, -C(S)N(R)-, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy-; L² is a covalent bond, or a Cns bivalent straight or branched hydrocarbon chain wherein 1-5 methylene units of the chain are independently and optionally replaced with -O-, -C(O)- , -C(O)O-, -OC(O)-, -N(R)-, -C(O)N(R)-, -(R)NC(O)-, -OC(O)N(R)-, -(R)NC(O)O-, - N(R)C(O)N(R)-, halogen, cycloalkyl, halogen, cycloalkyl, -S-, -SO-, -SO2-, -SO2N(R)-, - (R)NSO₂-, -C(S)-, -C(S)O-, -OC(S)-, -C(S)N(R)-, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy-; each -Cy- is independently a bivalent 6-membered arylene ring containing 0-2 nitrogen atoms, or a bivalent 5-membered heteroarylene ring with 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bivalent partially unsaturated 8-10 membered bicyclic heterocyclene ring with 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein -Cy- is optionally substituted with 1 or 2 substituents independently selected from C1-4 alkyl or -OR; and n is independently 0, 1, 2, 3, 4, or 5; wherein said compound is other than compound 4-[(l S)-l-({5-chloro-6-[(2-oxo-l,3- oxazolidin-5-yl)methoxy]-lH-indol-2-yl}formamido)-2 hydroxyethyl]benzoic acid and 4- [(1 S)-l -[[5-Chl oro-6-[2-(2 -hydroxy ethylamino)-2-oxo-ethoxy]-lH-indole-2-carbonyl] amino]-2-hydroxy-ethyl]benzoic acid.

In one preferred embodiment, the invention relates to the compound of formula (I) as above defined for use in the prevention and/or treatment of IPF. In other preferred embodiment, the invention relates to the compound of formula (I) as above defined for use in the prevention and/or treatment of renal fibrosis.

All the listed groups for each of the variable moieties R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, L, L¹, L², n and Cy of the compounds of the invention have to be intended as alternatives and may be combined with each other in embodiments which are included in the scope of the invention.

In one embodiment, R¹ is hydrogen or C1-4 alkyl. In a preferred embodiment, R¹ is C1-4 alkyl. In another preferred embodiment, Ri is hydrogen or methyl or ethyl. In a more preferred embodiment, R¹ is methyl.

In one embodiment, R² is selected from the group consisting of F, Cl, -CF3, -OCF3,

In a preferred embodiment, R² is selected from the group consisting of F, Cl, -CF3-OCF3, - OCHF2, -OCH2PI1, -OCH3, -CN, -CH3. In a more preferred embodiment, R² is selected from the group consisting of

In one embodiment, R³ is halogen, -OR, -CN, C1-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R⁸; or R² and R³ are optionally taken together with the carbon atoms to which they are attached and any intervening atoms to form a 5-8 membered partially unsaturated ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In a preferred embodiment, R³ is halogen or -OR. In one preferred embodiment, both R² and R³ are halogen or -OCH3. In even more preferred embodiment, both R² and R³ are Cl or -OCH3

In one embodiment, R⁴ is hydrogen, halogen, -OR⁵, -CN, Ci-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R. In a preferred embodiment, R⁴ is hydrogen, halogen, -OR⁵, methyl. In a more preferred embodiment, R⁴ is hydrogen, halogen, methyl or -OCH3. In a more preferred embodiment, R⁴ is hydrogen.

In one embodiment, R⁷ is hydrogen, -CO2R, C1-6 optionally substituted aliphatic group, or a bivalent 3-7 membered ring. In a preferred embodiment, R⁷ is hydrogen or -CO2R. in a more preferred embodiment, In a preferred embodiment, R⁷ is hydrogen or -CO2H.

