BR112017028516B1 - CRYSTALLINE FUMARATE SALT, ITS USE, PREPARATION PROCESS AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

CRYSTALLINE FUMARATE SALT OF (S)-[3,4-DIFLUORO-2-(2-FLUORO-4- IODOPHENYLAMINO)PHENYL][3-HYDROXY-3-(PIPERIDIN-2-IL) AZETIDIN-1-YL]- METHANONE. This disclosure relates to the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl] [3-hydroxy-3-(piperidin-2-yl)azetidin -1-yl]-methanone. The disclosure also relates to pharmaceutical compositions comprising the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -il) azetidin-1-yl]-methanone. The disclosure also relates to methods of treating cancer comprising administering to a patient in need thereof, the crystalline salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl fumarate ][3-hydroxy -3- (piperidin-2-yl) azetidin-1-yl]-methanone.

Description

Cross reference to related order

[001] This application claims priority to the United States Provisional Application with serial number 62/187,009, filed on June 30, 2015. The entire contents of the aforementioned application are incorporated herein.

Technical field

[002] This disclosure relates to the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone. The disclosure also relates to pharmaceutical compositions comprising the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -il)azetidin-1-il]-methanone. The disclosure also relates to methods of treating cancer comprising administering to a patient in need thereof the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl] [3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl]-methanone.

Foundation

[003] Traditionally, dramatic improvements in cancer treatment are associated with the identification of therapeutic agents acting through new mechanisms. One mechanism that can be explored in cancer treatment is the modulation of MEK (MAPK/ERK Kinase). MEK inhibition represents a promising strategy for the treatment of cancers caused by aberrant ERK/MAPK signaling (Solit et al., 2006; Wellbrock et al., 2004). The MEK-ERK signal transduction cascade is a conserved pathway that regulates cell growth, proliferation, differentiation, and apoptosis in response to growth factors, cytokines, and hormones. This pathway operates downstream of Ras, which is often upregulated or mutated in human tumors. MEK is a critical effector of Ras function. The ERK/MAPK pathway is upregulated in 30% of all tumors and oncogenic activating mutations in K-Ras and B-Raf have been identified in 22% and 18% of all cancer types, respectively (Allen et al., 2003; Bamford S, 2004; Davies et al., 2002; Malumbres and Barbacid, 2003). A large portion of human cancers, including 66% (B-Raf) of malignant melanomas, 60% (K-Ras) and 4% (B-Raf) of pancreatic cancer, 50% of colorectal cancer (colon in particular, K-Ras: 30%, B-Raf: 15%), 20% (K-Ras) of lung cancer, 27% (B-Raf) of papillary and anaplastic thyroid cancer and 10-20% (B-Raf ) of endometrioid ovarian cancers, carrying Ras and Raf activating mutations. Inhibition of the ERK pathway, and in particular inhibition of MEK kinase activity, results in anti-metastatic and anti-angiogenic effects largely due to a reduction in cell contact and cell motility, as well as downregulation of ERK expression. vascular endothelial growth factor (VEGF). Furthermore, expression of dominant negative MEK or ERK reduced the transforming capacity of mutant Ras as seen in cell culture and in primary and metastatic growth of human tumor xenografts in vivo. Therefore, the MEK-ERK signal transduction pathway is an appropriate route to achieve therapeutic intervention and compounds that target MEK have considerable therapeutic potential.

[004] A compound that specifically inhibits MEK is (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin- 1-yl]-methanone (Compound I), which has the chemical structure: WO 2007/044515 describes the synthesis of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1- il]-methanone (Example 22b, page 231) and also describes the therapeutic activity of this molecule to inhibit, regulate and/or modulate MEK (Biochemical Assay, page 268). Compound I has been approved in the United States, Europe and elsewhere for the treatment of melanoma in combination with vemurafenib (Zelboraf®).

[005] In addition to therapeutic efficacy, a drug developer strives to provide a suitable form of the therapeutic agent that has properties suitable for processing, manufacturing, storage stability and/or utility as a drug. Consequently, the discovery of a form that possesses some or all of these desired properties is important for drug development.

[006] Applicants have discovered a crystalline salt form of Compound I that has properties suitable for use in a pharmaceutical composition for the treatment of proliferative diseases such as cancer.

summary

[007] This disclosure relates to the crystalline fumarate salt of Compound I as described herein. The fumarate salt of Compound I has the following structure and has been identified as a hemifumarate:

[008] This disclosure also relates to pharmaceutical compositions comprising a crystalline fumarate salt of Compound I.

[009] This disclosure also lists the uses of the crystalline fumarate salt of Compound I.

Brief description of the figures

[0010] Figure 1 shows the infrared spectrum of the crystalline fumarate salt of Compound I, designated as Form A.

[0011] Figure 2 shows the 1H NMR spectrum in DMSO-d6 of the crystalline fumarate salt of Compound I, designated as Form A.

[0012] Figure 3 shows the 13C NMR spectrum in DMSO-d6 of the crystalline fumarate salt of Compound I, designated as Form A.

[0013] Figure 4 shows the solid-state 13C NMR spectrum of the crystalline fumarate salt of Compound I, designated as Form A.

[0014] Figure 5 shows the positive electrospray mass spectrum for the crystalline fumarate salt of Compound I, designated as Form A.

[0015] Figure 6 shows the negative electrospray mass spectrum for the crystalline fumarate salt of Compound I, designated as Form A.

[0016] Figure 7 shows the ultraviolet spectrum for the crystalline fumarate salt of Compound I, designated as Form A, in methanol.

[0017] Figure 8 shows the display of the differential scanning calorimetry trace for the crystalline fumarate salt of Compound I, designated as Form A.

[0018] Figure 9 shows the display of the differential scanning calorimetry trace for the fumarate salt of Compound I, designated as the amorphous form.

[0019] Figure 10 shows the XRPD diffactogram for the crystalline fumarate salt of Compound I, designated as Form A.

[0020] Figure 11 shows the XRPD difactogram for the fumarate salt of Compound I, designated as the amorphous form.

[0021] Figure 12 represents the dynamic moisture sorption/desorption isotherm for the crystalline fumarate salt of Compound I, designated as Form A, at 25°C.

[0022] Figure 13 represents the dynamic moisture sorption/desorption isotherm for the fumarate salt of Compound I, designated as the amorphous form, at 25°C.

Detailed Description

[0023] This description relates to a crystalline fumarate salt of Compound I. The invention also relates to new compositions comprising the described crystalline fumarate salt of Compound I. Therapeutic uses of the crystalline fumarate salt of Compound I, such as described, as well as therapeutic compositions containing the same, represent separate aspects of the disclosure. Techniques used to characterize the crystalline fumarate salt of Compound I are described in the examples below. These techniques, alone or in combination, can be used to characterize the crystalline fumarate salt of Compound I. The crystalline fumarate salt of Compound I can also be characterized by reference to the disclosed figures.

[0024] The crystalline fumarate salt of Compound I was found to be thermodynamically stable, was the only crystalline form identified after extensive experimentation, is non-hygroscopic and is consistently formed in manufacturing. In contrast, the amorphous form is non-crystalline, hygroscopic and converts to crystalline Form A. Furthermore, when attempting to produce salts of Compound I, only the fumarate provided a single crystalline form. Other salts that could be made were amorphous or a mixture of crystalline and amorphous materials.

Crystalline fumarate salt of Compound I

[0025] This disclosure relates to the crystalline fumarate salt of Compound I. This disclosure also relates to pharmaceutical compositions of the crystalline fumarate salt of Compound I. The fumarate salt can be prepared by combining (S)-[3 ,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl-methanone (Compound I) with fumaric acid, which forms a salt with stoichiometry of 2:1 Compound I: fumaric acid. The crystalline fumarate salt of Compound I can also be referred to as a hemifumarate.

[0026] Fumaric acid has the following structure:

[0027] There are several names for Compound I, including, XL518, GDC-0973, [3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]{3-hydroxy-3-[(2S) -piperidin-2-yl] azetidin-1-yl}methanone, cobimetinib and CotellicTM.

[0028] Compound I can be prepared according to any of several different methodologies, either on a gram scale (<1 kg) or on a kilogram scale (>1 kg). A gram scale method is presented in WO 2007/044515, which describes the synthesis of Compound I (Examples 22b), which document is incorporated herein by reference in its entirety. Alternatively, Compound I can be prepared on a kilogram scale in accordance with the procedure set forth in WO 2014/059422, which is also incorporated by reference herein in its entirety, and as provided in the Examples below.

[0029] Form A has a water solubility of 1.6 mg/mL at 25 °C. Under conditions of 25 °C/0% relative humidity (RH) and 25 °C/90% RH, Form A showed no change in the test, purity, humidity and dissolution. DSC showed that Form A was stable up to the melting point of 239 °C. No solvent losses were observed.

[0030] Form A as described herein may be characterized by at least one of the following: (i) a 1H NMR spectrum in DMSO-d6 substantially as depicted in Figure 2; (ii) a 13C NMR spectrum in DMSO-d6 substantially as depicted in Figure 3; (iii) a solid state 13C NMR spectrum with three or more selected peaks at 175.3, 173.6, 117.5, 155.5 and 153.5, ± 0.2 ppm; (iv) a solid state 13C NMR spectrum substantially as depicted in Figure 4; (v) a powder X-ray diffraction pattern (CuKα À = 1.5418Â) comprising three or more 2θ values selected from 4.6, 12.1, 13.2, 13.6 and 14.5 ± 0 .2° 2θ, where the crystalline form measurement is at room temperature; (vi) an X-ray powder diffraction (XRPD) pattern substantially in accordance with the pattern shown in Figure 10; and (vii) a differential scanning calorimetry thermogram substantially in accordance with Figure 8.

[0031] In one embodiment, Form A is characterized by at least two of (i), (ii), (iii), (iv), (v), (vi) or (vii).

[0032] In another embodiment, Form A is characterized by at least three of (i), (ii), (iii), (iv), (v), (vi) or (vii).

[0033] In another embodiment, Form A is characterized by at least four of (i), (ii), (iii), (iv), (v), (vi) or (vii).

[0034] In another embodiment, Form A is characterized by at least five of (i), (ii), (iii), (iv), (v), (vi) or (vii).

[0035] In another embodiment, Form A is characterized by at least six of (i), (ii), (iii), (iv), (v), (vi) or (vii).

[0036] In another embodiment, Form A is characterized by all of (i), (ii), (iii), (iv), (v), (vi) or (vii).

[0037] In one embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three, four or five selected peaks of 175.3, 173.6, 117.5, 155.5 and 153.5, ± 0.2 ppm in the solid state 13C NMR spectrum.

[0038] In another embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one or more selected peaks at 173.6, 117.5, 155.5 and 153.5, ± 0.2 ppm 13C NMR spectrum solid state.

[0039] In another embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 175.3, 117.5, 155.5 and 153.5, ± 0.2 ppm spectrum solid state 13C NMR.

[0040] In another embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 175.3, 173.6, 155.5 and 153.5, ± 0.2 ppm spectrum solid state 13C NMR.

