Salts of (s)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1h-pyrrolo[2,3-b]pyridin-3-YL)pyridin-2(1H)-one for use in the treatment of melanoma with BRAF v600 mutation

Abstract

The present invention concerns the use of pharmaceutically acceptable salts of (S)-1-(1-(3- chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3- yl)pyridin-2(1H)-one of the formula (I), wherein said salt is selected from a hydrochloride salt, a maleate salt, an esylate salt, an oxoglutarate salt, a malonate salt and an oxalate salt, in the treatment of melanoma with BRAF V600 mutation.

Description

[0001] SALTS OF (5)-l-(l-(3-CHLOROPHENYL)-2-(DIMETHYLAMINO)ETHYL)-4-(5- MORPHOLINO-lH-PYRROLO[2,3-B]PYRIDIN-3-YL)PYRIDIN-2(lH)-ONE FOR USE IN THE TREATMENT OF MELANOMA WITH BRAF V600 MUTATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to specific pharmaceutically acceptable salts, and solvates thereof, of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one (Compound 1), which are inhibitors of ERK kinases (ERK1 and ERK2), for use in the treatment of melanoma with BRAF V600 mutation.

[0004] The invention further relates to pharmaceutical compositions and medicaments comprising pharmaceutically acceptable salts and / or solvates of Compound 1, for use in the treatment of melanoma with BRAF V600 mutation.

[0005] BACKGROUND OF THE INVENTION

[0006] The ERK protein belongs to the RAS / RAF / MEKZERK pathway which plays a major role in cell cycle, proliferation, growth, and survival. The RAS / RAF / MEKZERK pathway is activated by growth factors through their receptor tyrosine kinase that allows activation of RAS GTPases. In turn, RAS activates RAF proteins. Then, RAF activates MEK, which activates ERK.

[0007] Finally, this enables phosphorylation of many substrates that have key roles in metabolism, protein synthesis, cell proliferation and survival.

[0008] RAF mutations lead specifically to an over-activation of this RAS / RAF / MEKZERK pathway and are responsible for 7% of all human cancers (Davies et al., Nature. 2002; Garnett et al., Cancer Cell. 2004).

[0009] Indeed, RAF mutations are frequently observed in melanomas (27-70%), thyroid cancers (36-53%), colorectal cancers (5-22%) and ovarian cancers (30%). Likewise, RAS mutations occur in almost 30% of cancers and are present in pancreatic (90%), lung (35%), colorectal (45%) and liver (30%) cancers (Downward, Nat. Rev. Cancer. 2003).

[0010] Thus, proteins of the RAS / RAF / MEKZERK pathway, and in particular upstream kinases such as RAF and MEK, represent targets of interest for cancer treatment. Substantial efforts in the past decades have led to the clinical success of BRAF and MEK inhibitors (Caunt et al, Nat. Rev. Cancer, 2015). However, the clinical benefits of these inhibitors are compromised by the frequently occurring acquired resistance due to cancer heterogeneity and genomic instability (Lito et al., Nat. Med. 2013).Moreover, most resistances to MEK or RAF inhibitors induce reactivation of ERK1 / 2, which are the most distal kinases in the MAPK signaling cascade, through different mechanisms such as MEK mutation, B-RAF amplification, C-RAF mutation (Little et al., Oncogene. 2013). Furthermore, RAF or MEK inhibition suppresses ERK negative feedback that restores upward signaling and finally ERK activity (Lito et al., Nat. Med., 2013).

[0011] Considering these resistance phenomena, it is essential to develop new therapeutic options. Targeting the ERK protein also appears to offer a promising prospect for cancer therapy by overcoming resistance to upstream targets (Kidger etal, Pharmacol Ther. 2018). Interestingly, inhibition of ERK1 / 2 can effectively inhibit MAPK signaling and induce anti-tumor activity, as demonstrated in preclinical studies.

[0012] The MAPK pathway is frequently activated in human cancers with RAS genes (Kirsten rat sarcoma viral oncogene homologue (KRAS), neuroblastoma rat sarcoma (NRAS) and Harvey rat sarcoma viral oncogene homologue (HRAS)) being the most frequently mutated oncogenes in all cancers (>30%) and V-RAF murine sarcoma viral oncogene homologue Bl (BRAF) mutated in -7% of all cancers (Davies et al., Nature. 2002). However, despite approval of BRAF and MEK and more recently of KRAS G12C inhibitors, clinically effective targeted therapies are still needed for most RAS mutant cancers (Yang et al., Cancer Commun. 2023; Conroy et al., Cancer Drug Resist. 2021).

[0013] A worldwide total of 325,000 new melanoma cases and 57,000 deaths was estimated for 2020 (Sung et al., CA Cancer J Clin. 2021). Approximately 50% of melanomas harbor mutations of BRAF and the most common mutation V600E accounts for -80% of BRAF mutations (Proietti etal., Cancers 2020).

[0014] BRAF inhibitors including vemurafenib, dabrafenib and encorafenib in monotherapy dramatically improved the response for patients with BRAF mutation, but the benefits were not durable with the rapid development of resistance through MAPK reactivation. Based on this mechanism of resistance, combined BRAF and MEK inhibition is now the SOC for patients with advanced or metastatic BRAF mutated melanoma. Targeted and immune checkpoint therapy have made great advances in patients with BRAF mutated, advanced melanoma, but the acquisition of resistance is still a major issue for further drug development.

[0015] Thus, the present invention relates to ERK inhibitors development to treat melanoma with BRAF V600 mutation.

[0016] Some ERK inhibitors are already described in the prior art. Thus, US 8,697,697 B2 describes substituted pyrazole derivatives as inhibitors of ERK2 kinase activity.Pyrrolo[2,3-b]pyrazine derivatives are also reported as ERK inhibitors in the international patent application WO 2014 / 060395 Al, and azaindole derivatives are reported as ERK inhibitors in the international patent application WO 2017 / 085230 Al.

[0017] (5)- 1 -( 1 -(3 -chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino- U / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one (Compound 1) of formula (I), depicted below, is an inhibitor of ERK kinases (ERK1 and ERK2). It may be used in particular as anticancer agent.

[0018]

[0019] (I)

[0020] However, despite its therapeutic potential, such a compound, which has been developed in a free base form, presents significant limitations related to its solubility.

[0021] Indeed, it is desired that such ERK inhibitor shows high solubility when it is used for a pharmaceutical formulation, in particular a pharmaceutical formulation for oral administration. In addition, it is desired that the solubility profile of such ERK inhibitor does not depend on the pH conditions. Indeed, the free base of such ERK inhibitor has a solubility profile that is strongly dependent on pH. Thus, at acidic pH the free base is slightly soluble, but its solubility decreases strongly when the pH is higher than 3, which is not favorable to an oral administration.

[0022] Further, improving the solubility of such compounds offers several advantages. Enhanced solubility results in better dissolution, which in turn increases the bioavailability of the active ingredient. This increases the likelihood of achieving the desired therapeutic dose while requiring less raw material, potentially reducing toxicity and side effects. Moreover, higher solubility enables the production of smaller tablets, leading to more convenient and less burdensome treatments for patients.

[0023] There is therefore a need to provide new means that address the solubility challenges associated with the free base form of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-U / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one.By addressing this need, the present invention provides an innovative solution that improves upon existing therapies for treating melanoma with BRAF V600 mutation, offering better treatment outcomes, fewer side effects, and a more patient-friendly therapeutic regimen.

[0024] The present invention is precisely directed to the use of specific salts of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one for treating melanoma with BRAF V600 mutation.

[0025] The compounds of the present invention are specific salts with at least enhanced antiproliferative activity, and good solubility in aqueous solutions.

[0026] The salts according to the invention are also characterized by their low toxicity, high permeability, and kinase inhibition selectivity.

[0027] Overall, the salts according to the invention are remarkable for their drug-like properties, for the treatment of melanoma with BRAF V600 mutation.

[0028] SUMMARY OF THE INVENTION

[0029] A first aspect of the invention relates to a pharmaceutically acceptable salt, and solvate thereof, of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-U / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one (Compound 1) of the formula (I) below:

[0030]

[0031] (I)

[0032] wherein said salt is selected from a hydrochloride salt, a maleate salt, an esylate salt, an oxoglutarate salt, a malonate salt and an oxalate salt, for use in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0033] After extensive searching and screening tests, the inventors have identified that these specific salts of the compound of formula (I) selectively target the active sites of the ERK kinases, act as effective inhibitors of ERK kinases activity, and are very effective in the treatment of melanoma with BRAF V600 mutation. Indeed, unexpectedly, the inventors have discovered that specific salts of Compound 1 effectively inhibit cell growth in a melanoma with BRAF V600 mutation.

[0034] Further, they have good solubility, as demonstrated in the following examples.Advantageously, the solubility of the salts according to the invention is increased in water, compared to the free base. The pH sensitivity of the solubility is further decreased, which allows a good absorption of the compound in the intestinal tract.

[0035] Furthermore, the obtention of salts according to the invention advantageously ensures the reproducibility of the compound, by defining specific crystal forms, and thus controlling its synthesis.

[0036] As will be seen below, these salts have utility in the treatment of melanoma with BRAF V600 mutation.

[0037] Another subject concerns the salts of the compound of formula (I) for their use especially in medicaments or in pharmaceutical compositions.

[0038] According to another aspect, the invention relates to a pharmaceutical composition comprising a specific salt and / or solvate thereof of Compound 1 as defined above, and at least one pharmaceutically acceptable excipient, for use in a method for treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0039] According to another aspect, the invention relates to a method of treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, comprising administering to the patient a composition comprising a specific salt and / or solvate thereof of Compound 1, as defined above.

[0040] Abbreviations and Definitions

[0041] In the context of the present invention, the following abbreviations and empirical formulae are used:

[0042] ACN Acetonitrile

[0043] ATP Adenosine 5 ’-triphosphate

[0044] AUC Area Under the Curve

[0045] BID Bis in die (twice a day)

[0046] Brij-35 Polyoxyethyleneglycol dodecyl ether

[0047] Cl 8 column Reversed-phase C18 column

[0048] CaCh Calcium Chloride

[0049] CMC Carboxymethyl Cellulose

[0050] DABCO 1,4-Diazabicyclo[2.2.2]octane

[0051] DCM Dichloromethane

[0052] DMF Dimethylformamide

[0053] DMEM Dulbecco’s Modified Eagle MediumDMSO Dimethylsulfoxide

[0054] DSC Differential Scanning Calorimetry

[0055] DVS Dynamic Vapor Sorption

[0056] °C Degree Celsius

[0057] ee Enantiomeric excess

[0058] EGTA Egtazic acid

[0059] Eq Equivalent

[0060] Et2O Diethyl ether

[0061] EtOAc Ethyl acetate

[0062] EtOH Ethanol

[0063] FaSSIF Fasted State Simulated Intestinal Fluid FaSSGF Fasted State Simulated Gastric Fluid

[0064] FBS Fetal bovine serum

[0065] FT-IR Fourier Transform Infrared Spectroscopy g gram(s)

[0066] h hour(s)

[0067] HBSS Hanks’ Balanced Salt Solution

[0068] HC1 Hydrochloric acid

[0069] HEPES (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid hERG Human Ether-a-go-go-Related gene

[0070] HPLC High performance liquid chromatography

[0071] IR Infrared Spectroscopy

[0072] IMDM Iscove's Modified Dulbecco's Medium

[0073] K2CO3 Potassium carbonate

[0074] KC1 Potassium Chloride

[0075] KF Potassium Fluoride

[0076] KOH Potassium Hydroxide

[0077] LC / MS Liquid chromatography / mass spectrometry LiAlH4 Lithium aluminum hydride

[0078] LiHMDS Lithium bis(trimethylsilyl)amide

[0079] M Mole(s) per liter

[0080] MAPK Mitogen-Activated Protein Kinase

[0081] MeCN Acetonitrile

[0082] MeOH MethanolMEM Minimum Essential Medium

[0083] mg Milligram(s)

[0084] MH+ Pseudo-molecular ion (positive ion mode in mass spectrometry) MHz Megahertz

[0085] MS Mass Spectrometry

[0086] pL Microliter(s)

[0087] MgCh Magnesium Chloride

[0088] mL Milliliter(s)

[0089] mmol Millimole(s)

[0090] mol Mole(s)

[0091] MPA Mobile Phase A

[0092] MPB Mobile Phase B

[0093] NaCl Sodium chloride

[0094] Na2CO3Sodium carbonate

[0095] NaHCO3Sodium hydrogen carbonate

[0096] NaOH Sodium hydroxide

[0097] Na2SO4Sodium sulfate

[0098] NEAA Non-Essential Amino Acid

[0099] NH4C1 Ammonium chloride

[0100] NMR Nuclear Magnetic Resonance

[0101] OS Overall Survival

[0102] PDX Patient-derived xenograft

[0103] p.o. per os (orally)

[0104] PSD Particle Size Distribution

[0105] RH Relative Humidity

[0106] RuPhos 2-Di cyclohexylphosphino-2 ’, 6 ’ -dii sopropoxybiphenyl RuPhos Pd G2 Chloro(2-dicyclohexylphosphino-2’,6’-diisopropoxy-l,r-bi- phenyl)[2-(2’ -amino- 1,1’ -biphenyl)]palladium(II) SDS Sodium dodecyl sulfate

[0107] TBAF Tetrabutylammonium fluoride

[0108] TEA Triethylamine

[0109] TG Thermal Gravimetric

[0110] TGA Thermogravimetric Analysis

[0111] THF TetrahydrofuranUV Ultraviolet

[0112] XRPD X-ray powder Diffraction

[0113] SOC Standard Of Care

[0114] TGI Tumor Growth Inhibition

[0115] The articles “a” and “an” may be used herein to refer to one or to more than one (i.e., at least one) of the grammatical objects of the article. By way of example “a mutation” means one mutation or more than one mutation.

[0116] In the meaning of the present invention, a “kinase inhibitor” is intended to mean a compound that reduces or suppresses the activity of the targeted kinase, as compared with said activity determined without said inhibitor.

[0117] As used herein, the term “room temperature” refers to a temperature ranging from 15 °C to 30 °C, more particularly from 18 °C to 25 °C.

[0118] The term “pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes what is acceptable for veterinary as well as human pharmaceutical use.

[0119] A hydrochloride salt is a salt of hydrochloric acid.

[0120] A maleate salt is a salt of maleic acid.

[0121] An esylate salt is a salt of ethanesulfonic acid, also known as esylic acid.

[0122] An oxoglutarate salt is a salt of oxoglutaric acid, also known as 2-oxoglutaric acid or a-ketoglutaric acid.

[0123] A malonate salt is a salt of malic acid, also known as 2-hydroxybutanedioic acid.

[0124] Within the meaning of the invention, an oxalate salt is a salt of oxalic acid, also known as ethanedioic acid.

[0125] Within the meaning of the invention, a besylate salt is a salt of benzenesulfonic acid, also known as besylic acid.

[0126] Within the meaning of the invention, a napsylate salt is a salt of naphthalene sulfonic acid. All BRAF V600 mutations are Class I BRAF mutations, including especially V600A, V600D, V600E, V600G, V600K, V600L, V600M, and V600R mutations, preferably V600E and V600D, most preferably V600E.

[0127] “Pharmaceutically acceptable solvate” refers to a composition of matter that is the solvent addition form. In some embodiments, solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and are formed during the process of making withpharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, alcoholates are formed when the solvent is an alcohol. The compounds provided herein exist in either unsolvated or as solvated form.

[0128] As used herein, “pharmaceutically acceptable excipient(s)” include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically acceptable for administration to a mammal, such as a human being, while retaining the pharmacological activity of the active compound(s). Except insofar as any conventional excipient is incompatible with the active compounds, its use in a medicament or pharmaceutical composition of the invention is contemplated.

[0129] An “effective dose'' means an amount sufficient to induce a positive modification in the condition to be regulated or treated, but low enough to avoid serious side effects. An effective amount may vary with the pharmaceutical effect to obtain or with the particular condition being treated, the age and physical condition of the end user, the severity of the condition being treated, the duration of the treatment, the nature of other treatments, the specific compound or composition employed, the route of administration, and like factors.

[0130] As used herein, in the context of the present disclosure, the terms “treat”, “treatment” and the like refer to relief from or alleviation of pathological processes in human. In the context of the present disclosure, insofar as it relates to any of the other conditions recited herein, the terms “treat”, “treatment”, and the like refer to relieving or alleviating one or more symptoms associated with melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0131] Other features, properties and advantages of the invention will emerge more clearly from the description and examples that follow.

[0132] BRIEF DESCRIPTION OF THE FIGURES

[0133] Figure 1 is a X-ray powder diagram of a mono-hydrochloride anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-U / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one (see example 12).

[0134] Figure 2 is a X-ray powder diagram of a hydrochloride hydrate salt of (5 -l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-U / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one (see example 13).Figure 3 is a X-ray powder diagram of a maleate anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one (see example 14).

[0135] Figure 4 is a X-ray powder diagram of an esylate anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (see example 15).

[0136] Figure 5 is a X-ray powder diagram of an oxoglutarate anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(U7)-one (see example 16).

[0137] Figure 6 is a X-ray powder diagram of a malonate anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (see example 17).

[0138] Figure 7 is a X-ray powder diagram of an oxalate anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (see example 18).

[0139] Figure 8 is a X-ray powder diagram of a besylate anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (see example 19).

[0140] Figure 9 is a X-ray powder diagram of a napsylate anhydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (see example 20).

[0141] DETAILED DESCRIPTION

[0142] As explained above, the specific salts in accordance with the present disclosure demonstrate improved solubility in comparison to the solubility of free base form.

[0143] As mentioned above, an object of the present invention is the use of a pharmaceutically acceptable salt, and solvate thereof, of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (Compound 1) of the formula (I) below:

[0144]

[0145] (I)

[0146] wherein said salt is selected from a hydrochloride salt, a maleate salt, an esylate salt, an oxoglutarate salt, a malonate salt and an oxalate salt, in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0147] According to a preferred embodiment, the salt is a hydrochloride salt of (5)-l-(l-(3-chlorophenyl)-2-(dirnethylarnino)ethyl)-4-(5-rnorpholino-lZZ-pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(lJ7)-one.

[0148] According to a preferred embodiment, the salt is a mono-hydrochloride anhydrate salt or a hydrochloride hydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one.

[0149] According to a preferred embodiment, the salt is a maleate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one. According to a preferred embodiment, the salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one is selected from a hydrochloride salt and a maleate salt.

[0150] According to a preferred embodiment, the salt is an esylate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one. According to a preferred embodiment, the salt is an oxoglutarate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one.

[0151] According to a preferred embodiment, the salt is a malonate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one. According to a preferred embodiment, the salt is an oxalate salt of fS')- l-( l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one. According to a preferred embodiment, said pharmaceutically acceptable salt is anhydrous and is selected from anhydrous crystalline (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one hydrochloride salt, maleate salt, esylate salt, oxoglutarate salt, malonate salt and oxalate salt.According to a preferred embodiment, a pharmaceutically acceptable salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(U7)-one as defined in the present invention is selected from anhydrous crystalline hydrochloride salt, anhydrous crystalline maleate salt, anhydrous crystalline esylate salt, anhydrous crystalline oxoglutarate salt, anhydrous crystalline malonate salt and anhydrous crystalline oxalate salt.

[0152] According to a preferred embodiment, said pharmaceutically acceptable salt is crystalline (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one hydrochloride hydrate salt.

[0153] According to a preferred embodiment, a pharmaceutically acceptable salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one as defined in the present invention is selected from anhydrous crystalline hydrochloride salt, crystalline hydrochloride hydrate salt, anhydrous crystalline maleate salt, anhydrous crystalline esylate salt, anhydrous crystalline oxoglutarate salt, anhydrous crystalline malonate salt and anhydrous crystalline oxalate salt.

[0154] According to a preferred embodiment, a pharmaceutically acceptable salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one as defined in the present invention is selected from anhydrous crystalline hydrochloride salt and anhydrous crystalline maleate salt.

[0155] The pharmaceutically acceptable salts of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one may be for example characterized by X-Ray Powder Diffraction (XRPD) and by Differential Scanning Calorimetry (DSC).

[0156] According to a preferred embodiment, the present invention concerns the use of an anhydrous crystalline hydrochloride salt, preferably anhydrous crystalline mono-hydrochloride salt, of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-U / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(l J7)-one (Compound 1), in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0157] More particularly, anhydrous crystalline hydrochloride salt of (A')-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one has a powder X-ray diffractogram displaying peaks expressed as degree 2-Theta angle at 11.50; 13.00; 15.23; 15.72; 16.10; 16.55; 17.46; 17.86; 18.29; 19.99; 22.23; 22.99; 23.15; 25.18 and30.76 (each time ±0.2), as illustrated in figure 1 (powder X-ray diffractogram) and / or has a single endotherm with an onset temperature of 258.0°C (±2°C).

