6-Pyrimidine-isoindole derivatives as ERK1 / 2 inhibitors

Abstract

The present invention relates to the compound (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide, and in particular to a new physical form of the compound, a method for preparing the compound and a synthetic intermediate used in the method, a new preparation containing the compound and the therapeutic use of the compound.

Description

[0001] The present invention relates to the compound (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide, and in particular to a new physical form of the compound, a method for preparing the compound and a synthetic intermediate used in the method, a new preparation containing the compound and the therapeutic use of the compound. Background Art

[0002] MAPK signaling and the role of ERK1 / 2

[0003] Extracellular signal-regulated kinases (ERK1 / 2) are ubiquitously expressed protein serine / threonine kinases that comprise key components of the mitogen-activated protein kinase (MAPK) signaling pathway. The MAPK pathway is an evolutionarily conserved cell signaling pathway that regulates a variety of cellular processes, including cell cycle progression, cell migration, cell survival, differentiation, metabolism, proliferation, and transcription. The ERK / MAPK signaling pathway responds to extracellular stimulation of cell surface receptor tyrosine kinases (RTKs). Upon activation of RTKs, RAS GTPases (K-RAS, N-RAS, and H-RAS) are converted from an inactive GDP-bound state to an activated GTP-bound state. Activated RAS phosphorylates, thereby activating RAFs (A-RAF, B-RAF, and C-RAF), which in turn phosphorylate and activate the dual-specificity kinase MEK (MEK1 / 2). Activated MEK then phosphorylates and activates ERK1 / 2. Upon activation, ERK1 / 2 activates a variety of nuclear and cytoplasmic substrates. There are currently >200 known ERK1 / 2 substrates, including transcription factors, kinases, phosphatases, and cytoskeletal proteins (Roskoski, Pharmacol. Res. 2012; 66: 105-143).

[0004] Multiple ERK isozymes have been identified (ERK1, ERK2, ERK3 / 4, ERK5, ERK7), but the two most widely studied isozymes are ERK1 and ERK2: see R. Roberts, J. Exp. Pharm., The extracellular signal-regulated kinase (ERK) pathway: a potential therapeutic target in hypertension, 2012: 4, 77-83, and Cargnello et al., Microbiol. & Mol. Biol. Rev., Activation and Function of the MAPKs and Their Substrates, the MAPK-Activiated Protein Kinases 2011, 50-83.

[0005] Upregulation of ERK1 / 2 signaling in cancer

[0006] The activity of ERK1 / 2 is often upregulated in cancer due to activating mutations in upstream components of the MAPK pathway. Approximately 30% of human cancers contain activating RAS mutations (Roberts and Der, Oncogene. 2007; 26: 3291-3310). K-RAS is the most common mutated subtype, mutated in 22% of tumors. KRAS mutations are particularly common in pancreatic cancer (70-90%), non-small cell carcinoma (10-20%), and colorectal cancer (25-35%) (Neuzillet et al., 2014. Pharmacol. Ther. 141; 160-171). N-RAS mutations and H-RAS mutations occur in 8% and 3% of cancers, respectively (Prior et al., Cancer Res. 2012; 72 (10); 2457-2467). It is worth noting that activating N-RAS mutations are reported in 15-20% of melanoma cases. In addition, activating B-RAF mutations occur in 8% of all tumors and are particularly prevalent in melanoma (50-60%), papillary thyroid cancer (40-60%), colorectal cancer (5-10%), and non-small cell lung cancer (3-5%) (Neuzillet et al., 2014. Pharmacol. Ther. 141; 160-171). In addition to the presence of activating RAS and RAF mutations, the MAPK signaling pathway is also upregulated in cancer through overexpression or mutational activation of upstream RTKS such as EGFR (Lynch et al., N Engl J Med. 2004; 350: 2129-2139), HER2 (Stephens et al., Nature. 2004; 431: 525-526), ​​and FGFR (Ahmed et al, Biochim. Biophys. Acta Mol. Cell. Res. 2012; 1823: 850-860).