In one embodiment, L¹ is a C 1-8 bivalent straight or branched hydrocarbon chain wherein 1- 5 methylene units of the chain are independently and optionally replaced with -O-,-C(O)-, -C(O)O- , -OC(O)-, -N(R)-, -C(O)N(R)-, -(R)NC(O)-, -OC(O)N(R)-, -(R)NC(O)O-, -N(R)C(O)N(R)-, halogen, cycloalkyl, -S-, -SO-, -SO2-, -SO2N(R)-, -(R)NSO2-, -C(S)-, -C(S)O-, -OC(S)-, - C(S)N(R)-, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy. In a preferred embodiment, L¹ is selected from the group consisting of

In a more preferred embodiment, L¹ is selected from the group consisting of

In a preferred embodiment, L² is a covalent bond or selected from the group consisting of

In a more preferred embodiment, L² is a covalent bond or selected from the group consisting of

In one embodiment, the present invention relates to a compound which is selected from Formulae Il-a, Il-b, II-c, Il-d, Il-e, II- f, or Il-g for use in prevention and/or treatment of fibrosis:

II-g or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁶, R⁷,

L², is defined above and described in embodiments herein.

In one preferred embodiment, the present invention provides a compound which is selected from Formulae Il-a, Il-b, II-c, Il-d, Il-e, Il-f, or II-g as above defined for use in the prevention and/or treatment of fibrosis selected from the group consisting of pulmonary fibrosis, familiar pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or cirrhosis. In one preferred embodiment, the present invention provides a compound which is selected from Formulae Il-a, Il-b, II-c, Il-d, Il-e, Il-f, or Il-g, as above defined for use in the prevention and/or treatment of IPF.

In an equally preferred embodiment, the present invention provides a compound which is selected from Formulae Il-a, Il-b, II-c, Il-d, Il-e, Il-f, or Il-g as above defined for use in the prevention and/or treatment of renal fibrosis.

According to preferred embodiments, the present invention relates to at least one of the compounds selected from those listed in the following Table 1 for use in the prevention and/or treatment of fibrosis:

Table 1 List of preferred compounds of Formula (I) (

It is to be understood that all the single deuterates, enantiomers, diastereoisomers and mixtures thereof, in any proportion, or pharmaceutically acceptable salts and solvates of the compounds of formula (I) are encompassed within the scope of the present invention.

In one preferred embodiment, the present invention relates to at least one of the compound selected from those listed in the above Table 1 for use in the prevention and/or treatment of fibrosis selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or Cirrhosis. In one more preferred embodiment, the present invention relates to at least one of the compounds selected from those listed in the above Table 1 for use in the prevention and/or treatment of IPF. In an equally more preferred embodiment, the present invention relates to at least one of the compounds selected from those listed in the above Table 1 for use in the prevention and/or treatment of renal fibrosis.

The preparation of compounds described in Table 1 and intermediates of synthesis was already described in WO 2017156179 (Raze Therapeutics), along with the PHGDH Activity Inhibition Data.

In one embodiment, the present invention refers to the Compound 15

Compound 15 for use in the prevention and/or treatment of fibrosis.

In one preferred embodiment, the present invention refers to the Compound 15 for use in the prevention and/or treatment of fibrosis selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or cirrhosis.

In a more preferred embodiment, the present invention refers to the Compound 15 for use in the prevention and/or treatment of IPF.

In an equally preferred embodiment, the present invention refers to a Compound 15 for use in the prevention and/or treatment of renal fibrosis.

The compounds of formula (I) are typically administered once or even more, for example twice daily optionally in combination with a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time, according to the skilled person knowledge.

The dosages of the compounds of the invention may 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.

In another embodiment, the invention relates to compounds of Formula (I), or a pharmaceutically acceptable salt thereof, for use for the prevention and/or treatment of fibrosis whereby said compounds is administered for days, weeks, months, or years, including indefinitely. In one aspect, the present invention also refers to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, in admixture with at least one or more pharmaceutically acceptable carrier or excipient for use in the prevention and/or treatment of fibrosis.

In one embodiment, the invention refers to a pharmaceutical composition of compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of fibrosis 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, intradermally and by infusion) and by inhalation.

Preferably, the compounds of the present invention for use in the prevention and/or treatment of fibrosis are administered orally or 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.

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) for use in the prevention and/or treatment of fibrosis is a liquid oral dosage forms such as aqueous and non-aqueous solutions, emulsions, suspensions, syrups. 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) for use in the prevention and/or treatment of fibrosis 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 jet or ultrasonic nebulizers known from the prior art or by soft-mist nebulizers.