[0158] A characteristic X-ray powder diffractogram of an anhydrous crystalline hydrochloride salt of (5)- 1 -( 1 -(3 -chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino- l / / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one can be given in figure 1 and its characteristic signals are summarized in the following table:

[0159] Table 1

[0160] Angle (2-Theta) Relative intensity

[0161] (each time ±0.2) (%)

[0162] 11.50 28.33

[0163] 13.00 23.40

[0164] 15.23 78.30

[0165] 15.72 42.35

[0166] 16.10 24.91

[0167] 16.55 26.60

[0168] 17.46 20.07

[0169] 17.86 31.95

[0170] 18.29 21.53

[0171] 19.99 47.64

[0172] 22.23 86.86

[0173] 22.99 100.00

[0174] 23.15 76.38

[0175] 25.18 34.60

[0176]

[0177] 30.76 19.26

[0178] According to a preferred embodiment, the anhydrous crystalline hydrochloride salt of (S)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(l 7 / )-one presents a powder X-ray diffractogram displaying at least one peak, in particular at least two peaks, preferably at least five peaks, and more preferably at least ten peaks, expressed as degree 2-Theta angle selected from 11.50; 13.00; 15.23; 15.72; 16.10; 16.55; 17.46; 17.86; 18.29; 19.99; 22.23; 22.99; 23.15; 25.18 and 30.76 (each time ±0.2). According to a preferred embodiment, the anhydrous crystalline hydrochloride salt of (S)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has an XRPD substantially similar to that depicted in figure 1.

[0179] According to a preferred embodiment, the present invention concerns the use of a crystalline hydrochloride hydrate salt of fS')-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (Compound 1), in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.More particularly, crystalline hydrochloride hydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one has a powder X-ray diffractogram displaying peaks expressed as degree 2-Theta angle at 8.71; 12.14; 13.15; 17.66; 18.22; 18.73; 20.64; 22.16; 23.19; 23.74; 24.39; 25.18; 25.71; 26.90 and 27.49 (each time ±0.2), as illustrated in figure 2 (powder X-ray diffractogram) and / or has an endotherm with onset temperatures at 85.3°C, 194.5°C and 255.5°C (±2°C).

[0180] A characteristic X-ray powder diffractogram of a crystalline hydrochloride hydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one can be given in figure 2 and its characteristic signals are summarized in the following table:

[0181] Table 2

[0182] Angle (2-Theta) Relative intensity

[0183] (each time ±0.2) (%)

[0184] 8.71 100.00

[0185] 12.14 14.91

[0186] 13.15 15.76

[0187] 17.66 51.56

[0188] 18.22 7.78

[0189] 18.73 10.09

[0190] 20.64 6.65

[0191] 22.16 8.89

[0192] 23.19 9.82

[0193] 23.74 5.37

[0194] 24.39 14.41

[0195] 25.18 11.70

[0196] 25.71 8.87

[0197] 26.90 23.02

[0198]

[0199] 27.49 6.62

[0200] According to a preferred embodiment, the crystalline hydrochloride hydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2( l 7 / )-one presents a powder X-ray diffractogram displaying at least one peak, in particular at least two peaks, preferably at least five peaks, and more preferably at least ten peaks, expressed as degree 2-Theta angle selected from 8.71; 12.14; 13.15; 17.66; 18.22; 18.73; 20.64; 22.16; 23.19; 23.74; 24.39; 25.18; 25.71; 26.90 and 27.49 (each time ±0.2).

[0201] According to a preferred embodiment, the crystalline hydrochloride hydrate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has an XRPD substantially similar to that depicted in figure 2.According to a preferred embodiment, the present invention concerns the use of an anhydrous crystalline maleate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one (Compound 1), in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation. More particularly, anhydrous crystalline maleate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one has a powder X-ray diffractogram displaying peaks expressed as degree 2-Theta angle at 3.80; 10.21; 15.42; 15.89; 16.55; 16.72; 17.42; 19.69; 19.91; 20.47; 21.98; 22.93; 24.65 and 25.26 (each time ±0.2), as illustrated in figure 3 (powder X-ray diffractogram) and / or has a single endotherm with an onset temperature of 185.0°C (±2°C).

[0202] A characteristic X-ray powder diffractogram of an anhydrous crystalline maleate salt of CS')- I-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one can be given in figure 3 and its characteristic signals are summarized in the following table:

[0203] Table 3

[0204] Angle (2-Theta) Relative intensity

[0205] (each time ±0.2) (%)

[0206] 3.80 25.98

[0207] 10.21 29.71

[0208] 15.42 100.00

[0209] 15.89 36.77

[0210] 16.55 19.14

[0211] 16.72 22.95

[0212] 17.42 39.08

[0213] 19.69 21.77

[0214] 19.91 24.48

[0215] 20.47 27.67

[0216] 21.98 66.78

[0217] 22.93 62.05

[0218] 24.65 34.85

[0219]

[0220] 25.26 30.99

[0221] According to a preferred embodiment, the anhydrous crystalline maleate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2( l 7 / )-one presents a powder X-ray diffractogram displaying at least one peak, in particular at least two peaks, preferably at least five peaks, and more preferably at least ten peaks, expressed as degree 2-Theta angle selected from 3.80; 10.21; 15.42; 15.89; 16.55; 16.72; 17.42; 19.69; 19.91; 20.47; 21.98; 22.93; 24.65 and 25.26 (each time ±0.2).According to a preferred embodiment, the anhydrous crystalline maleate salt of (5)-l-(l-(3-chlorophenyl)-2-(dirnethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(U7)-one has an XRPD substantially similar to that depicted in figure 3.

[0222] According to a preferred embodiment, the present invention concerns the use of an anhydrous crystalline esylate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3 -Z>]pyri din-3 -yl)pyridin-2(177)-one (Compound 1), in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0223] More particularly, anhydrous crystalline esylate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one has a powder X-ray diffractogram displaying peaks expressed as degree 2-Theta angle at 3.37; 11.43; 12.06; 12.51; 14.69; 15.31; 15.69; 17.18; 17.46; 17.69; 22.11; 22.88; 24.00; 25.08 and 25.91 (each time ±0.2), as illustrated in figure 4 (powder X-ray diffractogram) and / or has an endotherm with onset temperatures of 197.0°C and 247°C (±2°C).

[0224] A characteristic X-ray powder diffractogram of an anhydrous crystalline esylate salt of CS')- I-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one can be given in figure 4 and its characteristic signals are summarized in the following table:

[0225] Table 4

[0226] Angle (2-Theta) Relative intensity

[0227] (each time ±0.2) (%)

[0228] 3.37 14.66

[0229] 11.43 25.4

[0230] 12.06 38.81

[0231] 12.51 33.85

[0232] 14.69 57.01

[0233] 15.31 63.13

[0234] 15.69 40.5

[0235] 17.18 36.65

[0236] 17.46 21.05

[0237] 17.69 28.18

[0238] 22.11 35.39

[0239] 22.88 100

[0240] 24.00 77.25

[0241] 25.08 30.41

[0242]

[0243] 25.91 27.34

[0244] According to a preferred embodiment, the anhydrous crystalline esylate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2( l 7 / )-one presents a powder X-ray diffractogram displaying at least one peak, inparticular at least two peaks, preferably at least five peaks, and more preferably at least ten peaks, expressed as degree 2-Theta angle selected from 3.37; 11.43; 12.06; 12.51; 14.69; 15.31; 15.69; 17.18; 17.46; 17.69; 22.11; 22.88; 24.00; 25.08 and 25.91 (each time ±0.2).

[0245] According to a preferred embodiment, the anhydrous crystalline esylate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has an XRPD substantially similar to that depicted in figure 4.

[0246] According to a preferred embodiment, the present invention concerns the use of an anhydrous crystalline oxoglutarate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (Compound 1), in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation. More particularly, anhydrous crystalline oxoglutarate salt of (A')-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has a powder X-ray diffractogram displaying peaks expressed as degree 2-Theta angle at 9.90; 10.97; 11.62; 12.87; 14.91; 15.37; 16.42; 18.21; 18.77; 19.38; 18.89; 21.31; 22.03; 22.29; 23.35; 23.80 and 24.26 (each time ±0.2), as illustrated in figure 5 (powder X-ray diffractogram) and / or has an endotherm with onset temperatures of 177.0°C and 250°C (±2°C).

[0247] A characteristic X-ray powder diffractogram of an anhydrous crystalline oxoglutarate salt of (5)- 1 -( 1 -(3 -chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino- 17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one can be given in figure 5 and its characteristic signals are summarized in the following table:

[0248] Table 5Angle (2-Theta) Relative intensity

[0249] (each time ±0.2) (%)

[0250] 9.90 27.90

[0251] 10.97 36.00

[0252] 11.62 60.11

[0253] 12.87 44.30

[0254] 14.91 60.25

[0255] 15.37 100.00

[0256] 16.42 54.82

[0257] 18.21 24.92

[0258] 18.77 23.18

[0259] 19.38 25.06

[0260] 19.89 40.40

[0261] 21.31 44.59

[0262] 22.03 36.55

[0263] 22.29 33.08

[0264] 23.35 28.46

[0265] 23.80 55.01

[0266]

[0267] 24.26 48.80

[0268] According to a preferred embodiment, the anhydrous crystalline oxoglutarate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l7 / -pyrrolo[2,3- / i]pyri din-3-yl)pyridin-2(l 7 / )-one presents a powder X-ray diffractogram displaying at least one peak, in particular at least two peaks, preferably at least five peaks, and more preferably at least ten peaks, expressed as degree 2-Theta angle selected from 9.90; 10.97; 11.62; 12.87; 14.91; 15.37; 16.42; 18.21; 18.77; 19.38; 18.89; 21.31; 22.03; 22.29; 23.35; 23.80 and 24.26 (each time ±0.2). According to a preferred embodiment, the anhydrous crystalline oxoglutarate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has an XRPD substantially similar to that depicted in figure 5.

[0269] According to a preferred embodiment, the present invention concerns the use of an anhydrous crystalline malonate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one (Compound 1), in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation. More particularly, anhydrous crystalline malonate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has a powder X-ray diffractogram displaying peaks expressed as degree 2-Theta angle at 8.51; 12.48; 14.96; 15.30; 17.04; 18.69; 19.61; 23.07; 23.93; 24.54 and 26.80 (each time ±0.2), as illustrated in figure 6 (powder X-ray diffractogram) and / or has an endotherm with onset temperatures of 125.0°C and 259°C (±2°C).A characteristic X-ray powder diffractogram of an anhydrous crystalline malonate salt of fS')- l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l7 / -pyrrolo[2,3- / i]pyridin-3-yl)pyridin-2(U7)-one can be given in figure 6 and its characteristic signals are summarized in the following table:

[0270] Table 6

[0271] Angle (2-Theta) Relative intensity

[0272] (each time ±0.2) (%)

[0273] 8.51 46.24

[0274] 12.48 51.92

[0275] 14.96 56.83

[0276] 15.30 37.30

[0277] 17.04 44.16

[0278] 18.69 54.21

[0279] 19.61 34.44

[0280] 23.07 68.57

[0281] 23.93 100.00

[0282] 24.54 85.79

[0283]

[0284] 26.80 63.94

[0285] According to a preferred embodiment, the anhydrous crystalline malonate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l / Z-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(l 7 / )-one presents a powder X-ray diffractogram displaying at least one peak, in particular at least two peaks, preferably at least five peaks, and more preferably at least ten peaks, expressed as degree 2-Theta angle selected from 8.51; 12.48; 14.96; 15.30; 17.04; 18.69; 19.61; 23.07; 23.93; 24.54 and 26.80 (each time ±0.2).

[0286] According to a preferred embodiment, the anhydrous crystalline malonate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has an XRPD substantially similar to that depicted in figure 6.

[0287] According to a preferred embodiment, the present invention concerns the use of an anhydrous crystalline oxalate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one (Compound 1), in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation. More particularly, anhydrous crystalline oxalate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one has a powder X-ray diffractogram displaying peaks expressed as degree 2-Theta angle at 9.44; 11.14; 12.03; 12.48; 14.67; 15.28; 15.62; 17.17; 18.99; 19.36; 22.88; 23.13; 25.04; 26.25; 29.92 (each time ±0.2), as illustrated in figure 7 (powder X-ray diffractogram) and / or has an endotherm with onset temperatures of 204.0°C and 251°C (±2°C).A characteristic X-ray powder diffractogram of an anhydrous crystalline oxalate salt of CS')- I-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(U7)-one can be given in figure 7 and its characteristic signals are summarized in the following table:

[0288] Table 7

[0289] Angle (2-Theta) Relative intensity

[0290] (each time ±0.2) (%)

[0291] 9.44 60.07

[0292] 11.14 38.88

[0293] 12.03 24.82

[0294] 12.48 19.18

[0295] 14.67 30.45

[0296] 15.28 34.50

[0297] 15.62 47.21

[0298] 17.17 26.11

[0299] 18.99 25.25

[0300] 19.36 51.40

[0301] 22.88 64.22

[0302] 23.13 65.04

[0303] 25.04 44.18

[0304] 26.25 100.00

[0305]

[0306] 29.92 44.38

[0307] According to a preferred embodiment, the anhydrous crystalline oxalate salt of (5)-l-(l-(3-chlorophenyl)-2-(dirnethylamino)ethyl)-4-(5-morpholino-lJ / -pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(l 7 / )-one presents a powder X-ray diffractogram displaying at least one peak, in particular at least two peaks, preferably at least five peaks, and more preferably at least ten peaks, expressed as degree 2-Theta angle selected from 9.44; 11.14; 12.03; 12.48; 14.67; 15.28; 15.62; 17.17; 18.99; 19.36; 22.88; 23.13; 25.04; 26.25; 29.92 (each time ±0.2).

[0308] According to a preferred embodiment, the anhydrous crystalline oxalate salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-17 / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(177)-one has an XRPD substantially similar to that depicted in figure 7.

[0309] PREPARATION OF THE SALTS

[0310] Firstly, the compound of formula (I) may be prepared according to well-known methods by the skilled artisan, as illustrated in examples that follow.

[0311] Preferably, the compound of formula (I) may be prepared according to the process illustrated in example 1.Herein is further provided a method for preparing a pharmaceutically acceptable salt of CS')-I-(l-(3-chlorophenyl)-2-(dirnethylarnino)ethyl)-4-(5-rnorpholino-lZZ-pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(U7)-one used in the present invention, which comprises the following steps: a) dissolving (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-U / -pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(U7)-one in a solvent or in a mixture of solvents;

[0312] b) adding to the thus obtained mixture of step a) a counter ion under the form of an acid which may be itself already dissolved in a solvent or in a mixture of solvents so as to obtain a (5)-l-(l-(3-chlorophenyl)-2-(dirnethylarnino)ethyl)-4-(5-rnorpholino-lZZ-pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(U7)-one:counter ion molar ratio comprised between 3:1 and 1:2, particularly between 5:2 and 1:2; more particularly between 2: 1 and 1:2, and still more particularly is of 2: 1 or 1:1;

[0313] c) optionally evaporating the solvent(s) at a temperature comprised between 0°C and the boiling point of the selected solvent(s) or mixture of solvent(s) of step a) and step b), particularly between room temperature and 60°C, more particularly between room temperature and 50°C; d) optionally adding a solvent or a mixture of solvents,

[0314] e) applying a temperature program;

[0315] f) optionally filtrating; and

[0316] g) then optionally drying at a temperature comprised between room temperature and 60°C in order to obtain the desired salt of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyridin-3-yl)pyridin-2(lJ7)-one.

[0317] The solvent(s) used in step a), step b) and step d) is(are) any solvent conventionally used in crystallization step, particularly is(are) organic solvents, and may be selected from water, alcohol solvents such as 1 -propanol, 2-propanol, ethanol, methanol, 1 -butanol and 2-butanol, glycol solvents such as propylene glycol, ketone solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone, ether solvents such as 1,4-di oxane, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether and methyl tert-butyl ether, acetate solvents such as ethyl acetate and isopropyl acetate, aromatic solvents such as toluene, hydrocarbons solvents such as N-heptane, chlorinated solvents such as dichloromethane and chloroform, organosulfur solvents such as dimethyl sulfoxide (DMSO), amine and amide solvents such as N-methyl-2-pyrrolidone (NMP), dimethylacetamide and acetonitrile, and mixtures thereof. According to a preferred embodiment, the solvent(s) used in step a), step b) and step d) is(are) selected from 2-propanol, acetone, acetonitrile, ethanol, ethyl acetate, tetrahydrofuran, dichloromethane, methanol, 1,4-di oxane, and mixtures thereof.According to a preferred embodiment, the solvent used in step a), step b) and step d) is 2-propanol, acetone, acetonitrile, ethanol, ethyl acetate, tetrahydrofuran, dichloromethane, methanol, and mixtures thereof.

[0318] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is 2-propanol.

[0319] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is acetone.

[0320] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is acetonitrile.

[0321] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is ethanol.

[0322] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is ethyl acetate.

[0323] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is tetrahydrofuran.

[0324] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is dichloromethane.

[0325] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is DCM / Methanol.

[0326] According to a preferred embodiment, the solvent used in step a), step b) and / or step d) is 1,4-dioxane.

[0327] The skilled person would know how to determine the more appropriate solvent(s) in each step a), b) and d) so as to obtain the desired pharmaceutically acceptable salt.

[0328] According to one embodiment, the solvent(s) used in step a), step b) and / or step d) is(are) the same.

[0329] According to another embodiment, the solvent(s) used in step a), step b) and / or step d) is(are) different.

[0330] According to a preferred embodiment, the counter ion under the form of an acid of step b) is selected from hydrochloric acid, maleic acid, ethanesulfonic acid, ketoglutaric acid, malonic acid, and oxalic acid.

[0331] According to a preferred embodiment, the counter ion under the form of an acid of step b) is hydrochloric acid.

[0332] According to a preferred embodiment, the counter ion under the form of an acid of step b) is selected from maleic acid.According to a preferred embodiment, the counter ion under the form of an acid of step b) is ethanesulfonic acid.

[0333] According to a preferred embodiment, the counter ion under the form of an acid of step b) is ketoglutaric acid.

[0334] According to a preferred embodiment, the counter ion under the form of an acid of step b) is malonic acid.

[0335] According to a preferred embodiment, the counter ion under the form of an acid of step b) is oxalic acid.

[0336] According to a preferred embodiment, a process for the preparation of a hydrochloride salt according to the invention comprises the step of adding hydrochloric acid to a compound of formula (I) in a solvent chosen from 2-propanol, acetone, acetonitrile, ethanol, ethyl acetate, 1,4-di oxane and tetrahydrofuran, in particular chosen from 2-propanol, acetone, acetonitrile and ethanol, and preferably chosen from 2-propanol and acetone.

[0337] According to a preferred embodiment, a process for the preparation of a mono-hydrochloride anhydrous salt according to the invention comprises the step of adding hydrochloric acid to a compound of formula (I) in a solvent chosen from 2-propanol, acetone, acetonitrile, ethanol, ethyl acetate, and tetrahydrofuran, in particular chosen from 2-propanol, acetone, acetonitrile and ethanol, and preferably chosen from 2-propanol and acetone.

[0338] According to a preferred embodiment, a process for the preparation of a hydrochloride hydrate salt according to the invention comprises the step of adding hydrochloric acid to a compound of formula (I) in a solvent chosen from 2-propanol, acetone, ethanol, 1,4-di oxane and tetrahydrofuran.

[0339] According to a preferred embodiment, a process for the preparation of a hydrochloride salt according to the invention comprises the step of adding hydrochloric acid to a compound of formula (I) in a solvent chosen from 2-propanol, acetone, acetonitrile, ethanol, ethyl acetate, 1,4 dioxane and tetrahydrofuran, in particular chosen from 2-propanol, acetone, acetonitrile and ethanol, and preferably chosen from 2-propanol and acetone.

[0340] According to a preferred embodiment, a process for the preparation of a maleate salt according to the invention comprises the step of adding maleic acid to a compound of formula (I) in a solvent chosen from 2-propanol, acetone, acetonitrile and ethyl acetate, in particular chosen from 2-propanol, acetone and acetonitrile, and preferably in 2-propanol.

[0341] According to a preferred embodiment, a process for the preparation of an esylate salt according to the invention comprises the step of adding ethanesulfonic acid to a compound of formula (I)in a solvent chosen from acetone, acetonitrile, ethyl acetate and tetrahydrofuran, in particular chosen from acetone and ethyl acetate, and preferably in acetone.