[0041] In another embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 175.3, 173.6, 117.5 and 153.5, ± 0.2 ppm spectrum solid-state 13C NMR analysis.

[0042] In one embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 175.3, 173.6, 117.5 and 155.5, ± 0.2 ppm spectrum solid-state 13C NMR analysis.

[0043] In one embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three, four or five selected peaks of 4.6, 12.1, 13.2, 13.6 and 14.5 ± 0 .2°2θ in the X-ray diffraction pattern (CuKα À = 1.5418Â).

[0044] In another embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 12.1, 13.2, 13.6 and 14.5 ± 0.2 °2θ in the standard X-ray diffraction (CuKα À = 1.5418Â).

[0045] In another embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin-1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 4.6, 12.1, 13.6 and 14.5 ± 0.2 °2θ in the diffraction pattern of X-rays (CuKα À = 1.5418Â).

[0046] In another embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 4.6, 13.6 and 14.5 ± 0.2 °2θ in the X-ray diffraction pattern ( CuKα À = 1.5418 Â).

[0047] In one embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -yl)azetidin- 1-yl-methanone designated as Form A is characterized by one, two, three or four selected peaks of 4.6, 12.1, 13.2 and 13.6 ± 0.2 °2θ in the standard X-ray diffraction (CuKα À = 1.5418Â ).

[0048] Other solid state properties that can be used to characterize the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3 -(piperidin-2-yl)azetidin-1-yl-methanone designated as Form A are shown in the figures and discussed in the examples below. In one embodiment, the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl) azetidin-1-yl-methanone designated as Form A is characterized by unit cell parameters approximately equal to the following: Crystal system: Tetragonal Space group: P43212 Crystal shape: Plates Unit cell dimensions a = 7.8825 Â b = 7.8825 Â c = 76.846 Â α = 90° β = 90° Y = 90° Temperature: 293 K (25 °C) Cell volume: 4774.7 Â3 Molecules in unit cell: 8 Density: 1.637 g/cm3 The parameters of the Form A unit cell were measured at a temperature of approximately 25 °C, e.g., ambient or room temperature.

[0049] In another embodiment, the disclosure refers to Form A as described herein in any of the aspects and/or embodiments, in substantially pure form.

[0050] The disclosure also relates to a process for preparing the crystalline fumarate salt of Compound I designated as Form A. The preparation, solid state properties and characteristics of the crystalline fumarate salt of Compound I designated as Form A are described in the examples below.

Pharmaceutical compositions

[0051] Another aspect of this disclosure relates to a pharmaceutical composition comprising the crystalline fumarate salt of Compound I and one or more pharmaceutically acceptable excipients. The amount of the crystalline fumarate salt of Compound I may be a therapeutically effective amount. Another aspect of this disclosure relates to a solid or dispersed pharmaceutical composition comprising the crystalline fumarate salt of Compound I, or combinations thereof and a pharmaceutically acceptable excipient.

[0052] In one embodiment, the formulation is a tablet formulation. Tablets are generally formed from the active drug, filler, disintegrant and lubricant by mixing, granulation and tableting.

[0053] Fillers are known in the art and include, for example and without limitation, sugars and sugar alcohols, cellulosics and other fillers. Non-limiting examples of suitable sugars and sugar alcohols include dextrates, dextrin, dextrose, lactose, maltodextrin, mannitol, isomalt, sorbitol, sucrose, sugar beads, xylitol, fructose, lactitol, erythritol, maltitol, xylose, glucose, mannose, galactose, maltose, cellobiose, trehalose and raffinose. Non-limiting examples of cellulosics include microcrystalline cellulose ("MCC") and silicified MCC. Non-limiting examples of other fillers include calcium carbonate, calcium sulfate, calcium silicate, chitin, chitosan, dibasic calcium phosphate dihydrate, glyceryl palmitostearate, hydrogenated vegetable oil, kaolin, magnesium aluminum silicate, magnesium carbonate , magnesium oxide, polymethacrylates, potassium chloride, cellulose powder, pregelatinized starch, sodium chloride, starch, talc and di- and tri-basic calcium phosphate. In some aspects of the disclosure, the filler is lactose, MCC, silicified MCC, dibasic calcium phosphate, mannitol, isomalt, pregelatinized starch, and combinations thereof.

[0054] Disintegrants are known in the art. Non-limiting examples include: modified starches such as sodium carboxymethyl starch (sodium starch glycolate); cross-linked polyvinylpyrrolidones such as crospovidone; modified celluloses such as croscarmellose sodium; cross-linked alginic acid; gums such as gellan gum and xanthan gum; calcium silicate. In some aspects of the disclosure, the disintegrant is croscarmellose sodium, crospovidone, sodium starch glycolate, and combinations thereof. In some aspects of the disclosure, the disintegrant is Croscarmellose sodium, sodium starch glycolate, and combinations thereof.

[0055] Lubricants are known in the art. Non-limiting examples include magnesium stearate, calcium stearate, stearic acid, sodium stearyl fumarate, hydrogenated vegetable oils, polyethylene glycol (4000-6000), and sodium lauryl sulfate. In some aspects of the disclosure, the lubricant is magnesium stearate, sodium stearyl fumarate, and combinations thereof.

[0056] In one aspect of manufacturing a tablet of the present disclosure, the filler, disintegrant and lubricant are diverted by passing through the screen to form the deagglomerated pre-mixed material. The deagglomerated premixed material is combined with an active drug in a mixing apparatus and mixed to form a premix. The premix is granulated in a dry granulation apparatus (e.g. by granulating, grinding and sieving) to form granules. The filler material, disintegrant and lubricant are present in granules as intragranular components. The additional disintegrant and lubricant are deagglomerated by passing through a screen to form a deagglomerated material which is combined with the granules in a mixing apparatus and mixed to form a final mixture. The final mixture is compressed in a tableting apparatus to form core tablets. Core tablets are coated with a coating mixture in a film coating apparatus to form coated tablets.

[0057] As used herein, intragranular refers to a component that is added prior to granulation such that the component is incorporated within the granules. As used further herein, extragranular refers to a component that is combined with granules prior to compression, such as in a tablet press.

[0058] In another tablet manufacturing process of the present disclosure, the intragranular filler and the intragranular disintegrant are deagglomerated by screening and combined with an active drug in a mixing apparatus. The components are then mixed to form a primary premix. The intragranular lubricant is deagglomerated by screening and is combined with the primary premix in a mixing apparatus. The components are then mixed to form the premix. The premix is dry granulated in a granulating apparatus by granulating, grinding and screening to form granules. The extragranular disintegrant is deagglomerated by screening and is combined with the granules in a mixing apparatus. The components are then mixed to form a final primary mixture. The extragranular lubricant is deagglomerated by screening and is combined with the final primary mixture in a mixing apparatus. The components are then mixed to form a final mixture. The final mixture is compressed in a tableting apparatus to form core tablets. A solid film coating mixture is combined with water and suspended in a suspension apparatus to form a film coating mixture. The core tablets are coated with the coating mixture in a film coating apparatus to form coated tablets.

[0059] A particular manufacturing aspect of the present disclosure comprises the following steps of premixing, granulation/grinding and screening, final mixing, tableting and coating. A premix is formed in two steps. In a first step, the intragranular lactose monohydrate, intragranular croscarmellose sodium and intragranular microcrystalline cellulose are sieved to be deagglomerated and loaded into a mixer. Deagglomeration can be done by methods known to those skilled in the art, such as passing the material through a 1.0 mm mesh screen, such as using a vibrating filter or an in-line sieve. Coimetinib hemifumarate Form A is then charged to the mixer and the contents of the mixer are mixed at a mixing speed of 6 rpm for 30 minutes. In a second step, the intragranular magnesium stearate is sieved to deagglomerate through a 0.5 mm mesh screen and charged to the mixer, and the contents are mixed at a mixing speed of 6 rpm for 8 minutes to produce the premix. In some of these aspects, a premix batch suitable for producing 420,000 tablets is manufactured in which the premix comprises 22.982 kg of microcrystalline cellulose, 15.322 kg of lactose monohydrate, 1.008 kg of croscarmellose sodium and 0.126 kg of stearate. of magnesium. The premix is dry granulated by roller compaction, milled and sieved through a 1 mm screen. In some such aspects, for an active drug with a particle size D [v, 0.5] less than 38 μm, the roller compaction force is set at 2 kN/cm and the opening size is 5 mm . In some other such aspects, for an active drug having a particle size D [v, 0.5] of at least 38 μm, the roller compaction is adjusted between 2 kN/cm to 4 kN/cm and the opening size is 4mm to 5mm. A final mixture is formed in two steps. In a first step, the extragranular croscarmellose sodium is sieved through a 1.0 mm screen for deagglomeration as described above and combined with the granulate in a mixer. The contents of the mixer are mixed at a mixing speed of 6 rpm for 10 minutes. In a second step, the extragranular magnesium stearate is sieved through a 0.5 mm screen to deagglomerate and charged to the mixer and the contents are mixed at 6 rpm for 8 minutes to form the final mixture. In those aspects where a suitable final blend batch is manufactured to produce 420 thousand tablets, the amount of extragranular croscarmellose sodium is 1.008 kg and the amount of extragranular magnesium stearate is 0.63 kg. The final mixture is compressed in a press, such as a rotary tablet press, at a main compression force of 14 kN to 19 kN to form tablet cores. Tablet cores are spray coated with a coating suspension using a pan coating apparatus known in the art. In some of these aspects, where a final batch batch suitable for producing 420,000 tablets is manufactured, the coating suspension comprises 0.806 kg of polyvinyl alcohol, 0.504 kg of titanium dioxide, 0.407 kg of Macrogol/PEG 3350, 0.298 kg of talc and an appropriate amount of purified water to form the coating suspension. In some other aspects, the coating composition is Opadry II White 85F18422. Batch sizes other than those suitable for preparing 420,000 tablets can be prepared with the same proportions of ingredients.

[0060] Suitable mixers are known in the art and include any apparatus typically used in the pharmaceutical industry to uniformly mix two or more components, including V-shaped mixers, double cone mixers, bin mixers and rotary drum mixers. The combined mixer volume, mixer fill, rotation speed and rotation time can be suitably determined by those skilled in the art, based on routine experimentation, to obtain an essentially homogeneous mixture of components. The volume of the mixer is suitably 50 L, 100 L, 200 L, 250 L or greater. Mixer fill selection allows for convection and three-dimensional material movement and is suitably about 25%, about 30%, about 35%, about 40%, about 50%, about 60% or about 70% and its ranges, such as from about 30% to about 60%, from about 45% to about 65%, from 32% to 53% or from 32% to 40%. The mixing time is appropriate, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min or more. The rotation rate is suitably, for example, 2 rpm, 3 rpm, 4 rpm, 5 rpm, 6 rpm, 7 rpm, 8 rpm, 9 rpm or 10 rpm.