In one aspect, the invention related to a device comprising a pharmaceutical composition comprising a compound of Formula (I) for use in the prevention and/or treatment of fibrosis according to the invention, in form of a single- or multi-dose dry powder inhaler or a metered dose inhaler.

The compounds of the invention for use in the prevention and/or treatment of fibrosis can be administered as the sole active agent or in combination with other pharmaceutical active ingredients.

In a further aspect, the present invention relates to the use of compounds of Formula (I), or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of fibrosis.

In one embodiment, the present invention relates to the use of compounds of Formula (I), or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of fibrosis selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or Cirrhosis.

In one preferred embodiment, the present invention relates to the use of compounds of Formula (I), or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of IPF.

In one preferred embodiment, the present invention relates to the use of compounds of Formula (I), or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of renal fibrosis.

In a more preferred embodiment, the present invention relates to the use of compound 19 for the prevention and/or treatment of IPF. In a further aspect, the present invention relates to a method for the prevention and/or treatment of fibrosis administering compounds of Formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a method for the prevention and/or treatment of fibrosis selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or Cirrhosis, administering compounds of Formula (I), or a pharmaceutically acceptable salt thereof.

In one preferred embodiment, the present invention relates to a method for the prevention and/or treatment of IPF administering compounds of Formula (I), or a pharmaceutically acceptable salt thereof.

In an equally preferred embodiment, the present invention relates to a method for the prevention and/or treatment of renal fibrosis administering compounds of Formula (I), or a pharmaceutically acceptable salt thereof.

In a more preferred embodiment, the present invention relates to a method for the prevention and/or treatment of IPF administering compound 15, or a pharmaceutically acceptable salt thereof.

All preferred groups or embodiments described above for compounds of formula I for use in the prevention and/or treatment of fibrosis may be combined among each other and apply as well mutatis mutandis.

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 COMPOUNDS

Chemical Names of the compounds were generated with Structure To Name Enterprise 10.0 Cambridge Software or are common chemical names. 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. When reference is made to the use of a “similar” or “analogous” procedure, as it will be appreciated by those skilled in the art, such a procedure may involve minor variations, for example reaction temperature, reagent/solvent amount, reaction time, workup conditions or chromatographic purification conditions, that will be appreciated by those skilled in the art.

All the compounds of the present invention are synthetized as reported in WO

2017156179. PHARMACOLOGICAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION Example 1

In vitro assay

TGFb-induced profibrotic markers release and Serum-induced Proliferation

The antifibrotic activity of compound of the present invention has been monitored measuring the expression of fibrotic mediators, collagen I (Col-I) and alpha smooth muscle actin (a-SMA) after treatment in lung cells with TGF-P in comparison with Nintedanib (see Frank Hilberg et al, Cancer Res 2008 Jun 15;68(12):4774-82\ and allosteric inhibitor NCT- 503 (see Hamanaka et al., Am J Respir Cell Mol Biol. 2018 May; 58(5):585-593).

Cells cultured

Human normal lung fibroblasts (NHLFs) were purchased from Lonza (Basel, Switzerland). NHFLs were cultured in Eagle’s Minimum Essential medium (EMEM) w/o serine and glycine, supplemented with 10% Fetal Bovine Serum (FBS), antibiotics (50 U/ml penicillin and 0.05 mg/ml streptomycin) and 2 mM L-glutamine, in an atmosphere of 95% air and 5% CO2 at 37°C. NHLFs cultures were used between passage 1 and 9. Before all of the experiments, cells were incubated overnight in serum-free medium.