[0342] According to a preferred embodiment, a process for the preparation of an oxoglutarate salt according to the invention comprises the step of adding ketoglutaric acid to a compound of formula (I) in a solvent chosen from 2-propanol, acetone, acetonitrile, ethanol, ethyl acetate, dichloromethane, methanol, and a mixture of dichloromethane and methanol, in particular chosen from ethanol, 2-propanol, acetonitrile, dichloromethane and a mixture of dichloromethane and methanol, and preferably chosen from dichloromethane and a mixture of dichloromethane and methanol.

[0343] According to a preferred embodiment, a process for the preparation of a malonate salt according to the invention comprises the step of adding malonic acid to a compound of formula (I) in a solvent chosen from dichloromethane, a mixture of dichloromethane and methanol, and 2-propanol, and preferably chosen from dichloromethane and a mixture of dichloromethane and methanol.

[0344] According to a preferred embodiment, a process for the preparation of an oxalate salt according to the invention comprises the step of adding oxalic acid to a compound of formula (I) in methanol.

[0345] According to a preferred embodiment, the process for the preparation of the salts according to the invention is carried out at a temperature ranging from 40°C to 80°C and more particularly ranging from 45°C to 60°C.

[0346] According to a preferred embodiment, the process for the preparation of the salts according to the invention comprises a step of cooling at a temperature ranging from -5°C to 30°C and more particularly from 5°C to room temperature.

[0347] Pharmaceutical

[0348]

[0349] As specified previously and clearly illustrated by the following examples, the salts according to the present invention are useful as inhibitors of the ERK kinases activity in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation. The present invention therefore provides a method for preventing and / or treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, comprising at least a step of administering to an individual in need thereof at least an effective amount of at least one salt as described above.

[0350] The present invention also provides the salts as described above for their use for preventing and / or inhibiting and / or treating, preferably for preventing and / or treating, more preferably fortreating, melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0351] The present invention also provides the use of salts as described above for preventing and / or inhibiting and / or treating, preferably for preventing and / or treating, more preferably for treating, melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0352] In certain embodiments, the salts described herein are administered as a pure chemical.

[0353] In other embodiments, the salts described herein are combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable or acceptable excipient, a physiologically suitable or acceptable excipient, or a physiologically suitable or acceptable carrier), selected on the basis of a chosen route of administration and standard pharmaceutical practice.

[0354] Provided herein is a pharmaceutical composition comprising the salts as described herein, together with one or more pharmaceutically acceptable carriers.

[0355] The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject or patient) of the composition.

[0356] One embodiment provides a method of preparing a pharmaceutical composition comprising mixing the salts described herein, or a pharmaceutically acceptable salt, and / or solvate thereof, and a pharmaceutically acceptable carrier.

[0357] The salts used according to the present invention may be used alone or combined with chemotherapeutic agents or radiotherapeutic regimen.

[0358] Thus, according to one embodiment, a method of the invention may comprise the step of administering a salt used according to the present invention, separately, sequentially, or simultaneously with a chemotherapeutic agent.

[0359] As examples of chemotherapeutic agents that may be suitable for the invention, one may mention chemotherapeutic agents chosen from alkylating agents, intercalating agents, antimicrotubule agents, antimitotics, antimetabolites, antiproliferative agents, antibiotics, immunomodulatory agents, anti-inflammatories, kinases inhibitors, anti -angiogenic agents, antivascular agents, oestrogenic and androgenic hormones.

[0360] A radiotherapeutic regimen may be administrated by exposing an individual in need thereof to a source of ionizing radiation such as X-ray, gamma-ray or beta-ray.According to another of its aspects, the present invention relates to a pharmaceutical composition comprising at least one salt used according to the present invention and at least one pharmaceutically acceptable excipient.

[0361] The salts used according to the present invention may be used for the preparation of medicaments, in particular of medicaments for treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0362] Thus, according to yet another of its aspects, the present invention relates to a medicament comprising at least one salt used according to the present invention.

[0363] The pharmaceutical compositions may contain more particularly an effective dose of at least one salt used according to the present invention.

[0364] A salt used according to the present invention may be administered in an effective dose by any of the accepted modes of administration in the art.

[0365] In one embodiment, a salt used according to the present invention may be used in a composition intended to be administrated by oral, nasal, sublingual, aural, ophthalmic, topical, rectal, vaginal, urethral, or parenteral injection route.

[0366] The route of administration and the galenic formulation will be adapted by one skilled in the art pursuant to the desired pharmaceutical effect.

[0367] In a preferred embodiment, a salt used according to the present invention may be used in a composition intended to be administrated by oral.

[0368] Provided herein is the method wherein the pharmaceutical composition is administered orally. One of ordinary skill in the art of therapeutic formulations will be able, without undue experimentation and in reliance upon personal knowledge, to ascertain a therapeutically effective dose of a salt used according to the present invention for a given indication.

[0369] A pharmaceutical composition may be formulated with any known suitable pharmaceutically acceptable excipients according to the dose, the galenic form, the route of administration and the likes.

[0370] A medicament or pharmaceutical composition may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, sprays, ointments, gels, creams, sticks, lotions, pastes, soft and hard gelatine capsules, suppositories, sterile injectable solutions, sterile packages powders and the like.

[0371] Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract.In some embodiments, suitable nontoxic solid carriers are used (see, e.g., Remington: The Science and Practice of Pharmacy (Gennaro, 21stEd. LWW Pub., Philadelphia, PA (2005)). The dose of the composition comprising the salts described herein, or a pharmaceutically acceptable salt, and / or solvate thereof, differs depending upon the subject or patient’s (e.g., human) condition.

[0372] In some embodiments, such factors include general health status, age, and other factors.

[0373] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented).

[0374] An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient’s disease, the particular form of the active ingredient, and the method of administration.

[0375] In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and / or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or a lessening of symptom severity).

[0376] Optimal doses are generally determined using experimental models and / or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient.

[0377] According to one embodiment, a pharmaceutical composition may be intended to be administered separately, sequentially, or simultaneously with an agent useful for the prevention and / or the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, said agent being different from the salts used according to the present invention.

[0378] The applications also include a novel kit-of-parts that is suitable for use in the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation. A kit-of-part may comprise (i) a salt used according to the present invention, and (ii) at least one agent useful for the prevention and / or the treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, said agent being different from said salt. An agent useful for the prevention and / or treatment of melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation may be a chemotherapeutic agent or a radiotherapeutic agent.

[0379] The present invention will be better understood by referring to the following examples which are provided for illustrative purpose only and should not be interpreted as limiting in any manner the instant invention.Medical Uses

[0380] In certain aspects, disclosed herein is a method of treating melanoma with BRAF V600 mutation in an individual in need thereof, comprising administering an effective amount of a salt and / or a solvate thereof described herein to the individual.

[0381] In certain aspects, disclosed herein is a salt and / or a solvate thereof described herein for use in treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0382] In certain aspects, disclosed herein is a salt and / or a solvate thereof described herein for use in the preparation of a medicament for treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0383] One embodiment provides a method of treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, in a patient in need thereof, comprising administering to the patient a salt and / or solvate thereof described herein.

[0384] Another embodiment provides the method, wherein the method is adjuvant therapy following surgical resection.

[0385] Another embodiment provides the method or a salt and / or a solvate thereof described herein for use in treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, wherein the patient has relapsed after prior therapy.

[0386] Another embodiment provides the method or a salt and / or a solvate thereof described herein for use in treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, wherein the patient has acquired resistance to prior therapy.

[0387] Another embodiment provides the method or a salt and / or a solvate thereof described herein for use in treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, wherein the patient is refractory to therapy.

[0388] Another embodiment provides the method or a salt and / or a solvate thereof described herein for use in treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, wherein the pharmaceutically acceptable salt and / or solvate thereof is administered orally.

[0389] One embodiment provides a method of treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation, in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a salt and / or solvate thereof described herein, and at least one pharmaceutically acceptable excipient.Another embodiment provides the method or combination comprising a salt and / or a solvate thereof described herein for use in treating melanoma with BRAF V600 mutation, in particular melanoma with BRAF V600E mutation.

[0390] In another embodiment, the method is adjuvant therapy following surgical resection.

[0391] In another embodiment, the patient has relapsed after prior therapy.

[0392] In another embodiment, the patient has acquired resistance to prior therapy.

[0393] In another embodiment, the patient is refractory to therapy.

[0394] In another embodiment, the pharmaceutical composition comprising a salt and / or solvate thereof described herein is administered orally.

[0395] In another embodiment, the oral administration occurs every other day, once per day, twice per day, or three times per day.

[0396] In a particular embodiment, the oral administration occurs twice per day.

[0397] EXAMPLES

[0398]

[0399] and methods used for the

[0400] Unless otherwise stated, the following equipments and analytical methods are used in the examples.

[0401]

[0402] 1. X-ray Powder Diffraction

[0403] XRPD analysis was carried out on a PANalytical Xpert pro with PIXcel detector (128 channels), scanning the samples between 3 and 35° 29. The material was gently ground with mortar and pestle to release any agglomerates and loaded onto a multi-well plate with Mylar polymer film to support the sample. The multi-well plate was then placed into the diffractometer and analyzed using Cu K radiation (α1 λ = 1.54060 Å; α2 = 1.54443 Å; β = 1.39225 Å; α1 : α2 ratio = 0.5) running in transmission mode (step size 0.0130° 29, step time 18.87s) using 40 kV / 40 mA generator settings. Data were visualized and images generated using the HighScore Plus 4.9 desktop application (PANalytical, 2020).

[0404]

[0405] 2.

[0406] The presence of crystallinity (birefringence) was determined using an Olympus BX53 microscope, equipped with cross-polarising lenses and a Motic camera. Images were captured using Motic Images Plus 3.0. All images were recorded using the 20 x objective, unless otherwise stated.3. Thermogravimetric Analysis / Differential Scanning Calorimetry (TGA / DSC) Approximately 5-10 mg of material was added into a pre-tared open aluminium pan and loaded into a TA Instruments Discovery SDT 650 Auto - Simultaneous DSC and held at room temperature. The sample was then heated at a rate of 10°C / min from 30°C to 400°C during which time the change in sample weight was recorded along with the heat flow response (DSC). Nitrogen was used as the sample purge gas, at a flow rate of 200 cm3 / min.

[0407] 4. Differential Scanning Calorimetry (DSC)

[0408] Approximately 1-5 mg of material was weighed into an aluminium DSC pan and sealed nonhermetically with an aluminium lid. The sample pan was then loaded into a TA Instruments Discovery DSC 2500 differential scanning calorimeter equipped with a RC90 cooler. The sample and reference were heated to a maximum of 300°C at a scan rate of 10°C / min and the resulting heat flow response monitored. The sample was re-cooled to 20°C and then reheated again to a maximum of 300°C all at 10°C / min. Nitrogen was used as the purge gas, at a flow rate of 50 cm3 / min.

[0409] 5. Infrared Spectroscopy (IR)

[0410] Infrared spectroscopy was carried out on a Bruker ALPHA P spectrometer. Sufficient material was placed onto the centre of the plate of the spectrometer and the spectra were obtained using the following parameters:

[0411] - Resolution: 4 cm-1;

[0412] - Background Scan Time: 16 scans;

[0413] - Sample Scan Time: 16 scans;

[0414] - Data Collection: 4000 to 400 cm-1;

[0415] - Result Spectrum: Transmittance;

[0416] - Software: OPUS version 6.

[0417] 6. Nuclear Magnetic Resonance (NMR)

[0418] NMR experiments were performed on a Bruker AVIIIHD spectrometer equipped with a DCH cryoprobe operating at 500.12 MHz for protons. Experiments were performed in deuterated dimethylsulfoxide and each sample was prepared to ca. 10 mM concentration.Chemical shifts expressed in parts per million (ppm), signals expressed as follows: s = singlet, d = doublet, t = triplet, q = quadruplet, sept = septuplet, dd = double doublet, dt = double triplet, m = multiplet or large singlet, br = broad, H = proton.

[0419] 7. Dynamic Vapour Sorption (DVS)

[0420] Approximately 10-20 mg of sample was placed into a mesh vapour sorption balance pan and loaded into a DVS Intrinsic or Advantage dynamic vapour sorption balance by Surface Measurement Systems. The sample was subjected to a ramping profile from 40 - 90% relative humidity (RH) at 10% increments, maintaining the sample at each step until a stable weight had been achieved (dm / dt 0.004%, minimum step length 30 minutes, maximum step length 500 minutes) at 25 °C. After completion of the sorption cycle, the sample was dried using the same procedure to 0 %RH and then a second sorption cycle back to 40 %RH. Two cycles were performed. The weight change during the sorption / desorption cycles were plotted, allowing for the hygroscopic nature of the sample to be determined. XRPD analysis was then carried out on any solid retained.

[0421] 8. High Performance Liquid Chromatography-Ultraviolet Detection (HPLC-UV) Column: Halo C18 100 x 3.0 mm, 2.7 μm

[0422] Column Temperature: 40°C

[0423] Flow Rate: 1.0 mL / min

[0424] Column Pressure at start of Run: 400 Bar

[0425] Injection Volume: 2 pL

[0426] Autosampler Temperature: Ambient (°C)

[0427] Detection parameters: UV at 219 nm

[0428] Sampling Rate: 10 Hz

[0429] Mobile Phase A: 0.1% formic acid in H2O

[0430] Mobile Phase B: 0.1% formic acid in ACN

[0431] Diluent: 50:50 MPA: MPB

[0432] Needle Wash: MPB

[0433] Gradient:

[0434] Table 8Time (minutes) MP A % MP B %

[0435] 0.0 90 10

[0436] 1.0 90 10

[0437] 7.0 0 100

[0438] 10.0 0 100

[0439] 10.1 90 10

[0440]

[0441] 15.0 90 10

[0442] 9. Mass Spectrometry

[0443] Column: X-Bridge C18, 50 mm × 3 mm, 3.5 μm

[0444] Column Temperature: 40°C

[0445] Flow Rate: 1.0 mL / min

[0446] Injection Volume: 1 μL

[0447] Autosampler Temperature: Ambient (°C)

[0448] Detection parameters: UV 210 nm Monitor only UV Scan 190 to 900 nm; MS + / - ESI Fragmentor 135 V

[0449] Mobile Phase A: 0.1% formic acid in water v / v

[0450] Mobile Phase B: 0.1% formic acid in acetonitrile v / v

[0451] Diluent: 50:50 %v / v Acetonitrile: Water

[0452] Gradient:

[0453] Time (minutes) MP A % MP B %

[0454] Table 9

[0455] Time (minutes) MP A % MP B %

[0456] 0 95 5

[0457] 8 5 95

[0458] 10 5 95

[0459] 10.1 95 5

[0460]

[0461] 14 95 5

[0462] Sample Preparation: Working Concentration: 0.1 mg / mL

[0463] 10. pKa Determination

[0464] An automated titrator system with an incorporated UV-Vis spectrometer (SiriusT3TM, Pion Inc.) was used to acquire the spectrometric and / or potentiometric data. The optical system consisted of a photodiode array detector with a deuterium lamp and a fibre optic dip probe. The titrator module consisted of a temperature controller (by Peltier device with in-situ thermocouple), pH electrode, an overhead stirrer, and motorised dispensers for the automatic delivery of assay titrants and reagents via capillaries. The instrumentation was operated usingSiriusT3 Control software (V2.0). Data processing and generation of the reported pKa values was carried out using SiriusT3Refine software (V2.0).

[0465] All experiments are carried out at a controlled temperature 25.0 ± 0.2 °C. The pH range of titration assays is set between pH 2.0 to pH 12.0 unless otherwise stated. Prior to use, 0.5 M KOH base titrant is standardised by the titration of approximately 15 mg of potassium hydrogen phthalate, in triplicate. 0.5 M HCl titrant is subsequently standardised against the base titrant. The assay media for pKa determination is kept at a constant ionic strength of 0.15 M KC1 and under argon atmosphere. The pH electrode is calibrated daily using the Avdeef-Bucher four-parameter equation2. For the determination of pKa values, both pH-metric and UV-metric methods are employed. pKa results will be reported for compounds from data acquired using the UV-metric technique where pKas are shown to exhibit UV activity and pH-metric titrations are performed to supplement UV data. pKa values determined by the pH-metric technique will be provided where pKas cannot be identified using the UV-metric assay. The pKa values obtained from spectrophotometric experiments agree excellently with those derived from potentiometric titrations.

[0466] When cosolvent is incorporated into the assay design, the aqueous pKa values are extrapolated from the apparent pKa values measured in the presence of cosolvents (psKa) using Yasuda-Shedlovsky extrapolation equation; where psKa versus reciprocal function of the dielectric constant (ε) at different cosolvent mixtures is represented. A minimum of three ratios of water / cosolvent are titrated to determine the aqueous pKa values.

[0467] 11. Particle Size Distribution (PSD)

[0468] Particle size distribution analysis was conducted using a Malvern Mastersizer 2000, stirring unit rinsed with 2-propanol before use. The following parameters were used:

[0469] PSD Parameters:

[0470] - Dispersant: 0.05 % w / v Span-85 in Heptane

[0471] - Dispersant Volume: 10 mL

[0472] - Sample Weight: 150 ± 3 mg

[0473] - Analysis Model: General Purpose

[0474] - Particle Shape: Irregular

[0475] - Sensitivity: Normal

[0476] - Particle RI: Fraunhofer

[0477] - Particle Absorption: Fraunhofer

[0478] - Dispersant RI: 1.39- Measurements: 3

[0479] - Background / Measurement Time: 10 seconds

[0480] - Circulation Time Pre-Measurement: 3 minutes

[0481] - Circulation Rate: 2800 rpm

[0482] - Sonication Time: N / A

[0483] - Obscuration Limits: 9-15 %

[0484] Sample Preparation: The bulk sample was gently inverted and rotated to achieve homogeneity. Ca. 150 mg of sample was weighed into a 20 mL scintillation vial and 10 mL of dispersant was added.

[0485] 12. Flash chromatography

[0486] Apparatus: Biotage SP with auto-collector and UV detection (2 wavelengths).

[0487] Normal phase columns: 10, 25 or 120 g Biotage external dry load cartridge kit, packed with Sigma- Aldrich 40-63 pm silica gel.

[0488] Reverse phase column: 30 g Biotage SNAP Cartridges, KP-C18-HS.

[0489] Chiral column: Daicel ChiralFlash IG 100 x 30 mm 20 pM.

[0490] 13. Thermodynamic Solubility Study 1

[0491] Prior starting the solubility test, the purity of the salts was analyzed by HPLC.

[0492] A thermodynamic solubility study was carried out on the salts as follows:

[0493] Approximatively 15-20 mg of each sample was weighed in 2 mL vials. A stirrer bar was added and the sample was placed at 37°C. While stirring the sample, 50 pL of water / buffer was added until dissolution of the salt was observed, or a total of 1 mL was added. Media used: Unbuffered water, FaSSIF (pH 6.5), FaSSGF (pH 1.6). If a clear solution was observed, additional corresponding salt was added until a slurry was obtained. At T = 0 and T = 4 h, the slurries were filtered, the filtrate analyzed by HPLC for concentration determination. pH measurements were taken at each timepoint when sufficient liquor volume permitted it. The solids were analyzed by XRPD material permitting.

[0494] 14. Thermodynamic Solubility Study 2

[0495] Prior to starting the solubility test, the purity of the salts was analyzed by HPLC.

[0496] A thermodynamic solubility study was carried out on the salts as follows:

[0497] Approximatively 25-30 mg of each sample, one sample per buffer, was weighed in 2 mL vials. A stirrer bar was added, and the samples were placed at ca. 37°C. While stirring the sample, 75pL of water / buffer was added until dissolution of the salt or until a total of 1.5 mL was added. If full dissolution was observed, more solid material was added until a slurry was obtained. Media used: Unbuffered water, FaSSIF (pH 6.5), FaSSGF (pH 1.6). At T = 0, T = 4 h and T = 24 h, aliquots of the slurries were filtered. The filtrate was analyzed by HPLC for concentration determination. pH measurements were taken at each timepoint when sufficient volume permitted it. The solids were analyzed by XRPD.

[0498] 15. Seven days stability study

[0499] The 7-day stability assessments were carried out as follows:

[0500] Ca. 10 mg of the corresponding salt was exposed to the following conditions for 7 days: 40°C / 75 %RH; 80°C; Ambient temperature, humidity and light; Slurry in miglyol (15 mg was suspended in 1 mL of miglyol (caprylic / capric triglycerides)).

[0501] After 7 days, the solid materials were analyzed by XRPD for solid form and HPLC for purity.

[0502] 16. Hydration Studies

[0503] The salt hydration studies of salts were carried out using the following procedure:

[0504] 400 pL of the appropriate ethanol: water mixture according to the following Table was added to ca. 10 mg of the corresponding salt. The mixtures were agitated at ambient temperature for 24 h. Solids were isolated by centrifuge filtration and analyzed by XRPD.