[0061] Dry granulation, grinding and screening equipment are known in the art and are commercially available from several manufacturers, including Gertis, Fitzpatrick® and Fruend-Vector. Such equipment usually provides for control of roller compaction force, opening width, roller speed and feed rate. The roller surfaces may be smooth, serrated, or one roller surface may be smooth and the other roller surface may be serrated. In any of several aspects, the premix is loaded into a roller compactor feed hopper. Roller compaction is carried out at a specified force and opening size, and the process is preferably carried out under control of the opening. The formed ribbons are milled through a screen to produce granules. In some aspects of the disclosure, the screen is an integral part of the mill. The size of the opening is suitably about 2 mm, about 3 mm, about 4 mm or about 5 mm, and ranges thereof, such as from about 2 mm to about 5 mm, from about 2 mm to about 4 mm, from about 3 mm to about 5 mm or from about 4 mm to about 5 mm. The compaction force of the roller is suitably about 1 kN/cm, about 2 kN/cm, about 3 kN/cm, about 4 kN/cm, about 5 kN/cm, about 6 kN/cm , about 7 kN/cm or about 8 kN/cm and ranges thereof, such as from about 1 kN/cm to about 8 kN/cm, from about 2 kN/cm to about 5 kN/cm or from about 2 kN/cm to about 4 kN/cm. The grinding screen size is suitably 0.5mm, 0.75mm, 1.0mm, 1.25mm, 1.5mm, 1.75mm, 2.0mm, 2.25mm or 2 .5 mm, and ranges thereof, such as from about 0.5 mm to about 2.5 mm, from about 0.5 mm to about 2.0 mm, from about 0.5 mm to about 1.5mm, from about 0.5mm to about 1.25mm, from about 0.75mm to about 2.5mm, from about 0.75mm to about 2.0mm, from about 0.75mm to about 1.5mm, from about 0.75mm to about 1.25mm. In some particular aspects of the disclosure, a 1.0 mm grinding screen is used.

[0062] Suitable tablet presses are known in the art and are commercially available from, for example, Riva-Piccola, Fette, Bosch Packaging Technology, GEA and Natoli Engineering Company. Generally, each tablet is made by pressing the granules into a matrix, made up of hardened steel. The die is a disk shape with a hole cut through the center. The powder is compressed in the center of the matrix by two hardened steel punches that fit at the top and bottom of the matrix, thus forming the tablet. Tablet compression can be done in two stages, with the first stage of pre-compression involving squeezing the powder and compacting the mixture slightly before applying the main compression force to form tablets. The tablet is ejected from the die after compression. In some aspects of the disclosure, the compression force is about 5 kN, about 6 kN, about 7 kN, about 8 kN, about 9 kN, about 10 kN, about 11 kN, about 12 kN, about 13 kN, about 14 kN, about 15 kN, about 16 kN, about 17 kN, about 18 kN, about 19 kN or about 20 kN, and ranges thereof, such as from about from 5 kN to about 20 kN, from about 14 kN to about 19 kN, from about 14 kN to about 18 kN, or from about 8 kN to about 13 kN. In some aspects of the disclosure, tablets comprising about 60 mg of active drug can be formed at a compression force of about 14 kN to about 18 kN. In other aspects of the disclosure, tablets comprising about 20 mg of active drug can be formed at a compression force of about 8 kN to about 13 kN.

[0063] In some aspects, the tablet core comprises the components and weight % concentration ranges as indicated in Table A. Table A

[0064] In some aspects of the present disclosure, the tablet core comprises the components and weight % concentration ranges as indicated in Table B based on a tablet containing 20 mg of active drug. For tablets comprising except 20 mg of active drug, e.g., 40 mg or 60 mg, the proportions of the various components described below for the 20 mg tablets are maintained.Table B

[0065] In some aspects of the present disclosure, the tablet core comprises the components and weight % concentration ranges as indicated in Table C based on a tablet containing 20 mg of active drug. Table C

[0066] In some other aspects of the present disclosure, the tablet cores comprise the components and weight % concentrations as indicated in Table D based on a tablet containing 20 mg of active drug. Table D

[0067] In some other aspects of the present disclosure, the coated tablet cores comprise the components and weight % concentrations as indicated in Table E based on a tablet containing 20 mg of active drug. The components and weight % concentrations of a film coating composition are shown in Table F. Table E Table F

Treatment methods

[0068] Another aspect of this disclosure relates to methods of treating cancers comprising administering to a subject in need thereof, the crystalline fumarate salt of Compound I. In a particular embodiment, the crystalline fumarate salt of Compound I is Form A. The amount of the crystalline fumarate salt of Compound I that is administered can be a therapeutically effective amount.

[0069] In another aspect of this disclosure, the method of treatment can be practiced by administering to a patient in need thereof, a pharmaceutical composition comprising the crystalline fumarate salt of Compound I as discussed above and a pharmaceutically acceptable excipient. Another aspect of this disclosure relates to methods of treating cancers, as discussed above, wherein the cancer treated is melanoma (including BRAF V600 mutant melanoma), breast cancer (including triple negative breast cancer), colorectal cancer (including KRAS mutant colorectal cancer) non-small cell lung cancer, acute myeloid leukemia and pancreatic cancer.

[0070] BRAF inhibitors have been used to treat melanoma, and vemurafenib is a BRAF inhibitor that is currently being used to treat melanoma. Thus, another aspect of this disclosure relates to a method of treating melanoma in a subject, the method comprising administering to the subject in need of treatment a therapeutically effective amount of the crystalline fumarate salt of Compound I alone or in combination with vemurafenib. . In one embodiment, the crystalline fumarate salt of Compound I is administered before or after, or concurrently with, vemurafenib. In another embodiment, the melanoma is BRAF V600 mutant melanoma. In a particular embodiment, the crystalline fumarate salt of Compound I is administered to a patient with unresectable or metastatic melanoma with a BRAF V600 mutation. Another aspect of this disclosure relates to a method of treating BRAF V600 mutant melanoma in a subject, the method comprising administering to the subject in need of treatment a therapeutically effective amount of the crystalline fumarate salt of Compound I alone or in combination with vemurafenib. In one embodiment, the crystalline fumarate salt of Compound I is administered before or after, or concurrently with, vemurafenib. In a particular embodiment, the crystalline fumarate salt of Compound I is administered in combination with Zelboraf® (vemurafenib) for the treatment of patients with unresectable or metastatic melanoma with BRAF V600 mutation.

[0071] Tyrosine kinase inhibitors have been used to treat non-small cell lung cancer (NSCLC). Gefitinib and erlotinib are angiogenesis inhibitors that target receptors for an epidermal growth factor called tyrosine kinase that are currently being used to treat NSCLC. Other compounds are in clinical development for the treatment of non-small cell lung cancer, such as MEHD7945A. Thus, another aspect of this disclosure relates to a method of treating non-small cell lung cancer (NSCLC) in a subject, the method comprising administering to the subject in need of treatment a therapeutically effective amount of the fumarate salt. crystalline compound I, optionally in combination with erlotinib or gefitinib. In another embodiment, the combination is with erlotinib. In another embodiment, the combination is with MEHD7945A.

[0072] Another aspect of this disclosure relates to a method of treating diseases or disorders associated with uncontrolled, abnormal and/or unwanted cellular activities comprising administering to an individual in need of a therapeutically effective amount of the crystalline fumarate salt of Compound I designated as Form A. This method of treatment can be practiced by administering a pharmaceutical composition of the crystalline fumarate salt of Compound I designated as Form A.

[0073] Another aspect of this disclosure relates to a use of the crystalline fumarate salt of Compound I designated as Form A in accordance with any of the above embodiments for the manufacture of a medicament for the treatment of a disease or disorder discussed. above. A pharmaceutical composition can be any pharmaceutical form that contains the crystalline fumarate salt of Compound I. The pharmaceutical composition can be, for example, a tablet, capsule, topical or transdermal. Pharmaceutical compositions generally contain from about 1% to about 99% by weight of the active compound(s), or a crystalline form of the active compound(s), and 99% to 1 % by weight of one or more suitable pharmaceutical excipients. In one example, the composition will be between about 5% and about 75% by weight of the active compound, the remainder being suitable pharmaceutical excipients, as discussed below.

[0074] A "therapeutically effective" amount of the crystalline fumarate salt of Compound I refers to an amount sufficient to treat a patient suffering from cancer. A therapeutically effective amount according to this disclosure is an amount therapeutically useful for treating or preventing the disease states and disorders discussed herein. The crystalline fumarate salt of Compound I disclosed herein has therapeutic activity to inhibit, regulate and/or modulate signal transduction of kinases, particularly MEK 1/2 as described in WO 2007/044515.

[0075] The actual amount required to treat any particular patient will depend on a variety of factors, including the state of the disease being treated and its severity; the specific pharmaceutical composition used; the patient's age, body weight, general health, sex, and diet; the mode of administration; administration time; the route of administration; and the rate of excretion of the active compound(s), or a crystalline form of the active compound(s), in accordance with this disclosure; the duration of treatment; any drugs used in combination or coincidence with the specific compound used; and other such factors well known in the medical arts. These factors are discussed in Goodman and Gilman "The Pharmacological Basis of Therapeutics", Tenth Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, which is incorporated herein by reference. The active compound(s), or a crystalline form of the active compound(s), according to this disclosure and pharmaceutical compositions comprising them, can be used in combination with anticancers or other agents that are generally administered to a patient being treated for cancer. They can also be co-formulated with one or more of these agents in a single pharmaceutical composition.

[0076] Depending on the type of pharmaceutical composition, pharmaceutically acceptable excipients can be chosen from any one or a combination of excipients known in the art. The choice of pharmaceutically acceptable excipients depends in part on the desired method of administration to be used. For a pharmaceutical composition of this disclosure, that is, one of the active compounds, or a crystalline form of the active compound(s), of this disclosure, an excipient must be chosen so as to substantially maintain the particular form of the ( s) active compound(s), whether crystalline or not. In other words, any excipient must not substantially change the form of the active compound(s). Nor should the carrier otherwise be incompatible with the form of the active compound(s), such as producing any undesirable biological effect or otherwise interacting in a detrimental manner with any other component(s) of the pharmaceutical composition. .

[0077] The pharmaceutical compositions of this disclosure can be prepared by methods known in the art of pharmaceutical formulation, for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990). In solid dosage forms, Compound I is admixed with at least one pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate or (a) other excipients such as fillers or extenders, such as, for example, starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as cellulose derivatives, starch, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia gum; (c) humectants, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates and sodium carbonate; (e) solution retardants, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate, magnesium stearate and the like; (h) adsorbents, such as kaolin and bentonite; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0078] Pharmaceutically acceptable excipients, typically called adjuvants, known in the art of pharmaceutical formulation can also be used in the pharmaceutical compositions of this disclosure. These include, but are not limited to, agents for preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying and distributing. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. If desired, a pharmaceutical composition of this disclosure may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and antioxidants, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate and butylated hydroxytoluene. .

[0079] Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain pacifying agents, and may also be of such composition that they release the active compound or compounds into a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0080] Suspensions, in addition to active compounds, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures of these substances, and the like.

[0081] Compositions for rectal administrations are, for example, suppositories that can be prepared by mixing the active compound(s), or a crystalline form of the active compound(s), with, for example, suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore melt while in a suitable body cavity and release the active component in it.