NHLFs were seeded in 12-well plates at cultured until 70% of confluency. To examine the expression of fibrotic markers, cells were treated with different concentrations of compound 15 and NCT-503 in EMEM medium for Ih followed by stimulation with TGF-P for 48h at 37 °C, 5% CO2. Following cell treatments, medium was removed and whole cell extracts were prepared by directly adding to the well the IX loading buffer (125mM Tris-HCl pH 6.8, 4% SDS, 0.2% Orange G, 50% glycerol, 2.5% P- mercaptoethanol, Li-cor Biosciences), boiled for 5 min and then electrophoresed on Mini protean TGX gel 4-12% (Biorad). After electrophoresis, proteins were transferred to a nitrocellulose membrane. Immunodetection of Col-I, a-sma and glyceraldeide 3 -phosphate dehydrogenase (GAPDH) were obtained by using mouse anti-CollAl (Santacruz), mouse anti-a-sma (sigma) and rabbit anti-GAPDH (Cell Signaling Technology) all diluted at 1 : 1000 in TBS-tween 0.1%. Secondary antibodies were goat anti rabbit and goat anti mouse IgG conjugated with IRDye800 and IRDye680 probes (Li-cor) respectively, both diluted at 1 : 15,000 in TBS-tween 0.1% containing 1% of skim milk. Detection and quantification were performed with Odyssey imaging system (Li-cor) using GAPDH as loading control.

The antiproliferative activity of compounds of the present invention has been monitored measuring the incorporation of bromodeoxyuridine (BrdU), a thymidine analog of DNA, in newly synthesized DNA after treatment in lung cells. Briefly, NHLFs were seeded in 96-well and treated with different concentrations of Compound 15 and NCT-503 in EMEM medium for Ih followed by stimulation with Fetal Bovine Serum (FBS) for 72h at 37 °C, 5% CO2. After cell treatments, 10 pl/well BrdU labeling solution was added for incorporation in proliferating cells for 2h at 37 °C, 5% CO2. After removing the culture medium, the cells are fixed, and the DNA is denatured by adding FixDenat followed by incubation with 100 pl/well Anti -BrdU -POD for detection. The reaction product is quantified by measuring the absorbance at the respective wavelength using a scanning multiwell spectrophotometer (ELISA reader).

The potency of the Compound 15 and NCT-503 is reported in Table 2. Compound activity is expressed as pICso (Log of the IC50, compound concentration producing 50% of inhibition of the fibrotic markers expression or of the FBS-induced cell proliferation).

Table 2 pIC50 values for Compound 15 in in vitro assay

C.I.= Confidence Interval; NA: not active

As can be appreciated in Figure 1, Compound 15 reduce the fibroblast to myofibroblast transition (FMT) induced by TGF-P as shown by decrease of Collagen protein expression.

As shown in Figure 2, Compound 15 (50uM) is able to inhibit the release of and a-sma in NHLF treated with TGF-P (lOng/ml) for 48h in a more efficacious manner than the NCT-503 (50uM) and even more than the Nintedanib (luM).

Furthermore, only Compound 15 is also able to reduce the cell proliferation induced by serum treatment in NHLFs (72h) in comparison with NCT-503, as shown in Figure 3.

Example 2

Phenotypic assay - High Content Screening optimization of aSMA staining in NHLF cells

Method

NHLF cells (Lonza #CC2512) were seeded as 2 000 cells/well in 384-well plates in MEM medium w/o serine and glycine (Sigma #M2279) + 1% pen/strep + 5 mM L-glutamine + 10% FBS and incubated overnight at 37°C, 5% CO?.

The next day, cells were washed with PBS and starvation MEM medium w/o serine and glycine (0% FBS) was added and cells were incubated overnight at 37°C, 5% CO2. The day after, cells were pre-incubated with compounds or vehicle (0.3% DMSO) for 1 h. Compounds were tested at 9 consecutive 3-fold dilutions starting from 30 pM. Each compound was tested in duplicate. Cells were further incubated for 72 h at 37°C, 5% CO2, 95% humidity. After 72h of incubation, aSMA expression was quantified using immunostaining:

- Fixation in 4% paraformaldehyde,

- Blocking with 3% BSA, 2% FBS, 0.2% Triton-X in PBS,

- Primary ab. anti-aSMA (AbCam #ab7817) diluted 500x in blocking buffer

- Anti-mouse secondary ab. AF488 (ThermoFisher #A11029)

% of aSMA positive cells was determined by High Content Imaging (Molecular Device), data analysis was performed using MetaXpress software (Molecular Devices) and % of aSMA positive was further used to calculate % of inhibition for tested compounds. Total cell number was determined by Hoechst staining and % of viability over TGFp stimulated controls was calculated.