[0505] Table 10: Solvent Selection for the Hydration Studies of selected salt Calculated Aw Solvent Mixture

[0506] 0.8 Ethanol: Water 73:27 % v / v

[0507] 0.5 Ethanol: Water 93:7 % v / v

[0508]

[0509] 0.2 Ethanol:Water 98.5:1.5 % v / v

[0510] 17. Salt Disproportionation Studies

[0511] The salt disproportionation studies of the salts were carried out using the following procedure: Ca. 10 mg of each salt was weighed into a 2 mL vial and 500 pL of water was added resulting in formation of a slurry. The vial was agitated at ambient temperature for 24 hours. Any solids were isolated by centrifuge filtration and analyzed by XRPD.

[0512] 18. Three Month Stability Study

[0513] The compounds were set down for a stability study to investigate the chemical and physical stability over a total of three months. The conditions assessed were:

[0514] - Ambient light and temperature (closed vial) at 2 weeks, 1 month and 3 months- 80°C (closed vial) at 2 weeks, 1 month and 3 months

[0515] - 25°C / 60 %RH (open vial) at 2 weeks, 1 month and 3 months

[0516] - 40°C / 75 %RH (open vial) at 2 weeks, 1 month and 3 months

[0517] The samples were analyzed by XRPD and HPLC at each timepoint.

[0518] Example 1: Synthesis of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-

[0519]

[0520] -lH-\,3- / >]pvridin-3- l-one

[0521] Step 1: l-Chloro-3-vinylbenzene

[0522] ax

[0523]

[0524] 10 g (71.1 mmol) of 3 -chlorobenzaldehyde are dissolved in 50 ml of dry THF and the solution is cooled to -10°C with an ice / acetone bath. 17.3 g (48.4 mmol, 1.2 eq) of methyltriphenylphosphonium bromide are added followed by 2.1 g (52.4 mmol, 1.3 eq) of sodium hydride (60% in paraffin oil). The suspension is then stirred at room temperature overnight under argon. The mixture is diluted with 100 ml of Et2O and the precipitate is filtrated on Celite. The filtrate is evaporated under reduced pressure to give an orange residue. Crude mixture is finally purified by flash chromatography using a silica gel column and an Et2O / pentane mixture as eluent (3 / 97). 4.05 g of the title compound are obtained.

[0525] Step 2: 2-(3-Chlorophenyl)oxirane

[0526] C!

[0527]

[0528] r j

[0529] 4.05 g (29.2 mmol) of 1 -chi oro-3 -vinylbenzene (described in the previous step) are dissolved in 6 ml of 1,4-dioxane and 18 ml of water. The solution is cooled to 0°C and 584 pl (10.2 mmol, 1 eq) of acetic acid are added, followed by 1.99 g (11.2 mmol, 1.1 eq) ofTV-bromosuccinimide.Reaction mixture is stirred at 0°C for 5 min then at room temperature for 2h. Mixture is then cooled again to 0°C and a solution of NaOH 2N in water (35.7 mmol, 3.5 eq) is slowly added. The solution is allowed to stir at room temperature for Ih. Reaction mixture is concentrated under reduced pressure and aqueous resulting phase is extracted 3 times with DCM. Combined organic layers are dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a DCM / hexane mixture as eluent (2 / 98). 3.85 g of the title compound are obtained.

[0530] Step 3: l-(3-Chlorophenyl)-2-(dimethylamino)ethan-l-ol

[0531] OH

[0532] S

[0533]

[0534] To a solution of 4.15 g (26.9 mmol) of 2-(3-chlorophenyl)oxirane (described in the previous step) in 14 ml of EtOH 96%, are added 7.38 ml (14.76 mmol, 2 eq) of a solution of dimethylamine (2M in THF). The clear resulting solution is heated under microwave irradiation at 80°C for 30 min. Reaction mixture is then concentrated under vacuum and diluted with water. The solution is extracted 3 times with DCM. Combined organic layers are dried over Na₂SO₄, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 4.06 g of the title compound are obtained.

[0535] Step 4: 2-Chloro-2-(3-chlorophenyl)-7V,7V-dimethylethan-l-amine

[0536]

[0537] 4.06 g (20.3 mmol) of l-(3-chlorophenyl)-2-(dimethylamino)ethan-l-ol (described in the previous step) are dissolved in 15 ml of DCM and placed at 0°C. 2.1 ml (15.1 mmol, 3 eq) of triethylamine are added, followed by 0.781 ml (10.1 mmol, 2 eq) of mesyl chloride. The reaction is stirred at 0°C under argon for 2h. Water is then added and the mixture is decanted. Aqueous layer is extracted 2 times with DCM. Combined organic layers are dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude compound is directly used in the next step without further purification. 4.41 g of the title compound are obtained.Step 5: 4-Bromo-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)pyridin-2(LH)-one

[0538] Br

[0539]

[0540] To a mixture of 0.744 g (4.28 mmol, 1 eq) of 4-bromopyridin-2-(177)-one and 1.39 g (4.28 mmol, 1 eq) of cesium carbonate in 10 ml of dry DMF, is added at 0°C a solution of 4.41 g (20.3 mmol) of 2-chloro-2-(3-chlorophenyl)-7V,7V-dimethylethan-l -amine (described in the previous step) in 5 ml of dry DMF. The solution is then stirred at room temperature for 2h. EtOAc is added, and the mixture is washed 4 times with water and once with brine. Organic layer is dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a deactivated silica gel column and an Hexane / EtOAcmixture as eluent. 5.02 g of the title compound are obtained.

[0541] Step 6: 1 -( 1 -(3-Chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino- 1 -tosyl- 1 / / -pyrrolo [2,3-6] pyridin-3-yl)pyridin-2(LH)-one

[0542]

[0543] 2 g (5.6 mmol) of 4-bromo-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)pyridin-2(177)-one (described in the previous step) and 2.45 g (5.01 mmol, 1.3 eq) of 4-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l-tosyl-U / -pyrrolo[2,3-Z>]pyridin-5-yl)morpholine (described in Step 3) are dissolved in 13 ml of MeCN under argon. Then 13 ml of a solution of Na₂CO₃ 2M are added to give a biphasic mixture which is bubbled with argon for 15 min. 135 mg (0.19 mmol, 0.05 eq) of bis(triphenylphosphine)palladium dichloride are added and the solution was bubbled with argon for another 15 min. The reaction is stirred at 70°C for 2h under argon. Reaction mixture is then diluted with water and EtOAc and then decanted. Aqueous layer is extracted 2 times with EtOAc. Combined organic layer are dried over Na₂SO₄, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using asilica gel column and a DCM / MeOH mixture as eluent. 2.68 g of the title compound are obtained.

[0544] Step 7: l-( l-(3-Chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-LH-pyrrolo[2,3- 6]pyridin-3-yl)pyridin-2(lH)-one

[0545]

[0546] 2.68 g (4.2 mmol) of l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-l-tosyl- l / / -pyrrolo[2,3 -Z>]pyri din-3 -yl)pyridin-2(177)-one (described in the previous step) are dissolved in 15 ml of dry THF under argon. Then 10 ml (10 mmol, 3 eq) of a solution of TBAF (IM in THF) are added and the reaction is stirred at 66°C for Ih under argon. Solvent is removed under reduced pressure and 100 ml of a saturated NaHCO₃ solution are added. Mixture is extracted 3 times with EtOAc. Combined organic layer are dried over Na₂SO₄, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 617 mg of racemate are obtained.

[0547] Step 8: (. S’)- 1 -( 1 -(3-Chloropheny 1 )-2-( dim et hy la ni ino )et hyl )-4-(5-morphol ino- 1 / / -pyrrolo [2,3-6] pyridin-3-yl)pyridin-2(LH)-one

[0548]

[0549] Enantiomers obtained in the previous step are separated by flash chromatography using a Chiralflash IG column and an Hexane / EtOH / DCM / 0.1%TEA mixture as the mobile phase. First fraction to be eluted is the (-) ( / / (-enantiomer, followed by the (+) fS')-enantiomer with ee > 98%. 227 mg of the title compound are obtained starting from 617 mg of racemate.

[0550] MH+: 478.5; 480.6 (M; M+2).'H NMR (DMSO-d6, 400 MHz): 5 12.06 (br s, 1H); 8.17 (d, J=2.4Hz, 1H); 8.10 (d, J=2.3Hz, 1H); 7.76 (d, J=8.0Hz, 1H); 7.70 (d, J=2.4Hz, 1H); 7.47 (s, 1H); 7.44-7.32 (m, 3H); 6.72-6.65 (m, 2H); 6.23-6.13 (m, 1H); 3.84-3.73 (m, 4H); 3.34-3.23 (m, 1H); 3.20-3.08 (m, 4H); 2.78-2.67 (m, 1H); 2.21 (s, 6H).

[0551] Example 2: Synthesis of comparative compound 6S)-l-(2-amino-l-(3-chlorophenyl)ethyl)- 4-(5-inorpholino- 1 / / -pvrrolo|2.3- / ?|pvridin-3- l-one

[0552] Step 1: 2-(3-Chlorophenyl)-2-((trimethylsilyl)oxy)acetonitrile

[0553]

[0554] To a solution of 5 g (36 mmol, 1 eq) of 3 -chlorobenzaldehyde in 50 ml of dry DCM under argon, are added 399 mg (36 mmol, 1 eq) of DABCO, followed by 4.45 ml (36 mmol, 1 eq) of trimethyl silyl cyanide and the resulting mixture is stirred at 40°C for 2h. Reaction mixture is then diluted with DCM and washed 2 times with water and once with brine. Organic layer is dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude compound is used in the next step without further purification. 7.80 g of the title compound are obtained.

[0555] Step 2: 2-Amino-l-(3-chlorophenyl)ethan-l-ol

[0556]

[0557] To a solution of 7.80 g (33 mmol, 1 eq) of 2-(3-chlorophenyl)-2-((trimethylsilyl)oxy)acetonitrile (described in the previous step) in 80 ml of dry Et2O, placed at 0°C with an ice / water bath, are added in portions 1.85 g (49 mmol, 1.5 eq) of LiAlH₄. The resulting mixture is stirred at 0°C for Ih. Then ice is slowly added into the reaction at 0°C until no more gas is formed and finally 100 ml of water are added. The mixture is stirred at room temperature for 30 min, the precipitate is filtrated on Celite and washed 2 times with Et2O.Filtrate is decanted and aqueous layer is extracted 2 times with Et2O. Combined organic layers are dried over ISfeSC, filtered, and evaporated under reduced pressure. Crude compound is used in the next step without further purification. 5.95 g of the title compound are obtained.

[0558] Step 3: Tert-butyl (2-(3-chlorophenyl)-2-hydroxyethyl)carbamate

[0559]

[0560] To a solution of 5.95 g (35 mmol, 1 eq) of 2-amino-l-(3-chlorophenyl)ethan-l-ol (described in the previous step) in 60 ml of THF, are added 8.32 g (38 mmol, 1.1 eq) of di-tert-butyl dicarbonate and the resulting mixture is stirred at room temperature for Ih. Solvent is then removed under reduced pressure and the mixture is diluted with 100 ml of EtOAc. Organic layer is washed 2 times with water and once with brine, dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 6.78 g of the title compound are obtained.

[0561] Step 4: 2-((Tert-butoxycarbonyl)amino)-l-(3-chlorophenyl)ethyl methanesulfonate

[0562]

[0563] 6.78 g (25 mmol) of tert-butyl (2-(3-chlorophenyl)-2-hydroxyethyl)carbamate (described in the previous step) are dissolved in 15 ml of DCM and placed at 0°C. 2.1 ml (15.1 mmol, 3 eq) of triethylamine are added, followed by 0.781 ml (10.1 mmol, 2 eq) of mesyl chloride. The reaction is stirred at 0°C under argon for 2h. Water is then added and the mixture is decanted. Aqueous layer is extracted 2 times with DCM. Combined organic layers are dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude compound is directly used in the next step without further purification. 9.28 g of the title compound are obtained.

[0564] Step 5: Tert-butyl (2-(4-bromo-2-oxopyridin-l(2ET)-yl)-2-(3-chlorophenyl)ethyl)carbamate

[0565]

[0566] To a mixture of 0.744 g (4.28 mmol, 1 eq) of 4-bromopyridin-2-(177)-one and 1.39 g (4.28 mmol, 1 eq) of cesium carbonate in 10 ml of dry DMF, is added at 0°C a solution of 8.73 g (25 mmol) of 2-((tert-butoxycarbonyl)amino)-l-(3-chlorophenyl)ethyl methanesulfonate (described in the previous step) in 5 ml of dry DMF. The solution is then stirred at room temperature for 2h. EtOAc is added, and the mixture is washed 4 times with water and once with brine. Organic layer is dried over Na₂SO₄, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a deactivated silica gel column and an HexaneZEtOAcmixture as eluent. 5.56 g of the title compound are obtained.

[0567] Step 6: 4-( 1 / / - Pyrrol o [2,3-6] pyridin-5-yl)morpholine

[0568]

[0569] In 487 ml of LiHMDS (IM in THF, 487 mmol, 2.4 eq) are dissolved 947 mg (2.03 mmol, 0.01 eq) of RuPhos and 1.58 g (2.03 mmol, 0.01 eq) of RuPhos Pd G2. Then are added under argon 40 g (203 mmol, 1 eq) of 5-bromo-U / -pyrrolo[2,3-Z>]pyridine and 21.1 ml (244 mmol, 1.2 eq) of morpholine and the solution is heated at 66°C for lh30. The reaction mixture is then cooled to room tempareature and dropped into 1.2 1 of a saturated NH4CI solution maintaining the temperature under 10°C with an ice water bath. The mixture is stirred for 10 min at this temperature and decanted. Aqueous layer is extracted 3 times with DCM. Combined organic layers are dried over ISfeSC, filtered, and evaporated under reduced pressure to give 44.4 g of a brown solid. Crude is triturated in 200 ml of a mixture of EtOAc and hexane (3 / 7) for Ih. The solid is filtrated, rinsed with 200 ml of a mixture of EtOAc and hexane (1 / 9) and dried under vacuum to give 38.98 g of a slightly brown powder.

[0570] Step 7: 4-( 1 -Tosyl- l / / -pyrrolo|2.3- / i|pyridiii-5-yl)niorpholine

[0571]

[0572] 38.98 g (192 mmol, 1 eq) of 4-(17 / -pyrrolo[2,3-Z>]pyridin-5-yl)morpholine (described in the previous step) are dissolved in 390 ml of dry DMF, under argon. The solution is cooled to 0°C, and 11.5 g (288 mmol, 1.5 eq) of sodium hydride (60% in paraffin oil) are slowly added. The mixture is stirred for 10 min at this temperature and then 40 min at room temperature. The mixture is cooled again to 0°C, 47.5 g (249 mmol, 1.3 eq) of tosyl chloride are slowly added under argon and the reaction mixture is stirred at 0°C for Ih followed by Ih at room temperature. The mixture is dropped into 800 g of ice / water and stirred for Ih. A precipitate is obtained, which is filtrated and rinsed several times with cold water. The precipitate is then dissolved with 1.2 1 of DCM, and the solution is washed 2 times with a saturated NaHCCh solution, 2 times with water and once with brine. The organic layer is dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude compound is triturated in 500 ml of a mixture of EtOAc and hexane (5 / 95) for 3h. The solid is filtrated, rinsed with hexane and dried under vacuum to give 62.56 g of an off-white solid.

[0573] Step 8: 4-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l-tosyl-LH-pyrrolo[2,3- / >]pyridin-5-yl)morpholine

[0574]

[0575] 62.56 g (175 mmol, 1 eq) of 4-(l-tosyl-17 / -pyrrolo[2,3-Z>]pyridin-5-yl)morpholine (described in the previous step) are suspended in 512 ml of Me-THF under argon. Then 48.9 g of bis(pinacolato)diboron (193 mmol, 1.1 eq), 1.88 g of 4,4’-di- / c / 7-butylbiphenyl (7 mmol, 0.036 eq) and 2.32 g of (l,5-cyclooctadiene)(methoxy)iridium(I) dimer (3.5 mmol, 0.018 eq) are added. The reaction is heated to reflux for 45 min under argon. The reaction mixture is then cooled to -10°C with an ice / acetone bath and quenched carefully with MeOH (350 ml). The solution is stirred at room temperature for 15 min and evaporated under vacuum to give a brownoil. Dark oil is then dissolved in 1 1 of DCM, washed 3 times with water and once with brine. Organic layer is evaporated under reduced pressure to give a black paste. 21 of Et2O are added and the mixture is stirred for 15 min at room temperature, filtrated on Celite and evaporated under reduced pressure to give 95 g of a brown solid foam. Crude mixture is finally purified by flash chromatography using a silica gel column and an EtOAc / hexane mixture as eluent. 75.5 g of the title compound are obtained.

[0576] Step 9: Tert-butyl (2-(3-chlorophenyl)-2-(4-(5-morpholino-l-tosyl-l / / -pyrrolo|2.3- / >]pyridin-3-yl)-2-oxopyridin-l(2Z / )-yl)ethyl)carbamate

[0577]

[0578] 2.8 g (6.50 mmol, 1 eq) of tert-butyl (2-(4-bromo-2-oxopyridin-l(2J7)-yl)-2-(3- chlorophenyl)ethyl)carbamate (described in the step 5), 3.16 g (6.50 mmol, 1 eq) of 4-(3- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l-tosyl-lH-pyrrolo[2,3-b]pyridin-5-yl)morpholine (described in the previous step) and 905 mg (6.50 mmol, 1 eq) of K2CO3 are placed in 140 ml of MeCN under argon. The mixture is bubbled with argon for 15 min and 459 mg (0.65 mmol, 0.1 eq) of bis(triphenylphosphine)palladium dichloride are added. The mixture is bubbled for another 15 min and then the reaction is stirred at 80°C for Ih under argon. Reaction mixture is diluted with 140 ml of EtOAc and washed 3 times with water. Organic layer is dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 4.79 g of the title compound are obtained.

[0579] Step 10: Tert-butyl (2-(3-chlorophenyl)-2-(4-(5-morpholino-l / / -pyrrolo|2.3- / ?|pyridin-3-yl)-2-oxopyridin-l(2Z / )-yl)ethyl)carbamate

[0580]

[0581] 1.0 g (1.4 mmol) of tert-butyl (2-(3-chlorophenyl)-2-(4-(5-morpholino-l-tosyl-l / / -pyrrolo[2,3- Z>]pyri din-3 -yl)-2-oxopyri din- I (27 / )-yl (ethyl (carbarn ate (described in the previous step) are suspended in 5 ml of MeCN and 2.5 ml of a Na2COs 2M solution. The mixture is stirred at 120°C under microwave irradiation in a sealed tube (150W) for Ih. The mixture is cooled to room temperature, diluted with EtOAc and washed 3 times with water. Organic layer is dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude product is finally purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 644 mg of racemate are obtained.

[0582] Step 11: 1 -(2- m ino- 1 -(3-chlorophenyl )et hyl )-4-(5-inorpholino- 1 / / -py rrolo [2,3- / >]pyridin-3-yl)pyridin-2(LH)-one

[0583]

[0584] To a solution of 644 mg (1.17 mmol, 1 eq) of tert-butyl (2-(3-chlorophenyl)-2-(4-(5- morpholino-lJ / -pyrrolo[2,3-Z>]pyridin-3-yl)-2-oxopyridin-l(2J7)-yl)ethyl)carbamate (described in the previous step) in 6 ml of DCM, placed at 0°C with an ice / water bath, are added 3 ml of trifluoroacetic acid. The solution is stirred at 0°C for lh30, then solvent is removed under reduced pressure and the mixture is diluted with 100 ml of a saturated NaHCCh solution. The solution is extracted 3 times with DCM. Combined organic layers are dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a C18 column and a water / MeOH mixture as eluent. 340 mg of the title compound are obtained.Step 12: (S)-l -(2-Amino-l -(3-chlorophenyl)ethyl)-4-(5-morpholino-l / / -pyrrolo|2.3- 6]pyridin-3-yl)pyridin-2(lH)-one

[0585]

[0586] Enantiomers obtained in the previous step are separated by flash chromatography using a Chiralflash IG column and an Hexane / EtOH / DCM / 0.1%TEA mixture as the mobile phase. First fraction to be eluted is the (-) ( / / (-enantiomer, followed by the (+) fS')-enantiomer with ee > 98%. 33 mg of the title compound are obtained starting from 120 mg of racemate.

[0587] MH+: 450.7; 452.7 (M; M+2).

[0588] 'H NMR (DMSO-d6, 400 MHz): 5 12.06 (br s, 1H); 8.17 (d, J=2.5Hz, 1H); 8.09 (s, 1H); 7.75 (d, J=8.1Hz, 1H); 7.70 (d,.7=2, 6 Hz, 1H); 7.44-7.29 (m, 4H); 6.74-6.67 (m, 2H); 5.90 (t, J=7.5Hz, 1H); 3.84-3.72 (m, 4H); 3.34-3.25 (m, 2H); 3.18-3.09 (m, 4H); 1.60 (br s, 2H).