[0007] There are multiple mechanisms by which abnormal ERK1 / 2 signaling promotes cancer progression. When activated, ERK1 / 2 phosphorylates and activates a wide range of transcription factors involved in promoting cell proliferation and differentiation, such as c-Fos (Murphy et al., Nat. Cell Biol. 2002: 4(8): 556-64) and ELK-1 (Gille et al., EMBO J. 1995; 14(5): 951-62). In addition, it is well known that ERK1 / 2 signaling promotes cell cycle progression through multiple mechanisms, including induction of D-type cyclins and inhibition of the cyclin-dependent kinase inhibitor p27. KIP1(Kawada et al., Oncogene. 1997; 15: 629-637, Lavoie et al., J. Biol. Chem. 1996; 271: 20608-20616). In addition, ERK1 / 2 signaling can promote cell survival by regulating a series of apoptotic proteins. Examples of such mechanisms include ERK1 / 2-dependent inhibition of pro-apoptotic BCL-2 family proteins BIM1 and BAD (She et al., J. Biol Chem. 2002; 277: 24039-24048. Ley et al., J. Biol. Chem. 2003; 278: 18811-18816) and ERK1 / 2-dependent stabilization of anti-apoptotic proteins such as MCL-1 (Domina et al., Oncogene. 2004; 23: 5301-5315).

[0008] Role of ERK1 / 2 in resistance to MAPK inhibitors

[0009] Extensive preclinical studies have shown that inhibition of the MAPK pathway inhibits the growth of cancer cell lines containing B-Raf or Ras mutations (Friday & Adjei, Clin. Cancer Res. 2008; 14: 342-346). The RAF inhibitors vemurafenib and dabrafenib, as well as the MEK inhibitor trametinib, are clinically approved for the treatment of BRAF mutant melanoma. These agents induce profound anti-tumor responses in most patients, although responses are short-lived due to the development of acquired resistance (Chapman et al., N. Engl. J. Med. 2011; 364: 2507-2516. Hauschild et al., Lancet. 2012; 380: 358-365. Solit and Rosen, N Engl J Med. 2011; 364(8): 772-774. Flaherty et al., N. Engl. J. Med. 2012; 367: 1694-1703). Multiple mechanisms of acquired resistance to B-RAF inhibitors have been identified. These mechanisms include upregulation or activation of alternative MEK activators such as C-RAF or COT1 (Villanueva et al., Cancer Cell. 2010; 18: 683-95. Johannessen et al., Nature. 2010; 468: 968-72), upregulation of RTK or NRAS signaling (Nazarian et al., Nature. 2010; 468: 973-7), and the occurrence of MEK activating mutations (Wagle et al., J Clin Oncol. 2011; 29: 3085-96). MEK inhibitor resistance mechanisms include the emergence of MEK mutations that reduce drug binding or enhance intrinsic MEK activity (Emery et al., Proc Natl. Acad. Sci. 2009; 106: 20411-20416. Wang et al., Cancer Res. 2011; 71: 5535-5545); and BRAF or KRAS amplification (Little et al., Biochem Soc. Trans. 2012; 40 (1): 73-8). A common feature of the resistance mechanism of RAF or MEK inhibitors is the reactivation of ERK1 / 2 signaling, which drives cell proliferation and survival in the presence of inhibitors. Based on this observation, it has been suggested that direct ERK1 / 2 inhibition may be an effective treatment to overcome acquired resistance to RAF or MEK inhibitors.Preclinical evidence suggests that inhibition of ERK1 / 2 overcomes acquired resistance to RAF or MEK inhibitors (Hatzivassiliou et al., Mol Cancer Ther. 2012; 11(5): 1143-54. Morris et al., Cancer Discov. 2013; 3(7): 742-50).

[0010] Other diseases

[0011] In addition to tumors, aberrant ERK1 / 2 signaling has been reported in other diseases, including cardiovascular disease (Muslin, Clin. Sci. 2008; 115: 203-218), Alzheimer's disease (Giovannini et al., Neuroscience. 2008; 153: 618-633), polycystic kidney disease (Omori et al., J Am Soc Nephrol. 2006; 17: 1604-1614), asthma (Duan et al., J Immunol. 2004; 172: 7053-7059) and emphysema (Mercer et al., J. Biol. Chem. 2004; 279: 17690-17696).

[0012] (2R)-2-(6-{5-chloro-2-[(oxacyclohexyl-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro- 1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide

[0013] Our earlier international patent application PCT / IB2016 / 001507 (the contents of which are incorporated herein by reference) discloses benzolactam compounds as ERK2 inhibitors. One of the compounds specifically disclosed in the application is a compound of formula (1) (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide:

[0014]

[0015] The preparation of this compound is described in Example 685 of PCT / IB2016 / 001507 and involves the reaction of a compound of formula (2) with a compound of formula (3):

[0016]

[0017] The reaction is carried out in dimethylformamide (DMF) and triethylamine in the presence of an amide bond-forming promoter, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU).