Fibroblast to myofibroblast transition (FMT) in vitro assay: Collagen I detection

NHLF cells were seeded as 80 000 cells/well in 12-well plates in MEM medium w/o serine and glycine (Sigma #M2279) + 1% pen/ strep + 5 mM L-glutamine + 10% FBS and incubated overnight at 37°C, 5% CO2. The assay was carried out as report above.

The potencies of the Compounds of this invention and NCT-503 are provided in Table 3. Compound activity is expressed as pIC50 (Log of the IC50, compound concentration producing 50% of inhibition of the fibrotic markers expression).

Table 3- pIC50 values in in vitro assay

Results

As reported in the Table 3, all tested compounds, dose-dependently, reduce the expression of aSMA, after 72 hours of incubation. On the contrary, the NCT-503 partially reduces the expression of aSMA. Furthermore, tested compounds dose-dependently inhibit the collagen I deposition, after 72h of treatment. These findings suggest that de novo synthesis of serine and glycine is required for pulmonary fibrosis; the inhibition of myofibroblast differentiation and collagen deposition is strongly correlated and necessary for the antifibrotic activity of the compounds.

Claims

1. A compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of fibrosis, wherein:

R¹ is hydrogen or C1-4 alkyl;

R² is selected from the group consisting of F, Cl, -CF3, -OCF3, -

R³ is halogen, -OR, -CN, C1-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R⁸; or R² and R³ are optionally taken together with the carbon atoms to which they are attached and any intervening atoms to form a 5-8 membered partially unsaturated ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each L is independently a C1-6 bivalent straight or branched hydrocarbon chain wherein 1-4 methylene units of the chain are independently and optionally replaced with -O-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(R)-, -(R)NC(O)O-, -C(O)N(R)-, - (R)NC(O)-, -N(R)-, -N(R)C(O)N(R)-, -S-, -SO-, or -SO2-; R⁸ is hydrogen, Ci-6 aliphatic, an optionally substituted 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

R⁴ is hydrogen, halogen, -OR⁵, -CN, Ci-6 aliphatic optionally substituted with 1, 2, or 3 halogens, or -L-R⁸;

R⁵ is hydrogen, -(CH2)_n-phenyl, or Ci-6 alkyl optionally substituted with 1, 2, or 3 halogens;

R⁶ is hydrogen or Ci-4 alkyl;

R⁷ is hydrogen, -CO2R, C1-6 optionally substituted aliphatic group, or a bivalent 3-7 membered ring;

L¹ is a C1-8 bivalent straight or branched hydrocarbon chain wherein 1-5 methylene units of the chain are independently and optionally replaced with -O-,- C(O)-, -C(O)O-, -OC(O)-, -N(R)-, -C(O)N(R)-, -(R)NC(O)-, OC(O)N(R)-, -(R)NC(O)O-, -N(R)C(O)N(R)-, halogen, cycloalkyl, -S-, -SO-, -SO2-, -SO₂N(R)-, -(R)NSO₂-, -C(S)-, -C(S)O-, -OC(S)-, -C(S)N(R)-, -(R)NC(S)-, - (R)NC(S)N(R)-, or -Cy-;

L² is a covalent bond, or a C1-8 bivalent straight or branched hydrocarbon chain wherein 1-5 methylene units of the chain are independently and optionally replaced with -O-, -C(O)-, -C(O)O-, -OC(O)-, -N(R)-, -C(O)N(R)-, -(R)NC(O)-, -OC(O)N(R)- , -(R)NC(O)O-, -N(R)C(O)N(R)-, halogen, cycloalkyl, halogen, cycloalkyl, -S-, -SO-, -SO2-, -SO₂N(R)-, -(R)NSO₂-, -C(S)-, -C(S)O-, -OC(S)-, -C(S)N(R)-, -(R)NC(S)-, - (R)NC(S)N(R)-, or -Cy-; each -Cy- is independently a bivalent 6-membered arylene ring containing 0-2 nitrogen atoms, or a bivalent 5-membered heteroarylene ring with 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bivalent partially unsaturated 8-10 membered bicyclic heterocyclene ring with 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein -Cy- is optionally substituted with 1 or 2 substituents independently selected from C1-4 alkyl or -OR; and n is independently 0, 1, 2, 3, 4, or 5; wherein said compound is other than compound4-[(lS)-l-({5-chloro-6-[(2-oxo-l,3- oxazolidin-5-yl)m ethoxy] -lH-indol-2-yl}formamido)-2 hydroxyethyl]benzoic acid and 4-[(lS)-l-[[5-Chloro-6-[2-(2-hydroxyethylamino)-2-oxo-ethoxy]-lH-indole-2- carbonyl] amino]-2-hydroxy-ethyl]benzoic acid.