[0589] Example 3: Synthesis of comparative compound 6S)-l-(l-(3,4-dichlorophenyr)-2- (methylamino)ethyl)-4-(5- -1 / / -,3-61 pyridin-3-yl)pyridin-2( FH)-one (compound B)

[0590] Step 1: 2-(3,4-Dichlorophenyl)-2-((trimethylsilyl)oxy)acetonitrile

[0591]

[0592] To a solution of 2.5 g (14.3 mmol) of 3, 4-di chlorobenzaldehyde in 50 ml of dry DCM under argon, are added 399 mg (36 mmol, 1 eq) of DABCO, followed by 4.45 ml (36 mmol, 1 eq) of trimethyl silyl cyanide and the resulting mixture is stirred at 40°C for 2h. Reaction mixture isthen diluted with DCM and washed 2 times with water and once with brine. Organic layer is dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude compound is used in the next step without further purification. 3.70 g of the title compound are obtained.

[0593] Step 2: 2-Amino-l-(3,4-dichlorophenyl)ethan-l-ol

[0594] NH,

[0595]

[0596] To a solution of 3.70 g (13.5 mmol) of 2-(3,4-dichlorophenyl)-2-((trimethylsilyl)oxy)acetonitrile (described in the previous step) in 80 ml of dry Et2O, placed at 0°C with an ice / water bath, are added in portions 1.85 g (49 mmol, 1.5 eq) of LiAlH₄. The resulting mixture is stirred at 0°C for Ih. Then ice is slowly added into the reaction at 0°C until no more gas is formed and finally 100 ml of water are added. The mixture is stirred at room temperature for 30 min, the precipitate is filtrated on Celite and washed 2 times with Et2O. Filtrate is decanted and aqueous layer is extracted 2 times with Et2O. Combined organic layers are dried over ISfeSCh, filtered, and evaporated under reduced pressure. Crude compound is used in the next step without further purification. 1.66 g of the title compound are obtained.

[0597] Step 3: Tert-butyl (2-(3,4-dichlorophenyl)-2-hydroxyethyl)carbamate

[0598]

[0599] To a solution of 1.66 g (8.03 mmol) of 2-amino-l-(3,4-dichlorophenyl)ethan-l-ol (described in the previous step) in 60 ml of THF, are added 8.32 g (38 mmol, 1.1 eq) of di-tert-butyl dicarbonate and the resulting mixture is stirred at room temperature for Ih. Solvent is then removed under reduced pressure and the mixture is diluted with 100 ml of EtOAc. Organic layer is washed 2 times with water and once with brine, dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 2.55 g of the title compound are obtained.

[0600] Step 4: 2-((Tert-butoxycarbonyl)amino)-l-(3,4-dichlorophenyl)ethyl methanesulfonate

[0601]

[0602] 2.46 g (8.03 mmol) of tert-butyl (2-(3,4-dichlorophenyl)-2-hydroxyethyl)carbamate (described in the previous step) are dissolved in 15 ml of DCM and placed at 0°C. 2.1 ml (15.1 mmol, 3 eq) of triethylamine are added, followed by 0.781 ml (10.1 mmol, 2 eq) of mesyl chloride. The reaction is stirred at 0°C under argon for 2h. Water is then added and the mixture is decanted. Aqueous layer is extracted 2 times with DCM. Combined organic layers are dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude compound is directly used in the next step without further purification. 3.23 g of the title compound are obtained.

[0603] Step 5: Tert-butyl (2-(4-bromo-2-oxopyridin-l(2ET)-yl)-2-(3,4-dichlorophenyl)ethyl)carbamate

[0604]

[0605] To a mixture of 0.744 g (4.28 mmol, 1 eq) of 4-bromopyridin-2-(177)-one and 1.39 g (4.28 mmol, 1 eq) of cesium carbonate in 10 ml of dry DMF, is added at 0°C a solution 3.09 g (8.03 mmol) of 2-((tert-butoxycarbonyl)amino)-l-(3,4-dichlorophenyl)ethyl methanesulfonate (described in the previous step) in 5 ml of dry DMF. The solution is then stirred at room temperature for 2h. EtOAc is added, and the mixture is washed 4 times with water and once with brine. Organic layer is dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a deactivated silica gel column and an Hexane / EtOAcmixture as eluent. 1.74 g of the title compound are obtained.

[0606] Step 6: Tert-butyl (2-(4-bromo-2-oxopyridin-l(2ET)-yl)-2-(3,4-dichlorophenyl)ethyl)(methyl)carbamate

[0607]

[0608] To a solution of 840 mg (1.82 mmol, 1 eq) of tert-butyl (2-(4-bromo-2-oxopyridin-l(2J7)-yl)-2-(3,4-dichlorophenyl)ethyl)carbamate (described in the previous step) in 9 ml of dry DMF, placed at 0°C with an ice / water bath, are added under argon87 mg (2.18 mmol, 1.2 eq) of sodium hydride (60% in paraffin oil), followed by 170 pl (2.73 mmol, 1.5 eq) of methyl iodide. The solution is stirred at 0°C for lh30, then the mixture is diluted with 100 ml of EtOAc. The solution is washed 4 times with water and once with brine. Organic layer is dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a EtOAc / Hexane mixture as eluent. 750 mg of the title compound are obtained.

[0609] Step 7: Tert-butyl (2-(3.4-dichlorophenyl )-2-(4-(5-niorpholino- 1 -tosyl- l / / -pyrrolo| 2.3- / >]pyridin-3-yl)-2-oxopyridin-l(2Z / )-yl)ethyl)(methyl)carbamate

[0610]

[0611] 750 mg (1.57 mmol) of tert-butyl (2-(4-bromo-2-oxopyridin-l(2J7)-yl)-2-(3,4-dichlorophenyl)ethyl)(methyl)carbamate (described in the previous step) and 2.45 g (5.01 mmol, 1.3 eq) of 4-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l-tosyl-17T-pyrrolo[2,3-Z>]pyridin-5-yl)morpholine (described in Step 3) are dissolved in 13 ml of MeCN under argon. Then 13 ml of a solution of Na2COs 2M are added to give a biphasic mixture which is bubbled with argon for 15 min. 135 mg (0.19 mmol, 0.05 eq) of bis(triphenylphosphine)palladium dichloride are added and the solution was bubbled with argon for another 15 min. The reaction is stirred at 70°C for 2h under argon. Reaction mixture is then diluted with water and EtOAc and then decanted. Aqueous layer is extracted 2 times with EtOAc. Combined organic layer are dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 1.27 g of the title compound are obtained.Step 8: Tert-butyl (2-(3,4-dichlorophenyl)-2-(4-(5-morpholino-lH-pyrrolo[2,3- / >]pyridin-3-yl)-2-oxopyridin-l(2Z / )-yl)ethyl)(methyl)carbamate

[0612]

[0613] 1.27 g (1.69 mmol) of tert-butyl (2-(3,4-dichlorophenyl)-2-(4-(5-morpholino-l-tosyl-l / / -pyrrolo[2,3-Z>]pyridin-3-yl)-2-oxopyridin-l(2J7)-yl)ethyl)(methyl)carbamate (described in the previous step) are suspended in 5 ml of MeCN and 2.5 ml of a Na2COs 2M solution. The mixture is stirred at 120°C under microwave irradiation in a sealed tube (150W) for Ih. The mixture is cooled to room temperature, diluted with EtOAc and washed 3 times with water. Organic layer is dried over Na2SO4, filtrated and evaporated under reduced pressure. Crude product is finally purified by flash chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 730 mg of racemate are obtained.

[0614] Step 9: 1 -( 1 -(3.4-l)ichlorophenyl)-2-(methylamino)ethyl)-4-(5-morpholino-l / / -pyrrolo [2,3-6] pyridin-3-yl)pyridin-2(lH)-one

[0615]

[0616] To a solution of 730 mg (1.20 mmol) of tert-butyl (2-(3,4-dichlorophenyl)-2-(4-(5-morpholino-U / -pyrrolo[2,3-Z>]pyridin-3-yl)-2-oxopyridin-l(2J7)-yl)ethyl)(methyl)carbamate (described in the previous step) in 6 ml of DCM, placed at 0°C with an ice / water bath, are added 3 ml of trifluoroacetic acid. The solution is stirred at 0°C for lh30, then solvent is removed under reduced pressure and the mixture is diluted with 100 ml of a saturated NaHCCh solution. The solution is extracted 3 times with DCM. Combined organic layers are dried over Na2SO4, filtered, and evaporated under reduced pressure. Crude mixture is purified by flash chromatography using a C 18 column and a water / MeOH mixture as eluent. 543 mg of racemate are obtained.Step 10: (5)-l-( l-(3,4-Dichlorophenyl)-2-(methylamino)ethyl)-4-(5-morpholino-LH-pyrrolo [2,3-6] pyridin-3-yl)pyridin-2(LH)-one

[0617]

[0618] Enantiomers obtained in the previous step are separated by flash chromatography using a Chiralflash IG column and an Hexane / EtOH / DCM / 0.1%TEA mixture as the mobile phase. First fraction to be eluted is the (-) ( / / (-enantiomer, followed by the (+) fS')-enantiomer with ee > 98%. 54 mg of the title compound are obtained starting from 150 mg of racemate.

[0619] MH+: 498.6; 500.5 (M; M+2).

[0620] 'H NMR (DMSO-d6, 400 MHz): 5 12.08 (br s, 1H); 8.17 (d, J=2.5Hz, 1H); 8.10 (d, J=2.6Hz, 1H); 7.76 (d, J=7.3Hz, 1H); 7.69 (d, J=2.5Hz, 1H); 7.66-7.61 (m, 2H); 7.37-7.30 (m, 1H); 6.74-6.66 (m, 2H); 6.08-5.98 (m, 1H); 3.84-3.73 (m, 4H); 3.34-3.24 (m, 1H); 3.22-3.14 (m, 1H); 3.14-3.09 (m, 4H); 2.30 (s, 3H); 1.94 (br s, 1H).

[0621] Example 4: ERK2 (MAPK1) Enzymatic assay

[0622] To assess the capacity of the compound N°1 according to example 1 to inhibit ERK2 enzymatic activity, Z’-Lyte biochemical assay from Life technologies was used according to manufacturer’s instructions. Briefly, black 384-well plates containing 100 nl of 100X compound solution in 100% DMSO, 2.4 pl kinase buffer, 5 pl 2X MAPK1 (ERK2) / Ser / Thr 03 mixture and 2.5 pl 4X ATP solution were used. Plates were shaken for 30 seconds and incubated for 60 minutes at room temperature. Then, 5 pl of a 1:1024 dilution of Development Reagent A was added. Plates were shaken for 30 seconds and incubated for 60 minutes at room temperature. A plate reader was used to read fluorescence. In this assay, ERK2 enzyme was used at a concentration of 0.4 pg / ml (5.74 nM) at ATP Km (100 pM). Kinase buffer consisted of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl₂, 1 mM EGTA. Compound IC50 were determined with a 3 -fold serial dilution (10 point titrations in duplicate).

[0623] Compound N°1 of example 1 has an ERK2 inhibitory activity (IC50) of 2.1 nM. Therefore, it exhibits a capacity to inhibit ERK2 enzymatic activity.5: Cell line assay

[0624] A cell line assay was used to determine the capacity of the compound N° 1 according to example 1 to inhibit cell proliferation. A375 cells (malignant melanoma) were grown to near 80% confluence and seeded at 3000 cells per 100 pl per well in DMEM with 10% FBS in 96-well flat bottom plates. Cells were incubated for 24 hours at 37°C under 5% CO2. 100 pl compound solutions were added to cells and incubated for 72 hours at 37°C. Total volume of media was 200 pl per well. The compound was screened in 0.15% DMSO (final) using 10 titration points in duplicate. Negative control wells consisted of vehicle only (0.15% DMSO in 10% FBS DMEM). After 72 hours of compound treatment, SDS 1% (final) was added to positive control wells for 15 minutes at 37°C. Then, medium was discarded and replaced by 100 pl per well of a MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma, Cat#M5655) at 0.5 mg / ml in 10% FBS DMEM. Cells were incubated for 4 hours at 37°C. MTT reaction was stopped and homogenized by the addition of 100µl per well of SDS 10% 0.01M HCl. After 16 hours at 37°C, absorbance was measured at 570 nm in a Bio-Tek plate reader (PowerWave HT). Percent of proliferation inhibition was calculated using negative controls (0.15% DMSO) as 0% growth inhibition and positive controls (1% SDS) as 100% growth inhibition. IC50 values (concentration inducing a half-maximal growth inhibition) were determined by non-linear regression analysis of the inhibition curve generated by mean replicate values (using a sigmoid dose-response with variable Hill Slope and constraining the top to a constant value of 100 and the bottom to a value between 0 and 50). Analysis was performed using GraphPad Prism software.

[0625] Cell proliferation inhibitory activity of the compound N° 1 according to example 1 (“MTT A375 IC50”) is 35 nM. Therefore, it exhibits a capacity to inhibit A375 cell proliferation.

[0626] Example 6: hERG channel inhibition assay

[0627] Evaluation of hERG channel inhibition of the compound N°1 according to example 1 has been performed. The compound was used as a lOmM stock in DMSO before dilution in HEPES-buffered saline to 30 pM. 6-point concentration -response curves were generated using 3.16-fold serial dilutions from the top test concentration. Electrophysiological recordings were made from a Chinese Hamster Ovary cell line stably expressing the full-length ion channel. Single cell ionic currents were measured in whole-cell configuration at room temperature (21-23 °C) using a Patchliner (Nanion Technologies). The internal solution for hERG contained (mM): 120 KF, 20 KC1, 10 EGTA, 10 HEPES and was buffered to pH 7.3. The external solution (HEPES-buffered saline, HEPES -buffered saline) contained (mM): 138 NaCl, 4.5 KC1, 1.8 CaCl₂, 1.0MgCh, 10 HEPES, 10 glucose, buffered to pH 7.4. Voltage protocol is given below. Currents were measured from the step and referenced to the holding current. The compound was then incubated for 2 minutes to achieve steady state prior to addition of the next concentration. Table 11

[0628] Channel hERG

[0629] Holding potential (mV) -80

[0630] Step potential (mV) +40, 2s then -40 for 2s Frequency (Pulse applied every X seconds) 10

[0631]

[0632] Cell background CHO

[0633] hERG channel inhibition properties of the compound N°1 according to example 1 (“hERG IC50”) 10.9 p. Therefore, it exhibits a safe hERG profile with patch clamp assay.

[0634] Example 7: CYP 3A4 inhibition assay

[0635] To assess the capacity of the compound N°1 according to example 1 to inhibit CYP 3A4 enzymatic activity, the test compounds (0.1 pM - 25 pM) were incubated with cryopreserved human hepatocytes for 10 min in the presence of the specific CYP3A4 probe substrate, midazolam.

[0636] 1 -Hydroxymidazolam was monitored by LC-MS / MS and a decrease in the formation of the metabolite compared to the vehicle control was used to calculate an IC50 value.

[0637] CYP 3A4 inhibitory activity of the compounds (“CYP 3A4 IC50”) is reported in the Table below:

[0638] Table 12

[0639] Compounds CYP 3A4 IC50 (pM) Comparative compound A (S)

[0640] 0.74

[0641] according to example 2

[0642] Compound n°l according to example

[0643] 7.8

[0644]

[0645] 1 (S)

[0646] Compound N°1 according to example 1 exhibits a low inhibition of CYP 3A4. Indeed, it has an IC50 value greater than 5 pM. By contrast, the comparative compound A has an IC50 value of 0.74 pM. Comparative compound A is therefore a highly potent inhibitor of CYP 3A4.

[0647] Example 8: Kinase panel

[0648] To assess the kinase selectivity of the compound n°l according to example 1 (S), Z’-Lyte biochemical assay, and Adapta / Lanthascreen binding assays from Life technologies were usedaccording to manufacturer’s instructions. Pourcentages of inhibition at 500 nM of test compounds were performed towards 58 kinases: ABL1, ACVR1B (ALK4), AKT2 (PKB beta), AMPK (A1 / B2 / G3), AURKA (Aurora A), AXL, BRAF, BTK, CAMK2B (CaMKII beta), CDK2 / cyclin A, CHEK1 (CHK1), CLK1, CSNK1A1 (CK1 alpha 1), CSNK2A1 (CK2 alpha 1), DAPK3 (ZIPK), DYRK1A, EGFR (ErbBl), EPHB3, ERBB2 (HER2), FGFR2, FLT3, FRAP1 (mTOR), GSK3B (GSK3 beta), IGF1R, IKBKB (IKK beta), INSR, IRAK4, JAK2, KDR (VEGFR2), KIT, LCK, MAP2K1 (MEK1), MAPK10 (JNK3), MAPK11 (p38 beta), MAPKAPK2, MARK2, MET (cMet), NEK2, NTRK1 (TRKA), PAK2 (PAK65), PDGFRB (PDGFR beta), PDK1 Direct, PHKG2, PIK3CA / PIK3R1 (pl 10 alpha / p85 alpha), PIM1, PLK1, PRKACA (PKA), PRKCA (PKC alpha), PTK2 (FAK), RET, ROCK1, RPS6KA1 (RSK1), RPS6KB1 (p70S6K), SRC, STK3 (MST2), SYK, TEK (Tie2), TYRO3 (RSE). The concentration of ATP was used at apparent Km for all kinases except for MAPK 10 (JNK3) which was at 100 mM and for BRAF and MAP2K1 (MEK1) which are binding assays.

[0649] The results are reported in the Table below:

[0650] Table 13

[0651] Kinase panel

[0652] Compounds

[0653] @500nM @500nM

[0654] hits > 80% hits > 50% Comparative compound A (S)

[0655] 15 / 58 26 / 58

[0656] according to example 2

[0657] Comparative compound B (S)

[0658] 10 / 58 20 / 58

[0659] according to example 3

[0660] Compound n° 1 according to example 1

[0661] 1 / 58 10 / 58

[0662]

[0663] (S)

[0664] Compound n°l according to example 1 exhibits a favorable kinase selectivity score in the representative kinase panel. Indeed, only 1 kinase over 58 was inhibited with a percentage of inhibition higher than 80%, and 10 kinases at most were inhibited with a percentage of inhibition higher than 50%.

[0665] By contrast, comparative compounds A and B exhibit a poor selectivity score. Indeed, comparative compounds A and B inhibited respectively 15 / 58 and 10 / 58 kinases with a percentage of inhibition higher than 80%, and 26 / 58 and 20 / 58 kinases with a percentage of inhibition higher than 50%.Example 9: Permeability assay

[0666] Caco 2 cell lines were used for the in vitro transport studies and were obtained from ATCC. Cells were split every other day at a split ratio of 1:3-1:5 and grown in Dulbecco’s Modified Eagle Medium (GlutaMAX I, 4,500 mg / L D-glucose, sodium pyruvate.) supplemented with 10% FBS in the presence of antibiotics. For transport studies, cells were seeded onto polycarbonate Transwell filter membranes (Millipore) at a density of 60,000 cells / well. After 24h post seeding, changed medium and cultured for another 21 days before transport experiments. For transport studies, donor solutions were prepared by diluting the stock solutions of test compounds in transport medium (HBSS buffer with 10 mM HEPES, pH 7.4). Receiver solutions were the same HBSS buffer with lOmM HEPES, pH 7.4. The transport of test compounds (5 pM) was measured in duplicate in two directions [apical to basolateral (A— B) and basolateral to apical (B— > A)].

[0667] The permeability coefficient for membrane transport of test compounds was determined using the following equation: Papp (cm / sec) = (Vr / CO) (1 / S) (dC / dt); Papp = apparent permeability, Vr = volume of medium in the receiver chamber, CO = PAR of the test drug in the receiver chamber, S = surface area of monolayer, dC / dt = drug PAR in the receiver chamber with time). Efflux Ratio was defined as: Efflux Ratio = Papp B-A / Papp A-B Bioanalysis was done on LC-MS / MS.

[0668] The results are reported in the Table below:

[0669] Table 14

[0670] Caco-2 permeability assay Compounds

[0671] Papp A-B

[0672] Efflux Ratio

[0673] (IO-6cm / s)

[0674] Comparative compound A (S)

[0675] 3.7 5.1

[0676] according to example 2

[0677] Comparative compound B (S)

[0678] 8.3 5.9

[0679] according to example 3

[0680] Compound n° 1 according to

[0681] 34.9 0.7

[0682]

[0683] example 1 (S)

[0684] Compound n°l according to example 1 exhibits excellent Caco-2 permeability parameters. Indeed, it has a Papp A-B value higher than 10.10'6cm / s. Moreover, it has an Efflux Ratio lower than 2.By contrast, comparative compounds A and B exhibit poor Caco-2 permeability parameters, with low Papp A-B values (lower than 10.10'6cm / s) and high Efflux Ratios (higher than 3).