[0082] As the active compound(s), or a crystalline form of the active compound(s), is maintained during its preparation, solid dosage forms are preferred for pharmaceutical composition of this disclosure. Solid dosage forms for oral administration, which include capsules, tablets, pills, powders and granules, are particularly preferred. In such solid dosage forms, the active compound(s) are mixed with at least one inert, pharmaceutically acceptable excipient. Administration of the active compound(s), or a crystalline form of the active compound(s), in pure form or in an appropriate pharmaceutical composition, may be carried out by either method. accepted from administration or agents to serve similar utilities. Thus, administration may be, for example, orally, nasally, parenterally (intravenously, intramuscularly or subcutaneously), topically, transdermally, intravaginally, intravesically, intracologically or rectally, in the form of solid, semisolid, lyophilized powder or dosage forms. liquids, such as, for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions or aerosols or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. A preferable route of administration is oral administration, using a convenient dosage regimen that can be adjusted according to the degree of severity of the disease state being treated.

Preparation of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl]-methanone (Compound I)

[0083] Compound I can be prepared as described in WO 2014/059422, the entire contents of which are incorporated herein by reference, and as generally represented in Scheme 1. Reaction of (3S,5R,8aS)-3-phenylhexa- commercially available hydro-oxazolo[3,2-a]pyridine-carbonitrile VII with commercially available tert-butyl-3-oxo-1-azetidinecarboxylate VIIa in the presence of base provides compound VI. Compound VI is treated with a hydride reducing agent such as sodium cyanoborohydride in the presence of acid, followed by treatment with aqueous sodium hydroxide, to provide compound V. Deprotection of V using acid gives compound IV, which is coupled to acid chloride IVa in the presence of a catalytic amount of pyridine to provide III. Hydrogenation of III gives piperidine derivative II. Finally, coupling II with 2-fluoro-4-iodo aniline IIa provides the desired compound. Scheme 1

General methods for preparing crystalline forms

[0084] Crystalline forms can be prepared by a variety of methods including, but not limited to, for example, crystallization or recrystallization from a suitable solvent mixture; sublimation; growth of a molten mass; solid state transformation of another phase; crystallization from a supercritical fluid; and jet spraying. Techniques for crystallizing or recrystallizing crystalline forms from a mixture of solvents include, but are not limited to, for example, evaporation of the solvent; decreasing the temperature of the solvent mixture; seeding crystals from a supersaturated solvent mixture of the compound and/or a salt thereof; crystal seeding a supersaturated solvent mixture of the compound and/or a salt thereof; freeze-dry the solvent mixture; and adding antisolvents (countersolvents) to the solvent mixture. High-yield crystallization techniques can be used to prepare crystalline forms including polymorphs.

[0085] Drug crystals, including polymorphs, methods of preparation and characterization of drug crystals are discussed in Solid-State Chemistry of Drugs, S.R. Byrn, R. R. Pfeiffer, and J. G. Stowell, 2nd edition, SSCI, West Lafayette, Indiana (1999).

[0086] In a crystallization technique in which solvent is used, the solvent(s) are typically chosen based on one or more factors including, but not limited to, for example, solubility of the compound; crystallization technique used; and vapor pressure of the solvent. Combinations of solvents can be used. For example, the compound may be solubilized in a first solvent to give a solution to which the antisolvent is then added to decrease the solubility of the compound in the solution and precipitate the formation of crystals. An antisolvent is a solvent in which a compound has low solubility.

[0087] In a method that can be used in the preparation of crystals, the fumarate salt of Compound I can be suspended and/or stirred in a suitable solvent to provide a suspension, which can be heated to promote dissolution. The term "suspension" as used herein means a saturated solution of the compound, wherein such solution may contain an additional amount of compound to provide a heterogeneous mixture of compound and solvent at a given temperature.

[0088] Seed crystals can be added to any crystallization mixture to promote crystallization. Seeding can be used to control the growth of a particular polymorph and/or to control the particle size distribution of the crystalline product. Therefore, the calculation of the quantity of seeds required depends on the available seed size and the desired size of an average particle of the product, as described, for example, in "Programmed Cooling Batch Crystallizers", J. W. Mullin and J. Nyvlt, Chemical Engineering Science, 1971, 26, 3690377. In general, small seed sizes are necessary to effectively control crystal growth in the batch. Small-sized seeds can be generated by sieving, grinding, or micronizing large crystals, or by microcrystallizing a solution. When grinding or micronizing crystals, care must be taken to avoid changing the crystallinity of the desired crystalline form (i.e., changing to an amorphous or other polymorphic form).

[0089] A cooled crystallization mixture can be filtered under vacuum and the isolated solid product washed with a suitable solvent, such as, for example, cold recrystallization solvent. After being washed, the product can be dried under a nitrogen purge to provide the desired crystalline form. The product may be analyzed by a suitable spectroscopic or analytical technique including, but not limited to, for example, differential scanning calorimetry (DSC); X-ray powder diffraction (XRPD); and thermogravimetric analysis (TGA) to ensure the crystalline form of the compound formed. The resulting crystalline form can be produced in an amount greater than about 70% by weight of isolated yield, based on the weight of the compound originally used in the crystallization process, and preferably greater than about 90% by weight of isolated yield. . Optionally, the product can be deagglomerated by being co-ground or passed through the mesh screen.

[0090] The characteristics and advantages of this description can be more easily understood by those skilled in the art after reading the detailed description below. It should be appreciated that certain features of the invention which are, for reasons of clarity, described above and below in the context of separate embodiments, may also be combined to form a single embodiment. On the other hand, various features of this disclosure that are, for the sake of brevity, described in the context of a single embodiment, may also be combined to form subsets thereof. The disclosure is further illustrated by the following examples, which should not be construed as limiting the disclosure in scope or spirit to the specific procedures described therein.

[0091] The definitions presented here prevail over the definitions presented in any patent, patent application and/or patent application publication incorporated herein by reference. All measurements are subject to experimental error and are within the spirit of the invention.

[0092] As used herein, "amorphous" refers to a solid form of a molecule and/or ion that is not crystalline. An amorphous solid does not exhibit a definitive X-ray diffraction pattern with sharp maxima.

[0093] As used herein, the term "substantially pure" means that Form A contains at least about 90% by weight based on the weight of such crystalline form. The term "at least about 90% by weight", while not intended to limit the applicability of the doctrine of equivalents to the scope of the claims, includes, but is not limited to, for example, about 90, about 91, about 92 , about 93, about 94, about 95, about 96, about 97, about 98, about 99 and about 100% by weight, based on the weight of said crystalline form. The remainder of Form A may comprise other fumarate salt form(s) of Compound I and/or reaction impurities and/or processing impurities that arise, for example, when the crystalline form is prepared. The presence of reaction impurities and/or processing impurities can be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear magnetic resonance spectroscopy, mass spectroscopy and/or infrared spectroscopy.

[0094] In another aspect, the invention relates to a process for preparing the crystalline fumarate salt of Compound I designated as Form A, comprising: adding fumaric acid dissolved in a solvent to a mixture of Compound I dissolved in a solvent to form the crystalline fumarate salt of Compound I designated as Form A; and collecting the resulting crystals of the crystalline fumarate salt of Compound I designated as Form A.

[0095] In this embodiment, the solvents that are used are polar solvents. Depending on the solubility of fumaric acid and/or Compound I in a particular solvent, gentle heating (40-80°C) may be necessary to ensure complete dissolution. For example, fumaric acid can be dissolved in a polar protic solvent such as an alcohol (for example, methanol, ethanol, n-propanol or isopropanol or the like), alone or in mixture with other solvents or with water. Alternatively, fumaric acid can be dissolved in an aprotic solvent such as tetrahydrofuran, dichloromethane or the like. Similarly, Compound I can be dissolved in dichloromethane or in a polar solvent such as an alcohol (for example, methanol, ethanol, n-propanol or isopropanol or the like), alone or in admixture with one or more other solvents or with water. The fumaric acid solution is then added to the Compound I solution and the resulting mixture is allowed to stand until a precipitate forms. In some cases, to speed up crystal formation, the resulting mixture is cooled or a seed crystal is added. In other cases, an antisolvent such as a non-polar hydrocarbon solvent such as heptane or the like is used to expedite crystal formation.

[0096] Thus, in another aspect, the invention relates to a process for preparing the crystalline fumarate salt of Compound I designated as Form A, comprising: dissolving Compound I in a first solvent to form a first mixture; dissolving the fumaric acid in a second solvent to form a second mixture; adding the first mixture to the second mixture with cooling to form the crystals as a precipitate; and collecting crystals of the crystalline fumarate salt of Compound I designated as Form A.

[0097] As in the previous aspect, the solvents that are used are polar solvents. In a particular embodiment, the first and second solvents are the same and are a mixture of isopropanol and water. In one embodiment, the ratio of isopropanol to water is 9:1. In another embodiment, the ratio of isopropanol to water is 4:1. In another embodiment, the ratio of isopropanol to water is 85:15. Typically, approximately 7 to 11 weight equivalents of the first solvent are used for each weight equivalent of Compound I, and 2.0 to 3.0 weight equivalents of the second solvent are used for each weight equivalent of fumaric acid. More particularly, approximately 8 to 10 weight equivalents of the first solvent are used for each weight equivalent of Compound I, and 2.4 to 2.7 weight equivalents of the second solvent are used for each weight equivalent of fumaric acid. .

[0098] One molecule of fumaric acid forms a salt with two molecules of Compound I to form the hemifumarate salt of Compound I. Thus, about 0.5 equivalents of fumaric acid are used for each equivalent of Compound I. Typically, 0 .51 to .53 equivalents are used for each equivalent of Compound I.

[0099] In a typical example, before adding fumaric acid, Compound I dissolved in the first solvent is filtered, for example, through activated carbon. The fumaric acid dissolved in the second solvent is then slowly added to the solution of Compound I in the first solution with gentle heating to a temperature of approximately 4090 ° C; more preferably 60-85°C; and more preferably 75-80°C. In some cases, seed crystals can be added to the mixture of Compound I and fumaric acid in the propanol/water solvent. To complete the crystallization process, the mixture can be cooled to approximately 20 °C. The resulting crystals are isolated by filtration.

[00100] In another aspect, the invention relates to a process for preparing the crystalline fumarate salt of Compound I designated as Form A, comprising: adding fumaric acid dissolved in a solvent to a mixture of Compound I dissolved in a solvent to form the crystalline fumarate salt of Compound I designated as Form A as a precipitate.

[00101] In one embodiment of this aspect, the process further comprises adding seed crystals of the fumarate salt of Compound I designated as Form A into the mixture.

[00102] In a further embodiment, the invention relates to a process for preparing the crystalline fumarate salt of Compound I designated as Form A, comprising dissolving the amorphous form of Compound I in a solvent with gentle heating to 65-80°C and then allowing the resulting mixture to cool until crystals form. In one embodiment, seed crystals may be added to the mixture. In another embodiment, the mixture may be cooled to approximately 20 °C. The resulting crystals are then isolated by filtration.

Forms of embodiment

[00103] The invention is characterized by the following non-limiting embodiments.

[00104] Embodiment 1. A crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone designated as Form A.