[0018] In Example 685, it is disclosed that after work-up and partial purification by silica gel chromatography, the column eluent is evaporated to give a glassy material which is then triturated with ether to give compound (1) as an amorphous solid. Summary of the invention

[0019] In Example 685 of PCT / IB2016 / 001507, compound (1) is prepared in amorphous form. However, we have now found that a crystalline form of the compound of formula (1) can be prepared. Therefore, in a first aspect, the present invention provides a compound of formula (1) in substantially crystalline form.

[0020] The present invention also provides a novel method for preparing the compound of formula (1) and synthetic intermediates, and a novel preparation containing the compound of formula (1).

[0021] Crystalline form of the compound of formula (1)

[0022] In a first aspect, the present invention provides a compound of formula (1) (2R)-2-(6-{5-chloro-2-[(oxacyclohexyl-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide or a tautomeric form thereof in substantially crystalline form:

[0023]

[0024] Although the compound of formula (1) can form salts, the compound in crystalline form is referred to as a free base.

[0025] Where the context allows, the scope of the compounds of formula (1) includes all solvates, tautomers and isotopic variations thereof.

[0026] Amorphous solids lack the three-dimensional structure that is usually present in crystalline forms and, in amorphous forms, the positions of the molecules relative to each other are essentially random, see, for example, Hancock et al. J. Pharm. Sci. (1997), 86, 1).

[0027] The term "substantially crystalline" refers to a crystalline form in which the compound of formula (1) is 50% to 100%. Within this range, the compound of formula (1) may be at least 55% crystalline, or at least 60% crystalline, or at least 70% crystalline, or at least 80% crystalline, or at least 90% crystalline, or at least 95% crystalline, or at least 98% crystalline, or at least 99% crystalline, or at least 99.5% crystalline, or at least 99.9% crystalline, for example, 100% crystalline.

[0028] In one embodiment, the crystalline form is 95% to 100% crystalline, e.g., at least 98% crystalline, or at least 99% crystalline, or at least 99.5% crystalline, or at least 99.6% crystalline, or at least 99.7% crystalline, or at least 99.8% crystalline, or at least 99.9% crystalline, e.g., 100% crystalline.

[0029] The crystalline form of the compound of the present invention may be in a solvated (eg, hydrated) form or an unsolvated (eg, anhydrous) form.

[0030] In one embodiment, the compound is in anhydrous form.

[0031] The term "anhydrous" as used herein does not exclude the possibility that some water may be present on or in the compound (e.g., compound crystal). For example, there may be some water on the surface of the compound (e.g., compound crystal), or a small amount of water may be present in the compound (e.g., crystal). Typically, the anhydrous form contains less than 0.4 water molecules per molecule of the compound, more preferably less than 0.1 water molecules per molecule of the compound, for example, 0 molecules of water.

[0032] In another embodiment, the compound is in a solvated form, for example, a hydrated form. When the crystalline form is a hydrated form, they may contain, for example, no more than 3 molecules of crystal water, more typically no more than 2 molecules of water, for example, 1 molecule of water or 2 molecules of water. Non-stoichiometric hydrates may also be formed, wherein the number of water molecules present is less than 1, or is a non-integer. For example, when the number of water molecules present is less than 1, for example, the number of water molecules present per molecule of compound (1) may be 0.4 or 0.5 or 0.6 or 0.7 or 0.8 or 0.9.

[0033] In one embodiment, the crystalline form of Compound (1) is a monohydrate, wherein the crystalline form contains 1 molecule of crystal water.

[0034] Other solvates include alcoholates, for example, ethanolates and isopropanolates.

[0035] The crystalline forms, crystals thereof and their crystal structures described herein constitute further aspects of the invention.

[0036] Crystals and their crystal structure can be characterized using a variety of techniques, including X-ray powder diffraction (XRPD), single crystal X-ray diffraction, differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Crystal behavior under different humidity conditions can be analyzed using gravimetric vapor sorption studies (e.g., dynamic vapor sorption).