[0685] Example 10: Amorphization of the compound N°1 according to example 1

[0686] The compound was rendered amorphous in the following manner to provide a material free from the presence of crystalline seeds, prior to further experimentation:

[0687] The compound was weighed into a 20 mL vial and transferred into a 100 mL round bottom flask.

[0688] Dichloromethane: methanol (1:2 v / v) (15 mL) was added and a slurry was observed after gentle heating. Dichloromethane (15 mL) was added resulting in a clear solution. The solvent was rapidly removed using a rotary evaporator resulting in the appearance of yellow solids.

[0689] The solids were analyzed by XRPD to confirm the form. The resulting solids from the fast evaporation of a dichloromethane:methanol mixture were predominantly amorphous.

[0690] Characterization of the compound

[0691] The following results were obtained from the characterization of the compound:

[0692] XRPD analysis showed a crystalline material designated as Free base Pattern 1.

[0693] PLM analysis showed very small irregular shaped particles that displayed birefringence under cross-polarized light.

[0694] TGA / DSC indicated an anhydrous compound with no weight losses until the melt decomposition event. The DSC trace showed a melting endothermic event that was concurrent with the beginning of decomposition onset; 281°C, peak 285°C.

[0695] Standalone DSC analysis was in agreement with the initial melting event shown in the TGA / DSC analysis. The subsequent cooling and heating steps did not reveal any significant thermal events.

[0696] DVS analysis showed an uptake of 0.6 wt.% at 80% RH (relative humidity). Subsequent desorption and sorption steps showed very similar profiles indicating that the sample was slightly hygroscopic. No obvious indication of any form change was apparent. Post experimental XRPD analysis revealed that the same diffraction pattern was obtained.

[0697] FTIR analysis, obtained for comparative purposes with putative salt hits showed a profile consistent with the functional groups present in the molecule.

[0698] 1H NMR analysis also indicated that the material was consistent with the structure. A trace amount of di chloromethane was observed in the spectrum (< 0.01 equivalents).

[0699] HPLC-UV analysis indicated that the compound had purity of 98.56 %area.LC-MS analysis indicated that the material had a +ve ESI m / z of 478.1 [M+H]+. This was consistent with the expected mass of 477.2 g / mol.

[0700] pKa analysis revealed an acid pKa of 12.62 and two basic pKa values of 2.85 and 6.81. Thus, reaction of acids with a pKa below 4.81 should be suitable for salt formation.

[0701] Example 11: Dissolution and solubility of the compound according to example 1 The dissolution and solubility of the compound according to example 1 were determined in the Fasted State Simulated Gastric Fluid (FaSSGF) at pH = 1.6, in the Fed State Simulated Intestinal Fluid (FeSSIF) at pH = 5 and in the Fasted State Simulated Intestinal Fluid (FaSSIF) at pH = 6.5. Data were compared to the solubility values vs. pH obtained in buffers.

[0702] I. Manufacture of biorelevant media

[0703] FaSSGF medium (8.7 mM sodium lauryl sulphate, 34 mM NaCl, 0.03N HC1 at pH 1.6) was prepared with 251.57 mg of sodium lauryl sulphate, 200.20 mg of sodium chloride, 3 mL of HC1 IN, and diluted with purified water up to 100 mL. pH was measured at 1.53 and adjusted at 1.58.

[0704] FaSSIF medium was prepared with 8.336 g of concentrated FaSSIF buffer, 192.18 g of purified water and 0.448 g of FaSSIF powder. The mixture was maintained under magnetic stirring until complete dissolution then equilibrated for 2h at RT. pH was measured at 6.47.

[0705] FeSSIF medium was prepared with 20.345 g of concentrated FeSSIF buffer, 229.80 g of purified water and 2.80 g of FeSSIF powder. The mixture was maintained under magnetic stirring until complete dissolution. pH was measured at 4.98.

[0706] II. Dissolution study

[0707] The Microdissolutest pDISS Profiler TM was used according to AuPRO Instruction Manual 2018-2019 Pion Inc. PN IM10001 Rev D.

[0708] Stock solutions

[0709] First solutions (1) were prepared at 5 mg / mL in DMSO as follows:

[0710] - 1st study in FaSSIF as donor medium: 24.28 mg of compound according to example 1 was weighed into a 5 mL volumetric flask. 4 mL of DMSO was added, the mixture was stirred until complete dissolution of the active pharmaceutical ingredient. DMSO was added qs 5 mL up to the mark, the final mixture was stirred.

[0711] - 2nd study in FeSSIF as donor medium: 25.91 mg of compound according to example 1 was weighed into a 5 mL volumetric flask. 4 mL of DMSO was added, the mixture was stirred until complete dissolution of the active pharmaceutical ingredient. DMSO was added qs 5 mL up to the mark, the final mixture was stirred.The 1st solutions were diluted at 1 / 5 in DMSO, into a 5 mL volumetric flask, to obtain stock solutions (2) at 1 mg / mL.

[0712] Standards collection

[0713]

[0714] 1. In FaSSIF medium

[0715] The standards were collected as follows to plot the calibration curve in the donor compartments: The tested media were dispensed in glass vials as follows: 15 mL of tested donor medium (FaSSIF) in position 1, 3,5 and 7. Magnetic cross bars were inserted into glass vials and stirred at 150 rpm. The probes were equipped with 20 mm tips and were lowered into each medium without air bubbles. The 100% transmittance was read to normalize the detection system. Serial additions of the stock solution (1) were measured in each medium in the concentration range from 0.97 to 19.35 pg / mL. The spectra (OD vs. wavelength) were saved as “Blue standards”. The calculation parameters were selected from the standards spectra to obtain the most performant calibration curve.

[0716] 2. In ASB (Accentor Sink Buffer) medium (accentor compartments) from FaSSIF medium The same operating mode as the one described above was used except for the following items: Tested medium dispensed in glass: 15 mL of acceptor sink buffer (ASB) in position 2, 4, 6 and 8. The probes were equipped with 20 mm. Serial additions of the stock solution (2) were measured in each medium in the concentration range from 0.19 to 5.02 pg / mL.

[0717] 3. In FeSSIF medium (donor compartments)

[0718] The same operating mode as the one described for FaSSIF medium was used except for the following items: Tested medium dispensed in glass: 15 mL of FeSSIF in position 1, 3, 5 and 7. The probes were equipped with 10 mm tips. Serial additions of the stock solution (1) were measured in each medium in the concentration range from 5.18 to 86.62 pg / mL.

[0719] 4. In ASB medium (acceptor compartments) from FeSSIF medium

[0720] The same operating mode as the one described above was used except for the following items: Serial additions of the stock solution (2) were measured in each medium in the concentration range from 0.21 to 5.36 pg / mL.

[0721] 5. Dissolution experiments

[0722] The experiments were performed as follows: The probes equipped with the following tips were used: 20 mm tips in the donor compartments made of FaSSIF and the acceptor compartments (ASB). 10 mm in the donor compartments made of FeSSIF. The pFlux glassware pairs were assembled. The last pair of pFlux glassware was dedicated to the “Blank” measurement. 1.2 to 1.6 mg of compound according to example 1 was weighted in each donor compartment to obtain a donor concentration of 60 to 80 pg / mL. 25 pL of GIT lipid solution was laid on the PVDFhydrophobic membranes before connecting the pFlux pairs. 20 mL of ASB was added in the acceptor compartment and 20 mL of the tested medium was added in the donor compartment. Magnetic cross bars were inserted into each compartment, pFlux pairs were placed in the heater block and the stirring was set at 150 rpm. The probes equipped with tips were lowered into each medium without air bubbles. The UV spectra were collected for 24 h in donor and acceptor compartments. The maximal dissolution rate and the solubility at equilibrium were calculated. Solubility study

[0723] 1. Solubility in FaSSGF

[0724] 22 mg of compound according to example 1 was accurately weighed into a glass vial. 2 mL of FaSSGF pH 1.6 was added to obtain a concentration of about 11 mg / mL supposed to be higher than the actual solubility. The mixture was homogenized by a vortex mixing and then maintained at 37 °C under a magnetic stirring. Samples of about 500 pL were withdrawn at T30 min, T4h and T24h and were filtered through a GV-PVDF 0.22 pm membrane. Each filtrate was distributed in 2 vials. The final pH was measured in the first vial. The solution from the 2nd vial was diluted in the HPLC diluent and analyzed by HPLC. The appearance of the mixture was reported at each timepoint.

[0725] 2. Solubility in FeSSIF

[0726] As all the quantity of compound according to example 1 introduced in the donor compartments, during the dissolution study, was completely dissolved in the FeSSIF medium, an additional solubility study was performed in FeSSIF targeting a solubility value higher than the soluble concentration of 73 pg / mL. 7 mg of compound according to example 1 was accurately weighed into a glass vial. 2 mL of FeSSIF pH 5.0 was added to obtain a concentration of about 3.5 mg / mL supposed to be higher than the actual solubility. Then the solubility was determined as detailed above.

[0727] III. Results

[0728] Solubility in simulated intestinal and gastric fluids

[0729] 7. Dissolution in FaSSIF pH 6.5

[0730] The individual and average dissolution of compound according to example 1 obtained in the FaSSIF medium at pH 6.5 and at 37 °C was measured, and the solubility data of compound according to example 1 determined. The dissolution step lasts around Ih before attaining a plateau. The quantity of compound according to example 1 introduced in the donor compartments at a mean concentration of 61 pg / mL was not completely dissolved, it was thus possible to determine the solubility. The mean solubility at equilibrium and at 37 °C was determined at 3.44 + / - 0.17 pg / mL at the plateau from Ih to 3.3h.2. Dissolution in FeSSIF pH 5.0

[0731] The individual and average dissolution of compound according to example 1 obtained in the FeSSIF medium at pH 5.0 and at 37 °C was measured, and the solubility data of compound according to example 1 determined. The dissolution step lasts around 30min before attaining a maximum of soluble concentration. The quantity of compound according to example 1 introduced in the donor compartments at a mean concentration of 73 pg / mL was completely dissolved, therefore, the real solubility of compound according to example 1 in FeSSIF can be expected to be greater than 73 pg / mL.

[0732] 3. Solubility in FeSSIF pH 5.0 and in FaSSGF pH 1.6

[0733] The solubility data of compound according to example 1 and the final pH obtained after 30 min, 4h and 24h in FeSSIF pH 5.0 and in FaSSGF pH 1.6 are reported in the following table:

[0734] Table 15

[0735] Theoretical Soluble

[0736] Biorelevant Visual appearance cone. Timepoint concentration Final pH

[0737] medium before filtration (mg / mL) (mg / mL)

[0738] T30min 0.237 5.03 Cloudy yellowish FeSSIF

[0739] 3.6 T4h 0.324 5.04 Cloudy yellowish pH 5.0

[0740] T24h 0.328 5.07 Cloudy yellowish T30min 8.930 2.80 Milky yellow

[0741] FaSSGF T4h 6.947 2.88 Cloudy yellow

[0742] 11.1

[0743] pH 1.6 Clear yellow with some T24h 6.661 2.86

[0744]

[0745] particles

[0746] The compound according to example 1 presents a solubility of 328 pg / mL in FeSSIF pH = 5.0 after 24 h at 37°C. This value was already attained after 4h at the most and confirms the data obtained within the dissolution study (see >73 pg / mL).

[0747] In FaSSGF pH 1.6, a solubility of 6.7 mg / mL was obtained after 24 h at 37°C, this value is in the same range as the solubility obtained after 4 h (7.0 mg / mL), however, it is lower than the value obtained after 30 min (8.9 mg / mL) indicating a possible reprecipitation of the API at equilibrium which seems to be aligned with the visual observation (from a milky suspension at T = 30 min to a solution with particles at T = 24 h). At 6.7 mg / mL, compound of example 1 presents a buffering capacity that increases the initial FaSSGF pH from 1.6 to 2.9.

[0748] In the biorelevant media, the compound according to example 1 solubility is 20-fold higher at pH 1.6 than at pH 5.0.

[0749] 4. Solubility data comparison

[0750] The solubility data of the compound according to example 1 obtained in the biorelevant media and in buffers are reported vs. the pH, the medium composition and the ionisation state of thetwo pKa in the following table. The experimental and calculated pKa were considered to determine which of the two sets of data is the correct evaluation.

[0751] Table 16

[0752] Final Ionisation state (%) Medium Solubility pH at Experimental pKa Calculated pKa at 24h T24h Medium Surfactants

[0753] 2.85 6.81 5.8 8.5 (pg / mL)

[0754] HC1 USP buffer pH

[0755] 1.1 98 100 100 100 No >3000

[0756] 1.2

[0757] 2.9 47 100 100 100 FaSSGF pH 1.6 8.7 mM SLS 6661

[0758] HC1 USP buffer pH

[0759] 3.2 82 100 100 100 No >3000

[0760] 2.2

[0761] 3.5 31 100 100 100 Citrate buffer pH 3.4 No >3000

[0762] Acetate USP buffer

[0763] 4.6 18 99 94 100 No 149 pH 4.5

[0764] 15 mM NaTc

[0765] 5.1 0.6 98 83 100 FeSSIF pH 5.0 328

[0766] 3.75 mM lecithin

[0767] Acetate USP buffer

[0768] 5.5 0.2 95 67 100 No 13 pH 5.5

[0769] 6.5

[0770] 3 mM NaTc

[0771] (initial 0 67 17 99 FaSSIF pH 6.5 3.4

[0772] 0.75 mM lecithin

[0773] pH)

[0774] Phosphate USP

[0775] 6.7 0 56 11 98 No 1.2 buffer pH 6.8

[0776] Phosphate USP

[0777] 7.7 0 11 1 86 No 0.6

[0778]

[0779] buffer pH 7.8

[0780] The solubility of compound according to example 1 increases as a function of the pH decrease (behaviour expected for a base). The solubility profile is much more in agreement with the experimentally determined pKa values (2.85 and 6.81) than with the calculated values which are too high (5.8 and 8.5). In the biorelevant media, the solubility value is about 3-fold higher in FaSSIF at pH 6.5 (3.4 pg / mL vs. 1.2 pg / mL in phosphate buffer pH 6.7) and is about 8-fold higher in FeSSIF at pH 5.1 (328 pg / mL vs. a solubility estimated at around 40 pg / mL between the values of 13 pg / mL and 149 pg / mL obtained respectively in acetate buffers at pH 5.5 and 4.6). FeSSIF is 3 times more concentrated in surfactants than FaSSIF which could explain this difference in solubility improvement. In FaSSGF, compound according to example 1 presents a high solubility of 6.66 mg / mL and a buffering capacity increasing the initial pH from 1.6 to 2.9.

[0781] The compound according to example 1 is considered as “low soluble”.

[0782] Example 12: Mono-Hydrochloride anhydrate salt

[0783] The mono-hydrochloride salt of compound N°1 was prepared as follows:

[0784] Compound according to example 10 (800 mg) was weighed into a 20 mL vial and suspended in 2-propanol (15 mL). 1.05 equiv. of chlorohydric acid was added neat to the experiment. Theexperiment was then allowed to temperature cycle from 50 to 20°C at 0.1°C / min with an hour hold at each temperature with stirring for 48 hours.

[0785] After this time, a predominantly amorphous diffractogram was observed.

[0786] The solids were isolated by Buchner filtration under vacuum. 15 mL of acetone was added to the solids and the slurry was further cycled for 48 hours.

[0787] After this time, the diffractogram showed the hydrochloride salt had been formed.

[0788] The solids were isolated and allowed to dry under vacuum at 40°C for approximately 24 hours. Upon drying, no changes to the diffractogram were observed.

[0789] This salt was fully characterized as hydrochloride Pattern 1. The following observations and results were obtained:

[0790] - The isolated solids after 48 hours from 2-propanol were largely amorphous. The solubility of the hydrochloride salt was not sufficiently high at 50°C for efficient Ostwald ripening to occur to afford highly crystalline material. The re-slurry in acetone did however deliver crystalline material.

[0791] - XRPD analysis (figure 1) indicated that the material was consistent with hydrochloride Pattern 1. There was no change in form on drying.

[0792] - PLM analysis showed very small birefringent needles shaped crystals.

[0793] - TGA / DSC analysis: TGA analysis indicated that there was a no loss of weight until the melting point indicating an anhydrous salt. The melting point occurred with the beginning of molecular degradation / disproportionation. Simultaneous DSC analysis indicated that there was an endothermic event with an onset of 256°C (peak at 267°C).

[0794] - DSC analysis was carried out with the material sealed non-hermetically with a pierced lid. The first heat of the DSC analysis revealed that there was an endothermic event observed with an onset of 273°C with a peak at 275°C. A vitrification event (anticipated after the initial melt) was not observed during the cool cycle. Similarly, on the second heating step no clear thermal events were observed.

[0795] - DVS analysis indicated that hydrochloride Pattern 1 was slightly hygroscopic, with a moisture uptake of 0.97 wt.% at 80 % RH (0.4 mol eq. water). There was no evidence of form change during the DVS experiment, and equilibration at each step was rapid. XRPD analysis indicated that there was no change in form of hydrochloride Pattern 1 after the DVS experiment was completed.

[0796] - HPLC-UV analysis indicated that the hydrochloride salt had a purity of 98.39 %area.- HPLC-CAD analysis resulted in a chloride content of 7.0 %w / w (average of two replicates) which was consistent with a mono hydrochloride salt.

[0797] - FT-IR analysis was consistent with that expected. There was no indication of any hydration of the crystalline form.

[0798] -1H NMR analysis of hydrochloride Pattern 1 indicated that there was 0.06 mol eq. of 2-propanol (a trace amount) and the chemical shifts were in-line with salt formation.

[0799] - PSD analysis of hydrochloride salt Pattern 1 gave the following values:

[0800] • DIO - 0.824 pm

[0801] • D50 - 2.705 pm

[0802] • D90 - 11.635 pm

[0803] Thermodynamic solubility (according to study 2)

[0804] The thermodynamic solubility carried out on the salt showed:

[0805] Table 17

[0806] Buffer Timepoint (h) Solubility (mg / mL)

[0807] Water 0 15.3

[0808] 4 14.5

[0809] FaSSIF 0 1.8

[0810] (pH = 6.5) 4 2.3

[0811] FaSSGF 0 17.9

[0812]

[0813] (pH = 1.6) 4 20.3

[0814] The thermodynamic solubility assessment of hydrochloride Pattern 1 showed a solubility of ca.

[0815] 20 mg / mL across the study in water and in FaSSGF.

[0816] In FaSSIF the solubility was only around 3 mg / mL at each timepoint.

[0817] Furthermore, analysis of the XRPD diffractograms after 24 hours from water and FaSSGF showed that hydrochloride Pattern 1 was recovered unchanged, albeit a poorly crystalline Pattern 1 was observed after 24 hours in water.

[0818] In FaSSIF some small peaks that could be assigned to the free base were observed across the duration of the study.

[0819] 7 Day Stability Studies

[0820] The 7-day stability assessment of the salt showed that there was no change in form at ambient light, 40°C / 75% RH, 80°C or in miglyol and that the purity was maintained at each condition. These data underline the stability of the form at these conditions.

[0821] Hydration studiesThe hydrochloride Pattern 1 was observed at each of the investigated water activities indicating that hydrochloride pattern 1 is stable at least up to a water activity of 0.8. These data detail that the hydrochloride salt is stable to hydration.

[0822] Three Month Stability Study

[0823] After storage for two weeks at each condition, the purity of both hydrochloride Pattern 1 and the free base material were unchanged from the input purity (Hydrochloride Pattern 1: 98.39; free base: 98.56). The chemical stability was also unchanged, exemplified by the XRPD diffractograms showing free base Pattern 1 and hydrochloride salt Pattern 1 respectively. The diffractograms were unchanged, indicating that after one month the physical and chemical purity was stable.

[0824] At the three month time point, the purity of each sample remained unchanged with respect to the input material. The diffractograms were unchanged, indicating that after three months the physical and chemical purity was stable under the conditions studied.

[0825] Conclusion

[0826] Hydrochloride Pattern 1 was an anhydrous mono-hydrochloride salt with good thermal properties, which maintained form and crystallinity on drying, storage at 40°C / 75%RH and at elevated temperature.

[0827] The salt shows good solid-state chemical and physical stability profiles and maintained form and purity after one week at each accelerated aging conditions (80°C, 40°C / 75% RH and in miglyol).

[0828] The hydrochloride salt remained essentially intact after 24 hours. It shows its increased robust nature in biorelevant media.

[0829] A three-month stability study showed that the compound remained of high purity and retained their crystalline form.