[00105] Embodiment 2. The crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone of Embodiment 1, designated as Form A, wherein said salt is characterized by at least one of the following: (i) a 1H NMR spectrum in DMSO d6 substantially as depicted in Figure 2; (ii) a 13C NMR spectrum in DMSO-d6 substantially as depicted in Figure 3; (iii) a solid state 13C NMR spectrum with three or more selected peaks at 175.3, 173.6, 117.5, 155.5 and 153.5, ± 0.2 ppm; (iv) a solid state 13C NMR spectrum substantially as depicted in Figure 4; (v) a powder X-ray diffraction pattern (CuKα À = 1.5418Â) comprising three or more 2θ values selected from 4.6, 12.1, 13.2, 13.6 and 14.5 ± 0 .2 °2θ, where the measurement of the crystalline form is at room temperature; (vi) an X-ray powder diffraction (XRPD) pattern substantially in accordance with the pattern shown in Figure 10; and (vii) a differential scanning calorimetry thermogram substantially in accordance with Figure 8.

[00106] Embodiment 3. The crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone of Embodiment 1, designated as Form A, wherein said salt is characterized by a solid state 13C NMR spectrum with three or more peaks selected from 175.3, 173 .6, 117.5, 155.5 and 153.5, ± 0.2 ppm.

[00107] Embodiment 4. The crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone of Embodiment 1, designated as Form A, wherein said salt is characterized by a powder X-ray diffraction pattern (CuKα À = 1.5418 Â) comprising three or plus selected 2θ values of 4.6, 12.1, 13.2,13.6 and 14.5 ± 0.2°2θ, where the crystalline form measurement is at room temperature.

[00108] Embodiment 5. The crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone of embodiments 1-4, wherein said salt is at least 90% by weight of Form A, based on the weight of said salt.

[00109] Embodiment 6. A pharmaceutical composition comprising (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin) crystalline fumarate salt -2-yl)azetidin-1-yl]-methanone of any of Embodiment Forms 1-3, designated as Form A; and a pharmaceutically acceptable excipient.

[00110] Embodiment 7. Use of the crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2 -il)azetidin- 1-yl]-methanone of any one of Embodiment Forms 15, designated as Form A, for the manufacture of a medicine for the treatment of cancer.

[00111] Embodiment 8. The crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone of any of Embodiment Forms 15, designated as Form A, for use in therapy in the treatment of cancer.

[00112] Embodiment 9. The crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone designated as Form A, for use as a medicine for the treatment of cancer that is selected from the group consisting of melanoma (including BRAF V600 mutant melanoma), breast cancer (including breast cancer triple negative), colorectal cancer (including KRAS mutant colorectal cancer), non-small cell lung cancer, acute myeloid leukemia, and pancreatic cancer.

[00113] Embodiment 10. The use of Embodiment 9, wherein the cancer is BRAF V600 mutant melanoma.

[00114] Embodiment 11. The crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2- il)azetidin-1-yl]-methanone designated as Form A, in combination with vemurafenib for use as a medication for the treatment of melanoma.

[00115] Embodiment 12. A method of treating BRAF V600 mutant melanoma in a subject, the method comprising administering to the subject in need of treatment a therapeutically effective amount of the crystalline fumarate salt of Compound I alone or in combination with vemurafenib.

[00116] Embodiment 13. The method of Embodiment 12, in which the crystalline fumarate salt of Compound I is administered before or subsequently, or concurrently with vemurafenib.

[00117] Embodiment 14. A process for preparing the crystalline fumarate salt of Compound I designated as Form A, comprising adding fumaric acid dissolved in a solvent to a mixture of Compound I dissolved in a solvent to form the salt of crystalline fumarate of Compound I.

[00118] The following examples illustrate the scope of the invention. The following examples and preparations are provided to enable those skilled in the art to more clearly understand and practice the present invention. They should not be considered as limiting the scope of the invention, but only as being illustrative and representative thereof. Example 1 Synthesis of tert-butyl ester of 3-((3S,5R,8aS)-5-cyano-3-phenylhexa-hydro-oxazolo[3,2-a]pyridin-5-yl)-3- acid hydroxy-azetidine-1-carboxylic acid

[00119] A mixture of (3S,5R,8aS)-3-phenylhexa-hydro-oxazolo[3,2-a]pyridine-carbonitrile (20.0 g, 87.6 mmol, 1.0 eq) and dimethyltetrahydropyrimidone (DMPU, 11.3 g, 87.6 mmol, 1.0 eq.) in THF (95.1 mL) was stirred for 10 min until a clear solution was observed. The mixture was then cooled to -70 to -80°C and lithium diisopropylamide (28% solution in heptane, THF and ethylbenzene) (35.2 g, 92 mmol, 1.05 eq.) was added over 30 min while maintained the internal temperature between -70 to -80 °C. After complete addition, the mixture was stirred at -70 to -80 °C for a further 2 hours, followed by dosing with a solution of 3-oxo-azetidine-1-carboxylic acid tert-butyl ester (16.2 g, 94.6 mmol, 1.08 eq.) in THF (16.4 g) over 30 min while maintaining the internal temperature between -70 to -80 °C. After complete dosing, the reaction mixture was stirred at -70 to -80 °C for 1 hour.

[00120] In a separate vial, a solution of sodium chloride (10.3 g), deionized water (103.0 g) and acetic acid (5.29 g, 87.6 mmol, 1.0 eq.) was prepared and cooled to 0 °C. The reaction mixture was dosed onto the cooling mixture for 30 minutes while maintaining the internal temperature at less than 10°C. The reaction mixture vial was rinsed with THF (26.7 g) and the rinse was combined with the quenched mixture. After vigorous stirring for 20 min at 5 °C, stirring was stopped and the layers were allowed to separate. The lower aqueous phase was discarded. Ethyl acetate (61.8 g) and deionized water (68.5 g) were added to the organic phase. After vigorous stirring at 5 °C for 10 min, stirring was stopped, the layers were allowed to separate, and the lower aqueous phase was discarded. The washing procedure was repeated once with deionized water (68.5 g).

[00121] The organic phase was concentrated under reduced pressure (shell temperature approximately 4045 °C, pressure = 200-180 mbar (0.02-0.018 MPa)) until a total volume of approximately 120 mL of distillate was obtained resulting in a yellowish solution. The vacuum was released and heptane (102.0 g) was added over 10 min. Distillation under reduced pressure was continued (shell temperature approximately 35-40 °C, pressure approximately 250-110 mbar (0.025-0.011 MPa)) by adding heptane (177 g) at a rate such that the residual volume was maintained constant. After 10 minutes of distillation, a thick, stirred suspension was obtained. The vacuum was released and isopropanol (10.2 g) was added over 15 min at 35 °C. The suspension was heated to 45°C and stirred for 30 min. Subsequently, the suspension was cooled to 0 °C over 2 hours and maintained at 0 °C for 1 hour. The suspension was filtered over a glass filter. The flask and filter cake were rinsed with pre-cooled (approximately 5°C) heptane (46.6 g) and the wet cake was dried overnight at 40°C under reduced pressure until constant weight to yield the compound of title as slightly beige crystals. Purity by HPLC: area 91.9%. FOOT. (DSC): extrapolated peak: 151.80°C. 1H-NMR (600 MHz, CDCl3): δ 7.30 - 7.50 (m, 5 H), 4.17 - 4.27 (m, 3 H), 3.94 - 4.01 (m, 2 H), 4.11 - 4.1 (m, 2H), 4.09 (d, 1H), 3.95 (d, 1H), 3.87 (dd, 1H), 3.76 (dd, 1H), 3.54-3.70 (wide, 1H), 2,853.03 (br, 1H), 2.18 - 2.25 (m, 1H), 2.12 (wide, 1H), 1 .97 - 2.04 (m, 1H), 1.85 - 1.94 (m, 1H), 1.61 - 1.79 (m, 3H), 1.41 (s, 9H). MS(EI): m/z = 400.48 ([M + H]+, 100%). Example 2 Synthesis of 3-hydroxy-3-[(S)-1-((S)-2-hydroxy-1-phenyl-ethyl)-piperidin-2-yl]azetidine-1-carboxylic acid tert-butyl ester

[00122] A mixture of 3-((3S,5R,8aS)-5-cyano-3-phenylhexa-hydro-oxazolo[3,2-a]pyridin-5-yl)- acid tert-butyl ester 3-hydroxyazetidine-1-carboxylic acid (12.0 g, 30.0 mmol, 1.0 eq.) and sodium cyanoborohydride (3.18 g, 50.6 mmol, 1.68 eq.) in EtOH (70 mL) was heated to 30 °C and added slowly within 2 hours to a hot (70 °C) mixture of acetic acid (3.63 mL, 63.5 mmol, 2.1 eq.) in EtOH ( 20 mL). The resulting mixture was subsequently stirred for a further 3 hours at 70 to 75°C. After complete reaction, the mixture was cooled to 23 °C and slowly dosed within 30 min into a mixture of toluene (100 mL) and aqueous NaOH (60 g, 10%-w/w) and stirred for 15 min. The reaction flask was rinsed with the quenched mixture. The layers were separated and the organic phase was washed with toluene (30 ml). The combined organic phases were concentrated under vacuum (200 to 85 mbar (0.02 to 0.0085 MPa) at a jacket temperature of 35 to 40 °C) until 80 mL (70.82 g) of a product solution were obtained. yellowish. Purity by HPLC: area 97.6%.

[00123] For analytical purposes, the product solution was fully concentrated in the rotary evaporator, treated with EtOH and again fully concentrated resulting in 19.2 g of a foamy product. The residue was dissolved in a mixture of ethyl acetate (30 mL) and MeOH (15 mL) and purified by flash chromatography on 120 g of silica gel using ethyl acetate as eluent. Fractions 3 to 5 of 6 fractions of 100 mL each were combined and fully concentrated under vacuum on the rotary evaporator resulting in 14.6 g of colorless foam. This residue was dissolved again in a minimum of a mixture of heptane/ethyl acetate 2:1 (v/v) and purified by flash chromatography on 190 g of silica gel using heptane/ethyl acetate 2:1 (v/v). as eluent. After a 700 mL precursor, ten subsequent fractions (800 mL total) were combined, evaporated completely on the rotary evaporator under vacuum (bath temperature 35 °C, pressure > 20 mbar (0.002 MPa)) and the residue it was dried overnight at 35°C and under vacuum to constant weight to yield the title compound as a colorless solid. PE.(DSC): extrapolated peak: 220.9 °C (melting accompanied by exothermic decomposition). 1H-NMR (600 MHz, CDCl3): δ 7.38 - 7.41 (m, 2 H), 7.34 - 7.38 (m, 2 H), 7.27 - 7.30 (m, 1 H), 4.28 - 4.50 (broad, 1H), 4.19 (dd, 1H), 4.11 - 4.1 (m, 2H), 4.09 (d, 1H), 3 .95 (d, 1H), 3.87 (dd, 1H), 3.83 (t, 1H), 3.08 - 3.16 (m, 1H), 2.85 (ddd, 1H), 2, 57 (ddd, 1H), 1,761.84 (m, 1H), 1.68-1.75 (m, 1H), 1.53 - 1.58 (m, 1H), 1.41 - 1.48 (bs, 9 H), 1.31 - 1.41 (m, 2 H), 1.21 - 1.31 (m, 2 H). MS(EI): m/z = 377.24 ([M + H]+, 100%). EA for C21H32N2O4: calculated: C 66.99, H 8.57, N 7.44; found C 67.38, H 8.50, N 7.29. Example 3 Synthesis of 3-[(S)-1-((S)-2-hydroxy-1-phenyl-ethyl)-piperidin-2-yl]-azetidin-3-ol dihydrochloride

[00124] A solution of tert-butyl ester of 3-hydroxy-3-[(S)-1-((S)-2-hydroxy-1-phenyl-ethyl)-piperidin-2-yl]azetidine-1 acid -carboxylic acid (69.8 g, 29.6 mmol, 1.0 eq.) in toluene was treated at 23-27 °C for 12 minutes with a mixture of water (30.1 g) and HCl (37%, 7 .22 g, 73.3 mmol, 2.5 eq.) and stirred for 10 min. The resulting biphasic mixture was heated to 50°C for 30 minutes and kept stirring for 4 hours at 50°C. After complete conversion, the mixture was cooled to room temperature and the phases were allowed to separate. The aqueous phase was washed with toluene (36 ml) and the phases were allowed to separate, resulting in 44.2 g of a yellowish aqueous product solution. Purity by HPLC: area 96.3%.