2. The compound of formula (I) for use according to claim 1, wherein R¹ is methyl.

3. The compound of formula (I) for use according to claim 1-2, wherein R² is selected from the group consisting ofF, Cl, -CF₃, OCF₃, - OCHF2, -OCH₂Ph, -OCH3, -CN, -CH₃.

4. The compound of formula (I) for use according to claim 1-3, wherein R³ is halogen or -OR.

5. The compound of formula (I) for use according to any of claims 1 to 4, wherein both R² and R³ are halogen or -OCH3.

6. The compound of formula (I) for use according to claim 1-5, wherein R⁴ is hydrogen, halogen, methyl or -OCH3.

7. The compound of formula (I) for use according to claim 1-6, wherein R⁷ is hydrogen or -CO2R.

8. The compound of formula (I) for use according to claim 1-7, wherein L¹ is selected from the group consisting of

9. The compound of formula (I) for use according to claim 1-8, wherein L² is a covalent bond or selected from the group consisting of

10. The compound for use according to claim 1-9, wherein the compound is selected from at least one of

(±)-2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole-2-carbonyl)amino]-2-hydroxy- ethyl] phenyl]-2,2-dimethyl-acetic acid, (±)-2-[4-[(l S)-l-[(4-chloro-5 -methoxy- 1 -methyl-indole-2-carbonyl)amino]-2- hydroxy-ethyl] phenyl]-2,2-dimethyl-acetic acid,

(±)-2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole-2-carbonyl)amino]-2-hydroxy- ethyl] phenyl]-2-cyclopropyl-acetic acid,

(±)-2-[4-[(lS)-l-[(4,5-dichloro-6-methoxy-l-methyl-indole-2-carbonyl)amino]-

2-hydroxy- ethyl]phenyl]butanoic acid,

(±)-2-[4-[(l S)-l-[(4-chloro-5 -methoxy- 1 -methyl-indole-2-carbonyl)amino]-2- hydroxy-ethyl] phenyl]butanoic acid,

4-[l -[[(4, 5 -di chloro- 1 -methyl- lH-indol-2-yl)carbonyl]amino]-2-fluoroethyl]- benzoic acid,

4-[l -[[(4,5 -dichloro-6-methoxy-l -methyl- lH-indol-2-yl)carbonyl]amino] -2- hydroxy-ethyl]-3 -methyl-benzoic acid,

4-[(lS)-l-[[(4-chloro-6-methoxy- 1,5 -dimethyl- lH-indol-2-yl)carbonyl]amino]-

2-hydroxyethyl]-3-methyl-benzoic acid,

2-{3-chloro-4-[(lS)-l-[(4-chloro-5-methoxy-l-methyl-lH-indol-2- yl)formamido]-2-hydroxyethyl]phenyl} acetic acid,

2-{3-chloro-4-[(lS)-l-[(4,5-dichloro-l-methyl-lH-indol-2-yl)formamido]-2- hydroxyethyl]phenyl } -acetic acid,

4-[(lS)-l-[[(4-chloro-5-methoxy-l -methyl- IH-indol -2 -yl)carbonyl]amino]-2- hydroxy ethyl ] -3 -methyl -b enzoi c acid,

3-fluoro-4-[(lS)-l-[(4-chloro-6-methoxy-l,5-dimethyl-lH-indol-2- yl)formamido]-2-hydroxyethyl]benzoic acid,