[0830] Example 13: Hydrochloride hydrate salt

[0831] The hydrochloride hydrate salt of compound N°1 was prepared as follows:

[0832] Compound according to example 10 was added to 3.33 volume of EtOH to crystallizer under the protection of nitrogen. The slurry is heated to 70-75°C and kept for 0.5-1 h. 1.1 equivalent of chlorohydric acid is diluted with 1.67 volume of water. The obtained solution of chlorohydric acid is added to the slurry. Clear solution was obtained. This solution is cooled to 65-70°C, and 0.5 w% of seeds are added and aged for 2-4 h. The solution is then cooled to 20-25°C for 4.5-9h and aged for 1-2 h. 20 volumes of acetone are added over 5-10 h and aged for 2-4 h. The solution is then cooled to -5-0°C for 2.5-5 h and held at -5-0°C for 4-8 h. The slurry is filtered, and the wet cake is washed twice with 1 volume of acetone. The solids were isolated and allowed to dry under vacuum at 40-45°C under vacuum until constant weight.

[0833] This salt was fully characterized as hydrochloride Pattern 2. Based on the data below, it was shown that Pattern 2 is a hydrate. The following observations and results were obtained:

[0834] - XRPD analysis (figure 2) indicated that the material was consistent with hydrochloride Pattern 2. There was no change in form on drying.

[0835] X-ray powder diffraction data were collected under ambient conditions on a Bruker D2 PHASER diffractometer with a low power X-ray generator of 300 W. Powder patterns were collected on a zero background sample holder with a 0.15 s / step with a total step of 1837, two theta at 0.02° per step at 30 kV and 10 mA. The X-ray tube of Cu (Ka) was employed, with the Ka2 / Kal intensity ratio of 0.50 (1.54439 A / 1.5406 A).

[0836] - TGA analysis showed a weight loss of 11.4% up to 150°C (about 3.4% for one equivalent of water) and a weight loss of 2.7% from 150°C to 205 °C.

[0837] - DSC result showed three endotherms at 85.3 °C, 194.5 °C and 255.5 °C (peak temperature). - 'H NMR analysis of hydrochloride Pattern 2 showed the molar ratio of residual 1,4-dioxane / freebase was 0.05 (0.8 wt%).

[0838] 1H NMR data was taken using Bruker AVANCE NEO 400 MHZ in DMSO-t / 6 solvent.

[0839] - Ultra-Performance Liquid Chromatography (UPLC) and IC were tested on Pattern 2. The results showed the UPLC purity was 99.55 area% and Cl⁻ content was 5.9% which indicating the stoichiometric ratio of acid / base was 1.0.

[0840] UPLC was employed with the methods shown in the Table below:

[0841] Table 18

[0842] Instrument Waters H-Class UPLC or Equivalent System

[0843] Column Waters ACQUITY UPLC BEH C182.1*150 mm 1.7 pm,

[0844] P / N: 186002353

[0845] Mobile phase A: 0.1% FA in H2O (v / v), B: 0.1% FA in ACN (v / v) Gradient Time (min) A (%) B (%)

[0846] 0.0 98 2

[0847] 0.2 98 2

[0848] 3.0 0 100

[0849] 4.5 0 100

[0850] 4.8 98 2

[0851] 6.0 98 2

[0852] Detector UV, 220 nm

[0853] Column temperature 40 °C

[0854] Flow rate 0.6 mL / min

[0855] Injection volume 3 pLAutosampler 20 °C

[0856] temperature

[0857] Diluent MPA: MPB 9:1, v / v

[0858] Sample concentration 0.05 mg / mL

[0859] The data of chlorion content was collected with ion chromatography system of DIONEX ICS- 6000+ DP (Chromeleon 7.2 system) and the method was shown in the Table below:

[0860] Table 19

[0861] Items Parameters

[0862] Reagents H2O (Milli-Q water)

[0863] Standard solution of chloride (AR)

[0864] KOH (Eluent generator 500 KOH, PN:

[0865] 075778)

[0866] Column Dionex IonPac AS11-HC, 4×250 mm, Part No.

[0867] 052960

[0868] Guard column: Dionex IonPac AG11-HC, 4×50mm, Part No. 052962

[0869] Column temperature 30 °C

[0870] Flow rate 1.0 mL / min

[0871] Detector Electrical conductivity detector

[0872] Suppressor ASRS 3004 mm, Part No. SP6949

[0873] Mobile phase A: H2O, isocratic elution: 20 mM KOH

[0874] Injection volume 25 pL

[0875] Diluent H2O

[0876] Run time 20 min

[0877] Example 14: Maleate salt

[0878] Maleate salt of compound N°1 was prepared as follows:

[0879] Compound according to example 10 (800 mg) was weighed into a 20 mL vial and suspended in 2-propanol (16 mL).

[0880] 1.05 equiv. of maleic acid was added neat to the experiment.

[0881] The experiment was then allowed to temperature cycle from 20 to 50°C at 0.1°C / min with an hour hold at each temperature with stirring.

[0882] After 1 cycle, an aliquot was removed and checked by XRPD and found to be the correct form. The solids were isolated and dried at 40°C under vacuum for 16 hours and reanalyzed by XRPD. This salt was fully characterized as Maleate Pattern 1. The following observations and results were obtained:

[0883] - XRPD analysis (figure 3) indicated that the material was the correct form after one thermal cycle. After isolation XRPD analysis of the wet-cake indicated that the material was consistent with Maleate Pattern 1. There was no change in form on drying.

[0884] - PLM analysis showed very small birefringent irregular shaped crystals.- TGA / DSC analysis: TGA analysis indicated that there was a no loss of weight until the simultaneous melting / disproportionation event at ca. 170°C. At this point there was an 18.8 wt.% loss which was equivalent to 1.2 equivalents of maleic acid. Simultaneous DSC analysis indicated that there was an endothermic event with an onset of 177°C (peak at 184°C). A second endothermic event occurred at onset of 269°C (peak at 276°C) and this related to freebase melting.

[0885] - DSC analysis was carried out with the material sealed non-hermetically with a pierced lid. The first heat of the DSC analysis revealed that there was an endothermic event observed with an onset of 172°C with a peak at 178°C. This was immediately followed by an exothermic event indicating a possible recrystallisation. It is likely that the exothermic event was a recrystallisation of freebase generated during the disproportionation. A vitrification event (anticipated after the initial melt), was not observed during the cool cycle. The second heating step showed a glass transition with a midpoint at 128°C.

[0886] - DVS analysis indicated that maleate Pattern 1 was slightly hygroscopic, with a moisture uptake of 1.6 wt.% at 80% RH (0.5 mol eq. water). A decrease in weight (0.5 wt. % - 0.16 equiv. water) was observed in the first cycle between 70 to 80% RH which was most likely due some crystallization event, possibly induced in the high moisture environment. XRPD analysis indicated that there was no change in form of Maleate Pattern 1 after the DVS experiment was completed.

[0887] - HPLC-UV analysis indicated that the maleate salt had a purity of 98.32 %area.

[0888] - FT-IR analysis was consistent with that expected. There was no indication of any hydration of the crystalline form.

[0889] -1H NMR analysis of Maleate Pattern 1 indicated that there was 0.07 mol eq. of 2-propanol (a trace quantity) and the chemical shifts were in-line with salt formation. Moreover, there was 0.93 equivalents of maleic acid consistent with a mono salt.

[0890] - PSD analysis of Maleate Pattern 1 gave the following values:

[0891] • D10 - 2.470 pm

[0892] • D50 - 11.677 pm

[0893] • D90 - 34.125 pm

[0894] Thermodynamic solubility (according to study 2)

[0895] The thermodynamic solubility carried out on the salt showed:

[0896] Table 20Buffer Timepoint (h) Solubility (mg / mL)

[0897] Water 0 73

[0898] 4 104.3

[0899] FaSSIF 0 5.4

[0900] (pH = 6.5) 4 5.2

[0901] FaSSGF 0 78.4

[0902]

[0903] (pH = 1.6) 4 115.3

[0904] Maleate salt Pattern 1 showed a solubility of ca. 8 mg / mL in FaSSIF, however, the XRPD analysis at each timepoint identified freebase diffractograms.

[0905] In water the solubility was ca. 30 mg / mL. The XRPD analysis in this instance showed two new patterns over the time of the experiment. At T= 0 and 4 hours a new pattern (Pattern 2) was identified whereas at T = 24 hours a second new pattern (Pattern 3) was identified. These patterns are potentially hydrated forms. In FaSSGF the solubility was much higher (ca. 80 mg / mL) and the new patterns were once more observed. Pattern 2 at T = 0 and Pattern 3 at T = 4 hours and 24 hours.

[0906] 7 Day Stability Studies

[0907] The 7-day stability assessment of the salt showed that there was no change in form at ambient light, 40°C / 75% RH, 80°C or in miglyol and that the purity was maintained at each condition. These data underline the stability of the form at these conditions.

[0908] Salt disproportionation studies

[0909] A solution was maintained indicating that disproportionation did not occur during the study.

[0910] Conclusion

[0911] Maleate Pattern 1 was an anhydrous mono maleate salt, with good thermal properties. This maleate salt was stable physically and chemically stable and on drying, storage at 40°C / 75%RH and at elevated temperature.

[0912] The salt showed good solid-state chemical and physical stability profiles and maintained form and purity after one week at each accelerated aging conditions (80°C, 40°C / 75%RH and in miglyol).

[0913] The maleate salt showed high thermodynamic solubility.Example 15: Esylate salt

[0914] Esylate salt of compound N°1 was prepared as follows:

[0915] Approximately 250 mg of the compound according to example 10 was weighed into a 20 mL vial. 8.5 mL of acetone was added followed by a magnetic stirrer bar. 46 pL (1.05 equiv.) of ethanesulfonic acid was added neat. The solution was stirred at 40°C for 5 minutes.

[0916] The experiments were thermally cycled between 20 and 50°C at a rate of 0.1°C / min with a 1 hour hold at 20 and 50°C.

[0917] An aliquot of the mixture was taken after 72 hours, was isolated by centrifuge filtration and analyzed by XRPD.

[0918] The XRPD analysis showed that the Pattern 1 of the Esylate salt was obtained.

[0919] The solids were isolated by Buchner filtration (42.5 mm 0 Grade 1 Whatman), washed with cold acetone and dried under vacuum at 40°C for 16 h.

[0920] XRPD analysis was carried out on the dried solids. The esylate salt was characterized by PLM, TGA / DSC, DSC, 1H NMR and HPLC-UV for purity.

[0921] The following results were obtained for the Esylate Pattern 1:

[0922] - XRPD analysis (figure 4) of the material isolated from acetone showed a crystalline material consistent with Esylate Pattern 1.

[0923] - PLM showed birefringence under crossed polarised light. However, the crystals were small with irregular morphology.

[0924] - The TGA trace showed a 1.3 wt.% loss (equivalent to 0.43 eq. of surface moisture) in agreement with an anhydrous esylate salt.

[0925] - The DSC trace showed two endothermic events at onset: 190°C; peak: 197°C and at peak: 247°C. DSC analysis showed the melting event at onset: 189°C; peak: 197°C during the first heating step. A vitrification event was observed at ca. 63°C during the first cooling step. The second heating step showed a glass transition with a midpoint of ca. 70°C.

[0926] -1H NMR analysis was consistent with Esylate Pattern 1 with only trace solvent present. The spectrum indicated ca. 0.5 equivalents of ethanesulfonic acid indicating a hemi-salt.

[0927] - HPLC-UV analysis indicated that Esylate Pattern 1 had a purity of 99.44 %area.

[0928] Thermodynamic solubility (according to study 1)

[0929] A moderate / low solubility was observed for the esylate salt in unbuffered water and FaSSGF, where a solubility between 7 and 13 mg / mL was obtained. A high solubility was observed in FaSSIF, above 86 mg / mL.Example 16: Oxoglutarate salt

[0930] Oxoglutarate salt of compound N°1 was prepared as follows:

[0931] Approximately 250 mg of the compound according to example 10 was weighed into a 20 mL vial. 3 mL of dichloromethane was added followed by a magnetic stirrer bar. 81.2 mg (1.05 equiv.) of ketoglutaric acid was added neat.

[0932] The experiments were thermally cycled between 5 and 20°C at a rate of 0.1°C / min with a 1 hour hold at 5 and 20°C.

[0933] An aliquot of the mixture was taken after 48 hours, was isolated by centrifugation and analyzed by XRPD. The incorrect form was observed and the experiment was temperature cycled for a further 24 h.

[0934] The XRPD analysis after a total of 72 h showed that the Pattern 1 of the Oxoglutarate salt was obtained.

[0935] The solids were isolated by Buchner filtration (42.5 mm 0 Grade 1 Whatman) and dried under vacuum at 40°C for 16 h. XRPD analysis was carried out on the dried solids.

[0936] The oxoglutarate salt was characterized by PLM, TGA / DSC, DSC, 1H NMR and HPLC-UV for purity and organic acid content.

[0937] The following results were obtained for Oxoglutarate Pattern 1:

[0938] - XRPD analysis (figure 5) of the isolated from dichloromethane showed a crystalline material consistent with Oxoglutarate Pattern 1.

[0939] - PLM showed birefringence under crossed polarised light. The crystals were small and irregular shaped.

[0940] - The TGA trace showed an 11.7 wt.% loss from 170°C to 220°C (equivalent to 0.57 eq. ketoglutaric acid). However, the TGA mass loss beyond this point was continuous therefore a true picture of the salt stoichiometry was gained from the organic acid content determination by HPLC (vide infra).

[0941] - The DSC trace showed an endothermic event at onset: 177°C; peak: 182°C a second endothermic event was observed during degradation of the molecule at onset: 245°C; peak: 257°C. DSC analysis captured each of the endothermic events onset: 177°C; peak: 183°C and onset: 250°C; peak: 260°C. The first event is likely to be part of a concurrent melt / disproportionation and the second is likely free base melting.

[0942] -1H NMR analysis was consistent with salt formation and only trace DCM was present supporting that Pattern 1 was an anhydrous salt.

[0943] - Determination of the organic acid content by HPLC-UV showed 23.3 %w / w of ketoglutaric acid which was equivalent to 1.0 molar equivalents consistent with mono-salt.- HPLC-UV analysis indicated that the oxoglutarate salt Pattern 1 had a purity of 98.52 %area.

[0944] Thermodynamic solubility (according to study 1)

[0945] The thermodynamic solubility carried out on the salt showed:

[0946] Table 21

[0947] Buffer Timepoint (h) Solubility (mg / mL)

[0948] Water 0 9.6

[0949] 4 9.6

[0950] FaSSIF 0 11.2

[0951] (pH = 6.5) 4 7.2

[0952] FaSSGF 0 > 21.6

[0953]

[0954] (pH = 1.6) 4 > 26.5

[0955] A moderate solubility was observed for the oxoglutarate salt in all media, where a solubility between 7 and 11 mg / mL was obtained in unbuffered water and FaSSIF, and between 20 and 26 mg / mL in FaSSGF.

[0956] Example 17: Malonate salt

[0957] Mai onate salt of compound N°1 was prepared as follows:

[0958] Approximately 250 mg of the compound according to example 10 was weighed into a 20 mL vial. 3 mL of dichloromethane was added followed by a magnetic stirrer bar. 57.8 mg (1.05 equiv.) of malonic acid was added neat.

[0959] The experiments were thermally cycled between 5 and 20°C at a rate of 0.1°C / min with a 1 hour hold at 5 and 20°C.

[0960] An aliquot of the mixture was taken after 24 hours, was isolated by centrifugation and analyzed by XRPD. The XRPD analysis showed that the Pattern 2 of the Malonate salt was obtained. The solids were isolated by Buchner filtration (42.5 mm 0 Grade 1 Whatman) and dried under vacuum at 40°C for 16 h.

[0961] XRPD analysis was carried out on the dried solids.

[0962] The malonate salt was characterized by PLM, TGA / DSC, DSC, 1H NMR and HPLC-UV for purity.

[0963] The following results were obtained for Malonate Pattern 2:

[0964] - XRPD analysis (figure 6) of the material isolated from dichloromethane showed a crystalline material consistent with Malonate Pattern 2.

[0965] - PLM showed birefringence under crossed polarised light. The crystals were small and irregular shaped.- TGA / DSC analysis showed a 3.7 wt.% loss from 35 to 77°C (0.3 equiv. di chloromethane). This was followed by a gradual 12.4 % weight loss from 150 to 250 °C (0.8 equiv. malonic acid - consistent with a mono salt).

[0966] - The DSC trace showed an endothermic event at onset: 254°C; peak: 263 °C. DSC analysis showed a small endothermic event at onset: 125°C; peak: 128°C. The second endothermic event was at onset: 259°C; peak: 267°C. The first event is likely to be part of a concurrent melt / disproportionation and the second is possibly free base melting.

[0967] -1H NMR analysis was consistent with salt formation and only trace DCM was present. The counterion signal was likely obscured by the water peak in the spectrum.

[0968] - HPLC-UV analysis indicated that Malonate Pattern 2 had a purity of 98.43 %area.

[0969] Thermodynamic solubility (according to study 1)

[0970] The thermodynamic solubility carried out on the salt showed:

[0971] Table 22

[0972] Buffer Timepoint (h) Solubility (mg / mL)

[0973] Water 0 8.9

[0974] 4 8.9

[0975] FaSSIF 0 3.5

[0976] (pH = 6.5) 4 3.5

[0977] FaSSGF 0 > 23.4

[0978]

[0979] (pH = 1.6) 4 > 27.4

[0980] A low solubility was observed for the malonate salt in FaSSIF (ca. 3 mg / mL), moderate / low in unbuffered water (ca. 9 mg / mL) and moderate in FaSSGF (ca. 25 mg / mL).

[0981] Example 18: Oxalate salt

[0982] Oxalate salt of compound N°1 was prepared as follows:

[0983] Approximately 250 mg of the compound according to example 10 was weighed into a 20 mL vial. 5 mL of methanol was added followed by a magnetic stirrer bar. 75.1 mg (1.5 equiv.) of oxalic acid was added neat.

[0984] The experiments were thermally cycled between 5 and 20°C at a rate of 0.1°C / min with a 1 hour hold at 5 and 20°C.

[0985] An aliquot of the mixture was taken after 4 days, was isolated by centrifugation and analyzed by XRPD.

[0986] The XRPD analysis showed that a mixture of Oxalate salt Pattern 2 and free base of compound N°1 according to example 1 salt was obtained.An additional 0.5 equiv. (24.5 mg) of oxalic acid was added neat with 2 mL of methanol to prepare a mobile slurry. Experiment allowed to temperature cycle for a further 24 hours.

[0987] The solids were isolated by Buchner filtration (42.5 mm 0 Grade 1 Whatman) and dried under vacuum at 40°C for 16 h, affording Pattern 2. XRPD analysis was carried out on the dried solids.

[0988] The oxalate salt was characterized by PLM, TGA / DSC, DSC, 1H NMR and HPLC-UV for purity and organic acid content.

[0989] The following results were obtained for Oxalate Pattern 2:

[0990] - XRPD analysis (figure 7) of the material isolated from methanol showed a crystalline material consistent with Oxalate Pattern 2.

[0991] - PLM showed birefringence under crossed polarised light. The crystals were small and irregular shaped.

[0992] - The TGA trace did not show any mass losses until 180°C where 32.3 wt.% was lost which was equivalent to 2 molar equivalents of oxalic acid, indicating that Oxalate Pattern 2 is a bisoxalate salt.

[0993] - The DSC trace showed an endothermic event at onset: 204°C; peak: 209°C. A second endothermic event was observed after loss of oxalic acid at onset: 251°C; peak: 267°C relating to melting of the free base material. DSC analysis showed a small endothermic event at onset: 209°C; peak: 212°C. The cooling step did not reveal any clear vitrification events, however the second heating step showed a glass transition at 123 °C in agreement with formation of amorphous material upon melting in the first heat step.

[0994] -1H NMR analysis was consistent with salt formation and only trace methanol was present supporting that Pattern 2 was an anhydrous salt.

[0995] - Determination of the organic acid content by HPLC-UV showed 32.6 %w / w of oxalic acid which was equivalent to 2 molar equivalents and therefore consistent with bis-salt.

[0996] - HPLC-UV analysis indicated that the oxalate salt Pattern 2 had a purity of 98.72 %area.

[0997] Thermodynamic solubility (according to study 1)

[0998] The thermodynamic solubility carried out on the salt showed:

[0999] Table 23

[1000] Buffer Timepoint (h) Solubility (mg / mL)

[1001] Water 0 > 18.4

[1002] 4 27.4

[1003] FaSSIF 0 > 29.4

[1004]

[1005] (pH = 6.5) 4 45.6FaSSGF 0 > 22.1

[1006]

[1007] (pH = 1.6) 4 30.8

[1008] A moderate / high solubility was observed for the oxalate salt in all media, where a solubility between 20 and 45 mg / mL was obtained.