[00125] For analytical purposes, the product solution was fully concentrated in the rotary evaporator (bath temperature 45 °C). The yellow oily residue was dissolved in MeOH (190 mL) and again fully concentrated on the rotary evaporator under vacuum. The residue was taken up at least from a mixture of MeOH/ethyl acetate 1:1 (v/v) and purified by flash chromatography on silica gel (150 g) using a mixture of MeOH/ethyl acetate 1:1 (v /v) as eluent. A portion of 400 mL was removed and discarded and the subsequent fractions (1.5 L) were combined and completely concentrated in the rotary evaporator under vacuum (bath temperature 40 °C, pressure > 20 mbar (0.002 MPa)) resulting in an oil yellow which was dissolved in MeOH (20 mL). The oil was added dropwise at room temperature to ethyl acetate (80 mL), after which the product precipitated. The solids were filtered and rinsed with ethyl acetate (30 mL). Dry overnight at 30°C under vacuum until constant weight yields the title compound (22.0 g) as a colorless solid. PE (DSC): Initial 114.2°C, extrapolated peak: 123.4°C. 1H-NMR (600 MHz, DMSO-d6): δ 9.50 - 9.64 (br, 1H), 8.91 - 9.03 (br, 1H), 7.78 (s, 1H), 7 .62 - 7.56 (m, 2H) 7.41 - 7.52 (m, 3H), 6.03 (bs, 1H), 4.56-4.67 (m, 1H), 4.45 (dd, 1H), 4.25-4.33 (m, 2H), 4.23 (dd, 1H), 4.18 (dd, 1H), 3.95-4.05 (m, 1H) , 3.83 (dd, 1H), 3.45 - 3.54 (m, 1H), 3.26 - 3.40 (m, 1H) 1.67 - 1.86 (m, 4H), 1.55-1.65 (m, 1 H), 1.37 - 1.51 (m, 1 H). MS (EI): m/z = 277 ([M + H]+ free base, 100%). EA for C16H26N2O2Cl2, corrected for water (9.2% -w/w) and HCl (2.1 eq instead of 2.0 eq): calculated: C 49.44, H 7.80, N 7.21 , O 16.40, Cl 19.15; found C 48.76, H 7.48, N 7.36, O 16.44, Cl 19.11. Example 4 {3-Hydroxy-3-[(S)-1-((S)-2-hydroxy-1-phenyl-ethyl)-piperidin-2-yl]-azetidin-1-yl}- (2.3 ,4-trifluoro-phenyl)- Methanone

2,3,4-trifluoro-benzoyl chloride:

[00126] 2,3,4-Trifluorobenzoic acid (100 g, 568 mmol, 1.0 eq.) was suspended in toluene (1000 mL) and treated with pyridine (0.254 mL, 3.15 mmol, 0.0055 eq. .). The resulting suspension was heated to 60 to 70 °C, after which the mixture became a clear yellowish solution. At this temperature, oxalyl chloride (94.4 g, 729 mmol, 1.3 eq.) was added slowly over 156 minutes. After complete addition, the mixture was kept stirring for 10 min until complete. Toluene (360 mL) was partially removed by vacuum distillation (shell temperature: 60 to 70 °C, pressure: 200 to 100 mbar (0.02 to 0.01 MPa)). The solution was cooled to room temperature, resulting in 636 g of a yellowish and slightly cloudy solution that was stored under an N2 atmosphere and used in the subsequent step without any other treatment. Purity by HPLC: area 99.2%.

{3-Hydroxy-3-[(S)-1-((S)-2-hydroxy-1-phenyl-ethyl)-piperidin-2-yl]-azetidin-1-yl}-(2,3,4 -trifluoro-phenyl- methanone:

[00127] The aqueous solution of 3-[(S)-1- ((S)-2-hydroxy-1-phenyl-ethyl)-piperidin-2-yl]-azetidin-3-ol dihydrochloride (43.5 g) was treated with EtOH (24 ml) and stirred for 10 minutes at room temperature. To this mixture was added a solution of tripotassium phosphate (28.8 g, 136 mmol, 4.7 eq.) in 261 mL of water within 14 minutes at a batch temperature of 10 to 20°C and the mixture was stirred for 15 min at 15 °C (pH 11.9). To this solution was added via dropping funnel 34 g of the above-described 2,3,4-trifluoro-benzoyl chloride solution (34.0 g, 29.8 mmol, 1.0 eq.) over 32 minutes at a batch temperature of 10 to 20 °C while shaking vigorously. The dropping funnel was rinsed with toluene (1.2 mL) and the two-phase mixture was stirred at room temperature for 60 min. The layers were allowed to separate, and the aqueous phase was discarded. The organic phase was washed with a solution of sodium carbonate (3.36 g, 31.5 mmol, 1.09 eq.) in water (42 g) and stirred for 30 min at room temperature. The layers were allowed to separate, and the organic phase was washed with aqueous sodium chloride (30 g, 10% w/w). In the rotary evaporator (bath temperature 50°C, pressure <200 mbar (0.02 MPa)), the organic phase was concentrated to a volume of approximately 30%. The residue was taken up in EtOH (23 mL) and stirred for 5 min at 40 to 50 °C. The solution was concentrated again in the rotary evaporator (bath temperature 50 °C, pressure less than 200 mbar (0.02 MPa), 17 mL of distillate), resulting in a very viscous oil. The residue was again taken up in EtOH (23 mL) and stirred for 10 minutes and diluted again with EtOH (12 mL) to reach the target volume (53 mL, 46.06 g). Purity by HPLC: area 85.0%.

[00128] For analytical purposes, the product solution (90 mL) was filtered and the filter residue was washed with EtOH (15 mL). In the rotary evaporator (bath temperature 40 °C, pressure <150 mbar (0.015 MPa)), the solution was completely concentrated and the residue was taken up in MTBE (40 mL), subsequently fully concentrated again, then absorbed into an acetate mixture of ethyl (29 mL) and heptane (40 mL), then fully concentrated, then again in a mixture of MTBE (20 mL) and heptane (50 mL) and again fully concentrated resulting, finally, in a foamy solid (32.5 g). The solid residue (32.0 g) was dissolved in ethyl acetate (20 ml) and purified by flash chromatography on silica gel (150 g) using ethyl acetate as eluent. After a previous period of 200 mL, 6 fractions (800 mL) were combined and completely concentrated on the rotary evaporator (bath temperature: 40 °C, pressure ^ 20mbar (0.02 MPa)) resulting in 28.0 g of an oil slightly yellowish. At room temperature, the oily residue was taken up in dichloromethane (20 mL), diluted with heptane (150 mL) and again fully concentrated on the rotary evaporator, followed by dissolving the residue in MTBE (20 mL) and again by complete removal of the solvent in the rotary evaporator resulting in a rubber foam. This foam was dissolved in toluene (30 mL, room temperature) and dosed over 20 minutes and added dropwise via a dropping funnel at room temperature to heptane (400 mL), after which the product began to precipitate. The dropping funnel was rinsed with toluene (4 mL) and the suspension was kept under stirring for 1 hour at room temperature. The solids were removed by filtration and the reactor and filter cake were rinsed twice with the filtrate and subsequently with heptane (15 mL). Drying under vacuum at 35 °C until the weight remained constant resulted in 17.88 g of a colorless solid. Purity by HPLC: area 97.0%, residual solvents: toluene (1.2%-w/w) and heptane (2.3%-w/w). PE (visually): Initial: 55 - 73°C (melting accompanied by exothermic decomposition). 1H NMR (400 MHz, DMSO-d6, 120°C): δ 7.41 - 7.47 (m, 2 H), 7.27 - 7.32 (m, 2 H), 7.21 - 7, 26 (m, 2 H), 7.12 - 7.19 (m, 1 H), 5.21 (s wide, 1 H), 4.35 (bd, 1H), 4.22 (bs, 1H) , 4.05 (dd, 1H), 3.91 - 4.01 (m, 1H), 3.74 - 3.90 (m, 4H), 3.01 (dd, 1H), 2.75 - 2 .84 (m, 1H), 2.49 - 2.59 (m, 1H), 1.68 - 1.81 (m, 1H), 1.51 - 1.65 (m, 1H), 1, 23-1.50 (m, 3H), 1.09-1.22 (m, 1H). MS (EI): m/z = 435 ([M + H]+, 100%). EA for C23H25F3N2O3, corrected for residual toluene (1.2%-w/w) and heptane (2.3%-w/w): calculated: C 64.38, H 6.07, F 12.66, N 6 .22; found C 64.01, H 6.04, F 12.63, N 6.35. Example 5 Synthesis of ((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-(2,3,4-trifluoro-phenyl)-methanone hydrochloride