4-[(lS)-l-[[(4-chloro-5-methoxy-l-methyl-lH-indol-2-yl)carbonyl]amino]-2- hydroxyethyl]-3-fluoro-benzoic acid,

(±)-2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole-2-carbonyl)amino]-2-hydroxy- ethyl] phenyl]-2 -methyl-acetic acid,

(±)-2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole-2-carbonyl)amino]-2-hydroxy- ethyl]phenyl] butanoic acid,

3 -chi oro-4-[(l S)-l -[(4-chloro-6-methoxy- 1,5 -dimethyl- IH-indol -2- yl)formami do] -2-hydroxy ethyl ]benzoi c aci d,

3-chloro-4-[(lS)-l-[(4,5-dichloro-6-methoxy-l-methyl-lH-indol-2- yl)formami do] -2-hydroxy ethyl ]benzoi c aci d,

4-[(lS)-l-[[(4,5-dichloro-l-methyl-lH-indol-2-yl)carbonyl]amino]-2- hydroxyethyl] -3 -methyl-benzoic acid, 2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole-2-carbonyl)amino]-2-hydroxy- ethyl ] 3 -methyl -phenyl ] -acetic acid,

4-[( IS)- 1 -[[(4,5 -di chloro-6-methoxy-l -methyl- lH-indol-2-yl)carbonyl]amino] -

2-hydroxyethyl]-3-fluoro-benzoic acid,

3 -chi oro-4-[(l S)-l-[(4-chloro-5 -methoxy- 1 -methyl- lH-indol-2-yl)form amido]-

2-hydroxyethyl]benzoic acid,

4-[(lS)-l-[[(4,5-dichloro-l-methyl-lH-indol-2-yl)carbonyl]amino]-2- hydroxyethyl]-3-fluoro-benzoic acid,

2- {4-[(lS)-l -[(4-chloro-5 -methoxy- 1 -methyl- lH-indol-2-yl)form amido]-2- hydroxyethyl]phenyl (acetic acid,

2-[4-[(lS)-l-[(4-chloro-5-fuoro-6-methoxy-l-methyl-indole-2-carbonyl)amino]- 2-hydroxy-ethyl]-3-fluoro-phenyl]-acetic acid,

2-[4-[(lS)-l-[(4-chloro-6-methoxy-l,5-dimethyl-indole-2-carbonyl)amino]-2- hydroxy-ethyl]-3-fluoro-phenyl]-acetic acid,

3-chloro-4-[(lS)-l-[(4,5-dichloro-l-methyl-lH-indol-2-yl)formamido]-2- hydroxyethyl]benzoic acid,

2-{4-[(lS)-l-[(4,5-dichloro-6-methoxy-l-methyl-lH-indol-2-yl)formamido]-2- hy droxy ethyl] -3 -fluorophenyl ( aceti c aci d,

2- {4-[(lS)-l -[(4, 5 -di chloro- 1 -methyl- IH-indol -2 -yl)form amidolhydroxy ethyl]phenyl (acetic acid,

4-[l -[[(4, 5 -di chloro- 1 -methyl- lH-indol-2-yl)carbonyl]amino]ethyl] -3 -fluorobenzoic acid,

2-[4-[(l S)-l -[(4, 5-dichloro-l-methyl-indole-2-carbonyl)amino]ethyl]-3 -fluoro- phenyl]-acetic acid,

2- {4-[(lS)-l -[(4, 5 -di chloro- 1 -methyl- IH-indol -2 -yl)form amidolhydroxy ethyl] -3 -fluorophenyl ( aceti c aci d,

3-chloro-4-[l-[[(4,5-dichloro-l-methyl-lH-indol-2-yl)carbonyl]amino]ethyl]- benzoic acid,

(±)- 2-(4-(l -(4, 5-Dichloro- 1 -methyl- lH-indole-2-carboxamido)ethyl)phenyl) acetic acid,

4-[l -[[(4, 5 -dichloro- 1 -methyl- lH-indol-2-yl)carbonyl]amino]-l -methylethyl]- benzoic acid,