[1009] Example 19: Besylate salt (Counter-Example)

[1010] Besylate salt of compound N°1 was prepared as follows:

[1011] Approximately 250 mg of the compound according to example 10 was weighed into a 20 mL vial. 5 mL of 2-propanol was added followed by a magnetic stirrer bar. 88.8 mg (1.05 equiv.) of benzenesulfonic acid was added neat. The vial was washed with 3.5 mL of 2-propanol and the washes were added to the reaction mixture. The solution was stirred at 40°C for 5 minutes. The experiments were thermally cycled between 20 and 50°C at a rate of 0.1°C / min with a 1 hour hold at 20 and 50°C.

[1012] An aliquot of the mixture was taken after 72 hours, was isolated by centrifugation, and analyzed by XRPD. The XRPD analysis showed that the Pattern 1 of the besylate salt was obtained. The solids were isolated by Buchner filtration (42.5 mm 0 Grade 1 Whatman), washed with cold 2-propanol and dried under vacuum at 40°C for 16 h.

[1013] XRPD analysis was carried out on the dried solids.

[1014] The besylate salt was characterized by PLM, TGA / DSC, DSC, 1H NMR and HPLC-UV for purity.

[1015] The following results were obtained for Besylate Pattern 1:

[1016] - XRPD analysis (figure 8) of the material isolated from 2-propanol showed a crystalline material consistent with Besylate Pattern 1.

[1017] - PLM showed birefringence under crossed polarised light. However, the crystals were small with an irregular-shaped.

[1018] - The TGA trace showed a 1.2 wt.% loss (equivalent to 0.43 eq. of surface moisture) in agreement with an anhydrous besylate salt.

[1019] - The DSC trace showed an endothermic melting event at onset: 221 °C; peak: 224°C prior to decomposition onset. DSC analysis showed the melting event at onset: 221 °C; peak: 224°C during the first heating step. A vitrification event was observed at ca. 54°C during the first cooling step detailing an amorphous material. The second heating step showed a glass transition with a midpoint of ca. 62°C indicating no further recrystallisation from the amorphous material.

[1020] -1H NMR analysis was consistent with Besylate Pattern 1 with only trace solvent present. The spectrum indicated ca. 1 equivalent of benzenesulfonic acid indicating a mono-salt.- HPLC-UV analysis indicated that the Besylate salt Pattern 1 had a purity of 98.95 %area.

[1021] Thermodynamic solubility (according to study 1)

[1022] The thermodynamic solubility carried out on the salt showed:

[1023] Table 24

[1024] Buffer Timepoint (h) Solubility (mg / mL)

[1025] Water 0 2.1

[1026] 4 2.2

[1027] FaSSIF 0 < 0.1

[1028] (pH = 6.5) 4 1

[1029] FaSSGF 0 7.4

[1030]

[1031] (pH = 1.6) 4 8.2

[1032] A low solubility was observed for the besylate salt in all media, where solubilities below 10 mg / mL were obtained.

[1033] Example 20: Napsylate salt (Counter-example)

[1034] Napsylate salt of compound N°1 was prepared as follows:

[1035] Approximately 250 mg of the compound according to example 10 was weighed into a 20 mL vial. 5 mL of ethyl acetate was added followed by a magnetic stirrer bar. 117 mg (1.05 equiv.) of naphthalene-2-sulfonic acid was added neat. The vial was washed with 3.5 mL of ethyl acetate and the washes were added to the reaction mixture. The solution was stirred at 40°C for 5 minutes.

[1036] The experiments were thermally cycled between 20 and 50°C at a rate of 0.1°C / min with a 1 hour hold at 20 and 50°C.

[1037] An aliquot of the mixture was taken after 72 hours, was isolated by centrifugation and analyzed by XRPD. The XRPD analysis showed that the Pattern 1 of the Napsylate salt was obtained. The solids were isolated by Buchner filtration (42.5 mm 0 Grade 1 Whatman), washed with cold ethyl acetate and dried under vacuum at 40°C for 16 h.

[1038] XRPD analysis was carried out on the dried solids.

[1039] The napsylate salt was characterized by PLM, TGA / DSC, DSC, 1H NMR and HPLC-UV for purity.

[1040] The following results were obtained for the Napsylate Pattern 1:

[1041] - XRPD analysis (figure 9) of the material isolated from ethyl acetate showed a crystalline material consistent with pattern 1.

[1042] - PLM showed birefringence under crossed polarised light. The morphology was of smallfragmented plate like particles.- The TGA trace showed a 1.4 wt.% loss (equivalent to 0.54 eq. of surface water) in agreement with an anhydrous mono-napsylate salt.

[1043] - The DSC trace showed an endothermic melting event at onset: 201 °C; peak: 207°C. DSC analysis showed the melting event at onset: 199°C; peak: 206°C during the first heating step. A vitrification event was observed at ca. 78°C during the first cooling step. The second heating step showed a glass transition with a midpoint of ca. 94°C.

[1044] -1H NMR analysis was consistent with Napsylate Pattern 1 with only trace solvent present. The spectrum indicated ca. 0.8 equivalents of Naphthalensulfonic acid. Together with the TGA data napsylate pattern 1 could be considered as a likely mono-salt.

[1045] - HPLC-UV analysis indicated that Napsylate salt Pattern 1 had a purity of 98.44 %area.

[1046] Thermodynamic solubility (according to study 1)

[1047] The thermodynamic solubility carried out on the salt showed:

[1048] Table 25

[1049] Buffer Timepoint (h) Solubility (mg / mL)

[1050] Water 0 0.1

[1051] 4 < 0.1

[1052] FaSSIF 0 < 0.1

[1053] (pH = 6.5) 4 < 0.1

[1054] FaSSGF 0 2.9

[1055]

[1056] (pH = 1.6) 4 2.8

[1057] A very low solubility was observed for the napsylate salt in all media, where a solubility below 3 mg / mL was obtained.

[1058] Example 21: In vitro activity study in melanoma cell lines with BRAF V600 mutation Study overview

[1059] The objective of this study is to investigate the single agent effect of Compound 1 on cell viability of melanoma cell lines with BRAF V600 mutation.

[1060] Experimental design

[1061] Test article: Compound 1.

[1062] Doses: 10 dose points up to 10 pM, including the no treatment control.

[1063] Readout: CellTiter-Glo® (Promega Corp.) cell viability assay.

[1064] Protocol

[1065] Cells were seeded in 25 pL culture medium at the density specified in Table 27 in black 384-well tissue culture treated plates (Perkin Elmer). Cells were equilibrated in assay plates via centrifugation and placed at 37 °C, 5% CO2 for twenty -four hours before treatment. Compoundswere transferred to assay plates using Labcyte Echo acoustic liquid handling systems (Beckman Coulter). Cells were incubated with Compound 1 for 72 hours and ATP levels quantified at endpoint by adding CellTiter-Glo® 2.0 (Promega) and luminescence read on Envision plate readers (Perkin Elmer).

[1066] Inhibition percentages are calculated as a measure of cell viability with the following formula:

[1067] %Inh = 100 x (1-T / U)

[1068] where T is the signal measure for a test article, U is the untreated control measure.

[1069] The ability of Compound 1 to inhibit cell viability was expressed as EC50(concentration of compound required to gives half-maximal effect) that were obtained by plotting the effect values (% cell viability) for each data point of a dilution series and fitting the obtained curves using the Chalice analyzer software (Horizon Discovery Group, Ltd.).

[1070] Results

[1071] Cell lines are considered high responders, medium responders or low-responders depending on the level of response for EC50:

[1072] Table 26

[1073] <200 nM High responder

[1074] 200-500 nM Medium responder

[1075]

[1076] >500 nM Low responder

[1077] Table 27. Tested melanoma cell lines: origin tissue, cell culture conditions, and mutation status

[1078] Seeding Cell Line Tissue Origin Mutation(s) Cell culture Medium Density (cell / well) A101D Skin BRAF V600E DMEM with 10% FBS 500 A375 Skin BRAF V600E DMEM with 10% FBS 500 G-361 Skin BRAF V600E McCoy’s 5 A with 10% FBS 500 Hs 294T Skin BRAF V600E DMEM with 10% FBS 500 LOXIMVI Skin BRAF V600E RPMI with 10% FBS 500 RPMI with 10% FBS, 0.01

[1079] MDA-MB- Skin BRAF V600E mg / mL Bovine Insulin and 500 435S

[1080] 10 pg / mL Glutathione

[1081] RPMI-7951 Skin BRAF V600E DMEM with 10% FBS 500 EMEM with 10% FBS, 2

[1082] SK-MEL-28 Skin BRAF V600E mM Glutamine, 1%NEAA 500 and 1 mM Sodium Pyruvate

[1083] EMEM with 15% FBS, 1% NEAA, 1.5 g / L Sodium

[1084] SK-MEL-31 Skin BRAF V600E 500 Bicarbonate and 1 mM

[1085] Sodium Pyruvate

[1086] EMEM with 10% FBS, 2

[1087] WM-115 Skin BRAF V600E, V600D 500

[1088]

[1089] mM L-glutamine, 19%Seeding Cell Line Tissue Origin Mutation(s) Cell culture Medium Density (cell / well) NEAA and 1% Sodium

[1090] pyruvate

[1091] EMEM with 10% FBS, 2

[1092] mM L-glutamine, 19%

[1093] WM-266-4 Skin BRAF V600E 500 NEAA and 1% Sodium

[1094]

[1095] pyruvate

[1096] Table 28. Compound 1 growth viability potency values (ECso) for tested melanoma cell lines

[1097] Cell Line ECso Cell Line ECso Cell Line ECso (nM) (nM) (nM) A101D 164 LOXIMVI 406 SK-MEL-28 6 A375 58 MDA-MB- 784 SK-MEL-31 1088 G-361 131 435S WM-115 167

[1098]

[1099] Hs 294T 4213

[1100]

[1101] RPMI-7951 4539

[1102]

[1103] WM-266-4 33

[1104] Table 29. Compound 1 treatment response level based on EC50

[1105] Response Level

[1106] Cell category Low Medium High ECso >500 nM 2OO< ECso<5OO nM ECso<2OO nM

[1107]

[1108] BRAF V600 Melanoma (n = 11) 36% 9% 55%

[1109] Conclusions

[1110] Compound 1 showed a strong in vitro anti-proliferative activity in melanoma cell lines with BRAF V600 mutation, including 55% high and 9% medium responders.

[1111] Example 22: Patient-derived xenograft in vivo efficacy study

[1112] Study overview and objective

[1113] A mouse clinical trial was conducted to evaluate the therapeutic efficacy of Compound 1 in a large collection of patient-derived xenograft (PDX) models of melanoma with BRAF V600 mutation. The objective of this study was to evaluate preclinically the in vivo therapeutic efficacy of Compound 1 at 60 mg / kg in the treatment of PDX models.

[1114] Animals

[1115] 5-9 weeks old female NOD / SCID, NCG, and BALB / c Nude mice (animal strain for each model listed in Table 30) are maintained on 12 h light / dark cycle at 20-26 °C with ad libitum access to autoclaved, filtered, softened water, and standard (irradiated) rodent chow.Table 30. PDX model list

[1116] Mice

[1117] PDX Model Cancer Type Animal Strain Mutation (treatment+vehicle)

[1118] ME11974 Melanoma NOD / SCID 1+1 BRAF V600EME11982 Melanoma NOD / SCID 1+1 BRAF V600EME11995 Melanoma NOD / SCID 1+1 BRAF V600E

[1119] ME12026 Melanoma NOD / SCID 1+1 BRAF V600EME12035 Melanoma NOD / SCID 1+1 BRAF V600EME12053 Melanoma NOD / SCID 1+1 BRAF V600E ME12065 Melanoma NOD / SCID 1+1 BRAF V600E

[1120] ME12071 Melanoma NOD / SCID 1+1 BRAF V600EME12095 Melanoma NOD / SCID 1+1 BRAF V600E ME12120 Melanoma NOD / SCID 1+1 BRAF V600E ME12137 Melanoma NOD / SCID 1+1 BRAF V600E ME12168 Melanoma NOD / SCID 1+1 BRAF V600E ME12172 Melanoma NOD / SCID 1+1 BRAF V600E ME12179 Melanoma NOD / SCID 1+1 BRAF V600E ME12180 Melanoma NOD / SCID 1+1 BRAF V600E ME12182 Melanoma NOD / SCID 1+1 BRAF V600E ME12208 Melanoma NOD / SCID 1+1 BRAF V600E

[1121] ME13974 Melanoma NOD / SCID 1+1 BRAF V600E

[1122]

[1123] ME13989 Melanoma NOD / SCID 1+1 BRAF V600E

[1124] Materials

[1125] Vehicle group: 0.5% CMC in purified water BID p.o. (10 mL / kg dosing volume).

[1126] Treatment group: Compound 1 dosed at 60 mg / kg BID p.o. in vehicle for 21 days.

[1127] BID doses were separated by 8±1 h.

[1128] Study - Experimental design

[1129] For each PDX model, there were 2 mice included (one treated and one control).

[1130] Each mouse was inoculated subcutaneously at the right flank with primary human tumor xenograft model tumor fragment (2-3 mm in diameter) for tumor development.

[1131] Randomization was performed based on “Matched distribution” method (Study Director™ software, version 3.1.399.19) when mean tumor volume reached 150-200 mm3.

[1132] Treatment was initiated one day after randomization (day 1).

[1133] For each mouse, the tumor volume and the body weight were measured at least twice per week during approximatively 20 days. For all mice, baseline was defined as the day the treatment started, which was noted day 0.

[1134] Tumor volumes were measured in two dimensions using a caliper, and the volume was calculated using the formula: “V = (L x W x W) / 2, where V was tumor volume, L was tumorlength (the longest tumor dimension) and W was tumor width (the longest tumor dimension perpendicular to L).

[1135] Endpoints studied

[1136] • Tumor Growth Inhibition (TGI):

[1137] Ordinal: Non responder / Responder, Low / Moderate / High / Regression

[1138] Mean

[1139] The TGI is defined from the ratio between the change from baseline for the treated group at a given timepoint x (ATx) and the change from baseline for the control group at the same timepoint (ACx):

[1140] TGIx= 100 × (1 − (ΔTx / ΔCx))

[1141] The Mean TGI was calculated for each PDX model from day 8 included.

[1142] Ordinal Mean TGI were derived from Mean TGI by defining four categories:

[1143] Table 31

[1144] Mean TGI > 100% Regression

[1145] 70% < Mean TGI < 100% High response

[1146] 30% < Mean TGI < 70% Moderate response

[1147] Mean TGI < 30% Low response

[1148]

[1149] “Responders” according to statistical analyses were defined when Mean TGI > 70%.

[1150] Time to Reach at Volume (TTRV):

[1151] The TTRV is defined for one given volume, noted “xxx”, using the following formula:

[1152] Time after volume xxx — Time before volume xxx TTRVvvv= Time before volume xxx H - — - r -, \n / (TVZ. olume a£J_ x \n(Volume before xxx)Jfter xxx) —, ]n / (TV,ol, -ume x - —xx 7) — —,\n / (TV / ol, -ume b Hefi -

[1153]

[1154] ore xxx r) The volume thresholds were set for PDX models as follows:

[1155] Table 32

[1156] TTRV threshold

[1157] PDX category

[1158] (mm3)

[1159]

[1160] BRAF V600 Melanoma 200

[1161] Results

[1162] Mean TGI are reported for each PDX model in Table 33 below.

[1163] Table 33. Individual PDX models responsePDX Model Cancer Type Mutation Time Mean TGI

[1164] ME11974 Melanoma BRAF V600E 21 7

[1165] ME11982 Melanoma BRAF V600E 8 28.7

[1166] ME11995 Melanoma BRAF V600E 8 40.6

[1167] ME12026 Melanoma BRAF V600E 10 20.9

[1168] ME12035 Melanoma BRAF V600E 25 74.2

[1169] ME12053 Melanoma BRAF V600E 14 114.6

[1170] ME12065 Melanoma BRAF V600E 14 71.7

[1171] ME12071 Melanoma BRAF V600E 11 115.4

[1172] ME12095 Melanoma BRAF V600E 22 104.2

[1173] ME12120 Melanoma BRAF V600E 14 96.7

[1174] ME12137 Melanoma BRAF V600E 14 27.8

[1175] ME12168 Melanoma BRAF V600E 15 149.4

[1176] ME12172 Melanoma BRAF V600E 13 112

[1177] ME12179 Melanoma BRAF V600E 22 115.6

[1178] ME12180 Melanoma BRAF V600E 9 98.3

[1179] ME12182 Melanoma BRAF V600E 18 117.9

[1180] ME12208 Melanoma BRAF V600E 20 43.5

[1181] ME13974 Melanoma BRAF V600E 29 80.3

[1182] ME13989 Melanoma BRAF V600E 17 52.6

[1183]

[1184] To evaluate the antitumor activity of Compound 1 at 60 mg / kg BID, the percentage of PDX models in each response category was compiled based on Mean TGI (Ordinal), as reported in Table 34 below.

[1185] Table 34. Compound 1 treatment response level based on Mean TGI

[1186] Response Level

[1187] PDX category

[1188] Low Moderate High Regression

[1189]

[1190] BRAF V600E Melanoma (n = 19) 21% 16% 26% 37%

[1191] Conclusions

[1192] Compound 1 showed strong in vivo efficacy among all tested PDX models with melanoma BRAF V600 mutation, with a high % of Responders (i.e. 63%, including 37% in regression) using Mean TGI.The TTRV analysis showed a high survival for the treatment group for BRAF V600 Melanoma (p <0.001).

[1193] REFERENCES

[1194] Caunt CJ, Sale MJ, Smith PD, Cook SJ. MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road. Nat Rev Cancer. 2015 Oct;15(10):577-92. doi: 10.1038 / nrc4000 Conroy M, Cowzer D, Kolch W, Duffy AG. Emerging RAS-directed therapies for cancer. Cancer Drug Resist. 2021 Apr 8;4(3):543-558. doi: 10.20517 / cdr.2021.07

[1195] Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature.

[1196] 2002;417(6892): 949-954. doi: 10.1038 / nature00766

[1197] Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003 Jan;3(1):11-22. doi: 10.1038 / nrc969

[1198] Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell. 2004 Oct; 6(4):313-9. doi: 10.1016 / j.ccr.2004.09.022

[1199] Kidger AM, Sipthorp J, Cook SJ. ERK1 / 2 inhibitors: New weapons to inhibit the RAS-regulated RAF-MEK1 / 2-ERK1 / 2 pathway. Pharmacol Ther. 2018;187:45-60. doi: 10.1016 / j.pharmthera.2018.02.007

[1200] Lito P, Rosen N, Solit DB. Tumor adaptation and resistance to RAF inhibitors. Nat Med. 2013 Nov;19(11):1401-9. doi: 10.1038 / nm.3392

[1201] Little AS, Smith PD, Cook SJ. Mechanisms of acquired resistance to ERK1 / 2 pathway inhibitors. Oncogene. 2013 Mar 7;32(10): 1207-15. doi: 10.1038 / onc.2012.160

[1202] Proietti I, Skroza N, Bernardini N, et al. Mechanisms of Acquired BRAF Inhibitor Resistance in Melanoma: A Systematic Review. Cancers (Basel). 2020;12(10):2801. doi:10.3390 / cancersl2102801

[1203] Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin.

[1204] 2021;71(3):209-249. doi:10.3322 / caac.21660

[1205] Yang H, Zhou X, Fu D, et al. Targeting RAS mutants in malignancies: successes, failures, and reasons for hope. Cancer Commun (Lond). 2023;43(1):42-74. doi: 10.1002 / cac2.12377

Claims

CLAIMS1. A pharmaceutically acceptable salt, and solvate thereof, of (5)-l-(l-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-lZZ-pyrrolo[2,3-Z>]pyri din-3-yl)pyridin-2(lJ7)-one of the formula (I) below:(I),wherein said salt is selected from a hydrochloride salt, a maleate salt, an esylate salt, an oxoglutarate salt, a malonate salt and an oxalate salt, for use in the treatment of melanoma with BRAF V600 mutation.

2. A pharmaceutical composition comprising the pharmaceutical acceptable salt, and solvate thereof, as defined in claim 1 and at least one pharmaceutically acceptable excipient for use in the treatment of melanoma with BRAF V600 mutation.

3. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the melanoma with BRAF V600 mutation is melanoma with BRAF V600E mutation.

4. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is a hydrochloride salt.

5. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is is a mono-hydrochloride anhydrate salt.

6. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is a hydrochloride hydrate salt.

7. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is a maleate salt.

8. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is an esylate salt.

9. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is an oxoglutarate salt.

10. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is a malonate salt.

11. The pharmaceutically acceptable salt and / or solvate thereof for use according to claim 1, or the pharmaceutical composition for use according to claim 2, wherein the salt is an oxalate salt.

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