[00129] A 185 ml glass autoclave under argon was charged with Pd/C (3.37 g, 1.3 mmol, 0.04 eq, 60.2 wt% water, 10 wt% Pd in C), water (0.22 g) and a solution of {3-hydroxy-3-[(S)-1-((S)-2-hydroxy-1-phenyl-ethyl) - piperidin-2-yl] -azetidin-1-yl}-(2,3,4-trifluoro-phenyl)-methanone in EtOH (53 ml, 46 g, 29 mmol, 1.0 eq.). The mixture was treated with EtOH (13 mL), acetic acid (4.15 mL, 72 mmol, 2.5 eq.) and aqueous hydrochloric acid (2.5 mL, 37% w/w, 30 mmol, 1 ,0 eq.). The autoclave was rendered inert, pressurized with 2 bar (0.2 MPa) of H2 and the reaction was carried out at 2 bar of H2 pressure at 25 °C for 12 hours. The pressure was released from the autoclave and the suspension was treated with MeOH (25 mL) and kept stirring for 30 min and filtered under argon protection over filter paper. The autoclave and filter residue were rinsed with MeOH (4 mL). The combined filtrates were evaporated under reduced pressure to approximately 20-30 percent of the initial volume. The residue was treated with isopropanol (38.5 mL) at 30 to 35 °C, stirred for 1 hour, cooled to 20 to 25 °C and treated with water (0.58 g) and aqueous hydrochloric acid (2.5 mL, 37% wt/wt, 30 mmol, 1.0 eq.). The resulting suspension was concentrated under vacuum at 25 to 35 °C until it reached a volume of approximately 22 mL and MTBE (31 mL) was added at 25 to 35 °C. The final suspension was cooled to 5 to 10 °C, stirred for 1 hour and then filtered. The filter cake was rinsed with cold MTBE (12 mL) and dried under vacuum at 35°C to constant weight to yield the title compound (5.08 g) as a colorless solid. Purity by HPLC: area 99.6%. PE (DSC): Initial: 246.3 °C, extrapolated peak: 248.8 °C (melting accompanied by exothermic decomposition). 1H-NMR (400 MHz, DMSO-d6, 120°C): δ 8.59 (bs, 2 H), 7.14 - 7.48 (m, 2 H), 6.54 (bs, 1H), 4.39 (dd, 1H) 4.23 (dd, 1H), 3.85-3.97 (m, 2H), 3.27-3.35 (m, 1H), 3.20-3.27 (m, 1H), 2.80-2.95 (m, 1H), 1.78-1.88 (m, 2H), 1.64 - 1.78 (m, 2H), 1, 40 - 1.64 (m, 2 H). MS (EI): m/z = 315 ([M + H]+ free base, 100%). EA for C15H17F3N2O2 x HCl: calculated: C 51.36, H 5.17, N 7.99, F 16.25; found C 51.19, H 4.89, N 7.91, F 16.06. Example 6 Synthesis of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl]-methanone (Compound I)

[00130] To a solution of ((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-(2,3,4-trifluoro-phenyl)-methanone hydrochloride (15.0 g, 42.8 mmol, 1.0 eq) and 2-flouro-4-iodo-aniline (11.1 g, 47 mmol, 1.1 eq.) in THF (90 mL), a solution of LiHMDS in THF (149 g, 20.7% w/w, 184 mmol, 4.3 eq.) was dosed over 88 minutes at 20 to 30 °C. Stirring continued for 2 hours. After complete conversion, the mixture was dosed with a mixture of sulfuric acid (12.0 g, 96%-w/w, 118 mmol, 2.75 eq.) in water (75 mL) for 25 minutes and kept stirring for 1 hour. The layers were allowed to separate, and the organic phase was washed with a mixture of water (60 ml) and toluene (96 ml). The organic phase was concentrated under vacuum to a volume of approximately 150 mL. Toluene (250 mL) was added and residual THF was removed by distillation at a jacket temperature of 55 °C and a pressure of 84 mbar (8400 Pa) (keeping the batch volume constant by continuous dosing of toluene (400 mL). ), resulting in slow precipitation of the product. The batch temperature was then reduced to 10°C within 2 hours, and the suspension was kept under stirring overnight at 10°C. The product was removed by filtration and the cake was rinsed with cold toluene (150 mL). Drying overnight under vacuum at 35 °C until weight constant yielded the title compound (20.66 g) as a colorless product. HPLC purity: area 99.7 %. PE (DSC): Initial: 166.7 °C, extrapolated peak: 168.2 °C (91.5 J/g). 1H NMR (600 MHz, CDCl3): δ 8.28 - 8.48 ( br, 1H), 7.39 (dd, 1H), 7.32 (ddd, 1H), 7.09 - 7.14 (m, 1H), 6.75 - 6.86 (a), 6, 60 (ddd, 1H), 4.10 (d, 2H), 4.05-4.20 (br, 1H), 3.93 - 4.04 (wide, 1H), 3.09 (d, 1H) 2.70 (d, 1H), 2.56 - 2.67 (broad, 1H), 1.68 - 1.87 (m, 1H), 1.50 - 1.64 (m, 2H) , 1.25 - 1.38 (m, 2 H), 1.07 - 1.24 (m, 1 H). MS (EI): m/z = 532 ([M + H]+, 100%). EA for C21H21F3IN2O3: calculated: C 47.47, H 3.98, N 7.91, F 10.73; found C 47.68, H 4.00, N 7.66, F 10.80. Example 7 Preparation of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1 crystalline fumarate salt -yl]-methanone (Compound I) designated as Form A

[00131] O (S)-[3,4-Difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl]-methanone (80 kg) was dissolved in a mixture of 2-propanol/water 88:12 w/w (9 weight equivalents (wt eq.)) at 77 °C and filtered over activated carbon. Fumaric acid (0.52 eq.) was dissolved in a mixture of 2-propanol/water 88:12 w/w (2.6 eq. by weight). An initial amount of the fumaric acid solution (8% of the total amount) was added to the filtered solution of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy- 3-(piperidin-2-yl)azetidin-1-yl]-methanone at 77°C. Seed crystals (0.023 wt eq.) as a suspension in a mixture of 2-propanol/water 88:12 w/w (0.2 wt eq.) at 77 °C. The remaining amount of fumaric acid solution was added within 6 hours at the same temperature. To complete crystallization, the suspension was cooled to 20 °C within 7 hours. The crystalline fumarate salt Compound I Form A was isolated by centrifugation, washed with 2-propanol (e.g., 0.6 weight eq.), dried under reduced pressure to maximum temperature. 55 °C and deagglomerated. Characterization examples for crystalline fumarate salt of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1 -yl]-methanone designated as Form A

Elementary analysis

[00132] The results of the elemental analysis were calculated from a relative molecular mass of 1178.71 g/mol and a composition of C46H46F6I2N6O8 and are shown in Table 1. The results are consistent with the structure described above. Table 1: Analysis elementary C46H46F6I2N6O8

Infrared spectroscopy

[00133] An iS5 ThermoScientific Fourier transform infrared spectrophotometer (FTIR) with iD5 ATR accessory was used. The infrared (IR) spectrum was recorded as a measurement of the IR reflection in the range of 4000-650 cm-1 and is provided as Figure 1. The IR spectrum is consistent with the structure described above. A summary of the characteristic IR stretches is provided in Table 2. Table 2: IR assignments for the fumarate salt of Compound I

NMR spectrum in dimethyl sulfoxide solution

[00134] Nuclear magnetic resonance (NMR) measurements were performed on Bruker Avance 600 and 400 MHz spectrometers. The 600 MHz machine was equipped with a 5 mm CryoProbe, TCI, with Z gradient and the 400 MHz machine was equipped with a 5 mm probe, BBFO, z gradient. The sample was prepared by dissolving 6 mg of the crystalline fumarate salt of Compound I designated as Form A in 0.75 mL of DMSO-d6 (D, 99.8%) for all proton-detected experiments. For 13C-NMR and 19F-NMR, 62 mg were dissolved in 0.75 mL of DMSO-d6.

[00135] 1H NMR (600 MHz, DMSO-d6 at 25 °C): The 1H NMR spectrum at 600 MHz at 25 °C showed a 2:1 ratio of free base and fumarate taking the integral of the signals in 6 .62 and 6.42 ppm, proving the existence of a hemifumarate. There were eight proton signals for the azetidine ring, whereas only four were expected, indicating the existence of two sets of signals in a 1:1 ratio.

[00136] 1H NMR (DMSO-d6) is represented in Figure 2. δ 8.54 (s, 1H) 8.50 (s, 1H), 7.57 (dd, 2H), 7.37 (dd , 2H), 7.31 (m, 2H), 7.18 (m, 2H), 6.67 (t, 2H), 6.42 (s, 2H), 4.25 (d, IH), 4 .15 d, 1H), 4.09 (d, 1H), 4.01 (d, 1H), 3.92 (d, 1H), 3.86 (d, 1H) t 2H) 3.03 (d , 2H), 2.79 (m, 2H) 8.38 (t, 2H), 1.62 (m, 6H) 1.24 (m 6H) (2.50 quint 1.9 DMSO).

[00137] 13C NMR (DMSO-d6) is represented in Figure 3. δ 168.0, 167.5, 152.3, 151.4, 143.6, 135.2, 133.2, 131.3 , 130.5, 124.6, 123.7, 123.0, 122.7, 119.8, 119.6, 110.7, 81.6, 81.5, 70.3, 70.3, 63 ,0, 61.8, 60.7, 60.6, 59.0, 58.1, 45.5, 45.4, 24.1, 23.7, 23.1, 39.5 (DMSO-d6 ).

[00138] To support the results of liquid state NMR spectroscopy, solid state NMR spectroscopy was also performed for structure elucidation. In liquid state NMR spectroscopy, some of the signals are bent due to the constraints observed in free rotation, leading to rotameric mixing, whereas the solid state NMR spectroscopy result is not influenced by this steric hindrance. Therefore, a more unambiguous peak assignment is possible for the fumarate salt of Compound I using solid state NMR spectroscopy.

[00139] The 13C NMR of the solid state of Form A is represented in Figure 4. δ 175.3, 173.6, 168.9, 155.5, 153.5, 144.4, 142.5, 137.0, 136.0 135.5, 132.0, 130.5, 127.2, 125.0, 124.0, 117.9 m 108.0, 82.2, 71.7, 64.0, 59.3 , 56.2,45.0, 25.3, 24.0, 22.2.

[00140] Solid state 13C NMR of Form A confirms the results of other analytical technologies used to elucidate the structure of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl] [3-hydroxy-3-(piperidin-2-yl) azetidin-1-yl]-methanone. All carbons present in the free base structure, as well as in the counterion structure, are detected in the spectra.

[00141] 19F NMR (600 MHz, DMSO-d6 at 25 °C): 19-F NMR showed three different fluorine atoms.

Mass spectrometry

[00142] An Agilent 6520 QTOF spectrometer was used for ESI-positive CID MSMS and ESI-negative MS. The positive electrospray mass spectrum obtained for the fumarate salt of Compound I is shown in Figure 5. The [M + H]+ at m/z of 562.0714 is consistent with the formula for Compound I (the free base). The fragmentation behavior of M + H was studied by collision-induced dissociation (CID). Nitrogen was used as the collision gas. All fragments were in good correlation with the structure of Compound I.

[00143] The negative electrospray mass spectrum (Figure 6) obtained for the fumarate salt of Compound I [MH] - was found to be m/z = 115.0045 (calculated: m/z = 115.0037; difference m /z = 0.0008), indicative of the presence of fumarate counterion.

Analysis of the crystal structure of (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin- fumarate salt 1-yl]-methanone Form A by X-ray diffraction X-ray powder diffraction (XRPD) studies

[00144] A single crystal was assembled into a circuit and measured at room temperature. Data were collected on the Swiss Light Source beamline X10SA equipped with a DECTRIS Pilatus 6M detector with synchrotron radiation and data processed with the XDS program. The crystal structure was solved and refined with ShelXTL (Bruker AXS, Karlsruhe).