4-[2-cy clopropyl-1 -[[(4, 5 -dichloro- 1 -methyl- lH-indol-2- yl)carbonyl]amino]ethyl]-benzoic acid, 4-[l -[[(4, 5 -dichloro- 1 -methyl- lH-indol-2-yl)carbonyl]amino]methyl]-benzoic acid,

(±)-4-(l -(4, 5-Dichl oro-1 -methyl- lH-indole-2-carboxamido)-2, 2,2- trifluoroethyl)benzoic acid,

4-[( IS)- 1 -[[4,5 -Dichloro-6-[2-(2 -hydroxy ethylamino)-2-oxo-ethoxy]-l-methyl- indole-2- carbonyl]amino]-2-hydroxy-ethyl]benzoic acid,

4-[cy clopropyl [[(4, 5 -di chloro- 1 -methyl- lH-indol-2 -yl)carbonyl]amino]methyl]- benzoic acid,

4-[(lS)-l-[(4,5-dichloro-l-methyl-lH-indol-2-yl)formamido]-2- hydroxyethyl]benzoic acid,

4-[l -[[(4, 5 -di chloro- 1 -methyl- lH-indol-2-yl)carbonyl]amino]ethyl] -benzoic acid,

(±)-4-[3-[Acetylsulfamoyl(methyl)amino]-l -[(4, 5 -dichloro- l-methyl-indole-2- carbonyl)- amino]propyl]benzoic acid,

(±)-4-(2- Amino- 1 -(4, 5 -di chloro- 1 -methyl- lH-indole-2-carboxamido)ethyl) benzoic acid,

4-[3 -(Acetyl sulfamoylamino)- 1 -[(4, 5 -di chloro- 1 -methyl -indole-2 - carbonyl)amino] propyl]benzoic acid,

(±)-N-[3-(acetylsulfamoylamino)-l-[3-(hydroxymethyl)phenyl]propyl]-4,5- dichloro -l-methyl-indole-2-carboxamide,

(±)-3-[3-(Acetylsulfamoylamino)-l-[(4,5-dichloro-l-methyl-indole-2 carbonyl)amino]propyl] benzoic acid,

(±) - N-[3-(acetylsulfamoylamino)-l-phenyl-propyl]-4, 5-dichloro-l-methyl- indole-2-carboxamide,

Diastereoisomer 1 of 2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole-2- carbonyl)amino]-2-hydroxy-ethyl] phenyl]-3- methyl-butanoic acid,

Diastereoisomer 2 of 2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole- 2- carbonyl)amino]-2-hydroxy-ethyl]phenyl]-3- methyl-butanoic acid, (±)-2-[4-[(lS)-l-[(4,5-dichloro-l-methyl-indole-2-carbonyl)amino]-2-hydroxy- ethyl] phenyl]-3-methyl-butanoic acid.

11. The compound of formula (I) for use according to any one of claims 1 to 10, wherein said compound is selected from Formulae Il-a, Il-b, II-c, Il-d, Il-e, Il-f, or Il-g:

II-g or a pharmaceutically acceptable salt thereof.

12. The compound for use according to claim 10 or 11, wherein the compound is

Compound 15 or a pharmaceutically acceptable salt thereof.

13. The compound of Formula (I), or a pharmaceutically acceptable salt thereof, according to claim 1-12, for use in the prevention and/or treatment of fibrosis selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or Cirrhosis.

14. The compound of Formula (I), or a pharmaceutically acceptable salt thereof, according to claim 13 for use in the prevention and/or treatment of IPF.

15. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof according to claims 1 to 12, in admixture with one or more pharmaceutically acceptable carrier or excipient, for use in the prevention and/or treatment of fibrosis.

16. The pharmaceutical composition comprising a compound of formula (I) for use according to claim 15 wherein the fibrosis is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (CHP), hepatic fibrosis, kidney or renal fibrosis, ocular fibrosis, cardiac fibrosis, arterial fibrosis, sarcoidosis and systemic sclerosis, liver fibrosis or cirrhosis.

17. The pharmaceutical composition comprising a compound of formula (I) according to claim 16 for use in the prevention and/or treatment of IPF.

18. The pharmaceutical composition according to claims 15 to 17 for oral administration.