[00145] The structure of the fumarate salt of Compound I designated as Form A incorporates a chiral center with (S) configuration according to the Cahn-Ingold-Prelog convention. To prove the structure, a single crystal determination was carried out. Single crystals were crystallized from a 1:1 diluted acetonitrile/water solution by slow evaporation of the solvent. To prove that the conformation did not change through crystallization, chiral high-performance liquid chromatography (HPLC) was additionally performed. Crystal data is summarized in Table 3.Table 3: Form A crystal data

[00146] The molecular conformation and crystal structure parameters for Form A are presented in Table 3. In the Form A crystal structure, the piperidine nitrogen is protonated and the fumaric acid is deprotonated. Fumarate is coordinated by two piperidines and two OH groups from different active molecules. The crystal packing is characterized by infinite intermolecular hydrogen bonding chains. The configuration was confirmed to be (S) for the chiral carbon atom in the piperidine ring, as demonstrated by the absolute structure parameter (Flack parameter: 0.048, esd 0.013).

Ultraviolet spectrum for (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl fumarate salt ]-methanone

[00147] The UV-Vis absorption maxima around 200 nm, 239 nm, 276 nm and 310 nm are indicative of the π^π* transitions of the aromatic ring moieties and the n^π* lone pair electrons, respectively . The spectrum in Figure 7 is consistent with the structure of the fumarate salt of Compound I and shows the expected characteristics of the chromophores present in the structure.

Structure elucidation for (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1- fumarate salt il]-methanone: polymorphism

[00148] More than 15 salt forms of Compound I have been evaluated for their suitability for clinical development, including, for example, salts prepared from benzoic acid, malonic acid, fumaric acid, mandelic acid, acetic acid and orotic acid. When benzoic acid, malonic acid, mandelic acid, acetic acid and orotic acid formed amorphous salts, crystalline salts or mixtures of amorphous and crystalline salts depending on the solvent and conditions, the salt prepared from fumaric acid was the most desirable as described below .

[00149] Comprehensive sieving of crystalline solid forms of the fumarate salt of Compound I revealed a crystalline form (Form A) and an amorphous form (the amorphous form). Form A, which is solvent-free and non-hygroscopic, is the thermodynamically stable form and is the form consistently produced by the manufacturing process. The amorphous form is non-crystalline and hygroscopic. The amorphous form was observed to convert to Form A upon heating and by solvent-mediated phase transition in water. In approximately 3000 crystallization experiments performed during polymorphism examinations, no additional polymorphic forms were observed. Based on DSC measurements as well as temperature-controlled XRPD, no conversion of the polymorphic form is observable upon heating until fusion occurs. In a suspension experiment using process conditions (solvent 2-propanol/water 88:12, temperature: 20 °C), the amorphous form rapidly converted to Form A. Upon heating, the amorphous form began to convert to Form A between 90°C and 200°C.

[00150] Form A and amorphous can be distinguished by differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Furthermore, differences between Form A and amorphous were also demonstrated using Raman spectroscopy and 13C nuclear magnetic resonance (NMR) spectroscopy.

Differential scanning calorimetry (DSC)

[00151] DSC thermograms were recorded using a Mettler-Toledo instrument (DSC820/821e/1 sensor; FRS05). Approximately 2-6 mg of the sample was placed in aluminum pans and sealed with aluminum lids. The lids were automatically pierced before heating. Generally, samples under nitrogen were heated at a rate of 10 K/min to a maximum of 250 °C.

[00152] Crystalline Form A melted at 239.6 °C (Initial). Since fusion and decomposition are superimposed, the heat of fusion and Textra.pol. were not determined (Figure 8).

[00153] The amorphous form exhibited a glass transition at 116.2 °C (from milling) and 120.6 °C (from freeze-drying), followed by an exothermic event due to crystallization between 150 °C and 200 °C for Form A. Above approximately 225 °C, the material begins to melt (Figure 9).

Powder X-ray diffraction of Form A and the amorphous form

[00154] X-ray diffraction patterns were recorded under ambient conditions in the transmission geometry with a STOE STADI P diffractometer (Cu K radiation α [1.54 Â], primary monochromator, silicon strip detector, angular range of 3 ° to 42° 2-θ, approximately 30 minutes total measurement time). Samples were prepared and analyzed without further processing (e.g. grinding or sieving) of the substance.

[00155] As shown in Table 4, crystalline Form A is selectively identified by a set of characteristic diffraction peak positions expressed in 2-θ value. The XRPD diffractogram characteristics for the individual shapes are shown in Figure 10 and Figure 11. Table 4

Intrinsic dissolution rate of crystalline Form A and amorphous form

[00156] For each intrinsic dissolution measurement, a granule a was produced from the crystalline Form A or the amorphous form sample using an applied load of approximately 15 kN on a flat disk (surface area = 0.5 cm2). After compaction, each granule was checked by XRPD to confirm that there was no polymorphic transformation during the granulation process. The experimental conditions that were employed are summarized in Table 5.Table 5

[00157] Batches of Form A and the amorphous form (from freeze-drying) were used to determine the intrinsic dissolution rate of both solid forms. Intrinsic dissolution allows the characterization of different crystalline forms by exposing a constant surface area to the dissolution medium. The results are summarized in Table 6.Table 6

[00158] The intrinsic dissolution rate of both forms is very different. Based on the data, the amorphous form has an intrinsic dissolution rate approximately 35 times faster than Form A.

Hygroscopicity of crystalline Form A and amorphous form

[00159] Moisture adsorption/desorption data were collected on a DVS-1/DVS-HT/DVS-intrinsic moisture balance system (SMS Surface Measurements Systems). Sorption/desorption isotherms were measured stepwise over a range from 0% RH (relative humidity) to 90% RH at 25 °C. A weight change of <0.002 mg/min was chosen as the criterion for switching to the next RH level (with a maximum equilibration time of 24 hours if the weight criterion was not met). The data were corrected for the initial moisture content of the samples; that is, the weight after drying the sample with 0% RH was taken as the zero point. The hygroscopicity of a given substance is characterized by the increase in mass when the RH was increased from 0% RH to 90% RH as indicated in Table 7.Table 7

[00160] Moisture adsorption/desorption data for crystalline Form A are provided in Figure 12. During the time scale of standard dynamic vapor sorption experiments, no conversion was observed. Between 0% RH and 90% RH, Form A exhibits a minimal, reversible weight gain or loss of ±0.1% and is therefore classified as non-hygroscopic.

[00161] Amorphous moisture sorption/desorption data are provided in Figure 13 (from freeze-drying). During the time scale of standard dynamic vapor sorption experiments, no conversion was observed. Between 0% RH and 90% RH, amorphous exhibits a reversible weight increase or loss of ±12.1% and is therefore classified as hygroscopic.

[00162] The previous description has been described in some detail by way of illustration and example, for purposes of clarity and understanding. The invention has been described with reference to several specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention. It will be obvious to one skilled in the art that changes and modifications may be made within the scope of the appended claims. Therefore, it should be understood that the above description is intended to be illustrative and not restrictive. The scope of the invention must therefore be determined not with reference to the above description, but must instead be determined with reference to the following appended claims, together with the full scope of the equivalents to which such claims are entitled.

Claims (14)

1. Crystalline fumarate salt of Compound I, characterized in that Compound I is (S )-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3- ( piperidin-2-yl)azetidin-1-yl]-methanone, and wherein the crystalline fumarate salt of Compound I has the structure: wherein the crystalline fumarate salt of Compound I is designated as Form A. 2. Crystalline fumarate salt of Compound I, according to claim 1, characterized by the fact that it is designated as Form A, wherein the crystalline fumarate salt of Compound I is represented by at least one of the following: (i) a 13C NMR spectrum in DMSO-d6 having the following peaks: δ 168.0, 167.5, 152.3, 151.4, 143.6, 135.2, 133.2, 131.3, 130.5, 124.6, 123.7, 123.0, 122.7, 119.8, 119.6, 110.7, 81.6, 81.5, 70.3, 70.3, 63.0, 61, 8, 60.7, 60.6, 59.0, 58.1, 45.5, 45.4, 24.1, 23.7, 23.1, 39.5 (DMSO-d6); (ii) a solid-state 13C NMR spectrum with three or more selected peaks at 175.3, 173.6, 117.5, 155.5 and 153.5, ± 0.2 ppm; (iii) a powder X-ray diffraction pattern (CuKα À=1.5418Â) comprising three or more 2θ values selected from 4.6, 12.1, 13.2, 13.6 and 14.5 ± 0 .2°2θ, where the crystalline form measurement is at room temperature; and (iv) a differential scanning calorimetry thermogram showing melting at 239.6 °C (TOnset). 3. Crystalline fumarate salt of Compound I, according to claim 1, characterized by the fact that it is designated as Form A, wherein said salt is represented by a solid state 13C NMR spectrum with three or more peaks selected from 175.3, 173.6, 117.5, 155.5 and 153.5, ± 0.2 ppm. 4. Crystalline fumarate salt of Compound I, according to claim 1, characterized by the fact that it is designated as Form A, wherein said salt is represented by a powder X-ray diffraction pattern (CuKα À = 1, 5418 Â) comprising three or more 2θ values selected from 4.6, 12.1, 13.2, 13.6 and 14.5 ± 0.2 °2θ, wherein the measurement of the crystalline form is at room temperature. 5. Crystalline fumarate salt of Compound I, according to claim 1, characterized by the fact that it is designated as Form A, wherein said salt is represented by a powder X-ray diffraction pattern as set out in Table 4: Table 4 6. Crystalline fumarate salt of Compound I, according to any one of claims 1 to 5, characterized by the fact that said salt is at least 90% by weight of Form A, based on the weight of said salt. 7. Pharmaceutical composition, characterized by the fact that it comprises the crystalline fumarate salt of Compound I, as defined in any one of claims 1 to 5, and a pharmaceutically acceptable excipient. 8. Use of the crystalline fumarate salt of Compound I, as defined in any one of claims 1 to 5, characterized by the fact that it is for the manufacture of a medicine for the treatment of cancer. 9. Use according to claim 8, characterized in that the cancer is selected from the group consisting of melanoma (including BRAF V600 mutant melanoma), breast cancer (including triple negative breast cancer), colorectal cancer (including KRAS mutant colorectal cancer), non-small cell lung cancer, acute myeloid leukemia and pancreatic cancer. 10. Use according to claim 9, characterized by the fact that the cancer is melanoma. 11. Use according to claim 10, characterized by the fact that the cancer is BRAF V600 mutant melanoma. 12. Use according to claim 10 or 11, characterized in that the crystalline fumarate salt of Compound I is used in combination with vemurafenib. 13. Use according to claim 12, characterized in that the crystalline fumarate salt of Compound I is administered before or subsequent to, or simultaneously with, vemurafenib. 14. Process for preparing the crystalline fumarate salt of Compound I, designated as Form A, as defined in any one of claims 1 to 5, characterized in that it comprises: adding fumaric acid dissolved in a solvent to a mixture of Compound I dissolved in a solvent for forming the crystalline fumarate salt of Compound I designated as Form A; and collecting the resulting crystals of the crystalline fumarate salt of Compound I designated as Form A.