[0037] The crystal structure of the compound can be analyzed by solid-state techniques such as X-ray powder diffraction (XRPD). X-ray powder diffraction can be performed according to conventional methods, such as those described herein (see Example 4A) and in Introduction to X-ray Powder Diffraction (Ron Jenkins and Robert L. Snyder (John Wiley & Sons, New York, 1996). The presence of defined peaks in the diffraction pattern of X-ray powder diffraction (compared to random background noise) indicates that the compound has a certain degree of crystallinity.

[0038] The X-ray powder pattern of a compound is characterized by the diffraction angle (2θ) and interplanar spacing (d) parameters of the X-ray diffraction spectrum. These parameters are related by the Bragg equation: nλ=2d Sinθ (where n=1; λ=wavelength of the X-ray radiation; d=interplanar spacing; and θ=diffraction angle). Here, due to the characteristics of these data, interplanar spacing, diffraction angle and overall pattern, they are very important for the crystal identification of X-ray powder diffraction. Since the relative intensity may vary depending on the direction of crystal growth, grain size and measurement conditions, the relative intensity should not be strictly interpreted. In addition, the diffraction angles usually refer to those diffraction angles consistent with the range of 2θ±0.2°.

[0039] To date, two specific crystalline forms of the free base of the compound of formula (1) have been identified, referred to herein as "Form A" and "Form B". Of the two crystalline forms, Form B appears to be the most stable form. Characterization data for Form A and Form B are given in the Examples below.

[0040] The crystalline (2R)-2-(6-{5-chloro-2-[(oxacyclohexyl-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide of the present invention, Form A and Form B, were characterized by XRPD (see Examples 3 and 4 and Figure 1 and Figure 2 ).

[0041] In each case, the powder X-ray diffraction pattern can be described by the diffraction angle (2θ), the interplanar spacing (d), and the relative intensities of the peaks in the diffraction pattern.

[0042] Form A

[0043] As described in Example 3A below, the first crystalline form (Form A) of the free base of Compound (1) can be formed by disproportionation of a solution of the hydrochloride salt of Compound (1) in a water-propanol (3:1) solvent mixture. The XRPD diffraction pattern of Form A is shown in Figure 1 During the disproportionation, the acid separates from the free base and remains in solution, resulting in a suspension of the free base of compound (1).

[0044] Form A can also be prepared by suspending amorphous Compound (1) hydrochloride in 70°C water for a long time (eg, 96 hours) and then filtering out the crystalline material.

[0045] Form B

[0046] A second crystalline form (Form B) of the free base can also be prepared by stirring an aqueous suspension of an acid addition salt (e.g., hydrochloride, sulfate or hydrobromide) in purified water at a temperature lower than that used in the preparation of Form A for a long time, through the disproportionation of these salts. Thus, Form B can be prepared according to the method described in Examples 3B-3D below: an inorganic acid salt (e.g., hydrochloride, sulfate or hydrobromide) of amorphous Compound (1) is suspended in purified water at 18-23°C, and the mixture is then stirred at 45-50°C for 20 hours, followed by further stirring at 30-35°C for 96 hours, and then the crystals of Form B are filtered out. The XRPD diffraction pattern of Form B is shown in Figure 2 shown.

[0047] The process of disproportionation of the acid addition salt of the amorphous compound (1) to form the crystalline form B can be assisted by adding an alcohol co-solvent such as isopropanol to the reaction mixture.

[0048] In another method for preparing Form B, the amorphous free base form of compound (1) can be suspended in water, which can be unbuffered or buffered at pH about 2 to pH 7, and then stirred at a slightly elevated temperature (e.g., 30° C.) for a period of time (e.g., up to 6 days, e.g., about 5 days) sufficient to allow the amorphous compound (1) to convert to Form B.

[0049] The X-ray diffraction pattern of the crystalline form B of compound (1) exhibits peaks with the highest intensity at the diffraction angles listed in Table A (i.e., 14.0°, 20.6°, 24.0°, and 24.2° (±0.2°)).

[0050]

[0051] Thus, in another embodiment, the present invention provides (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide in substantially crystalline form (Form B), having an X-ray powder diffraction pattern characterized by major peaks appearing at diffraction angles (2θ) of 14.0° and / or 20.6° and / or 24.0° and / or 24.2° (±0.2°).

[0052] In another embodiment, the X-ray diffraction pattern is characterized by at least one peak occurring at a diffraction angle selected from 14.0°, 20.6°, 24.0°, and 24.2° (±0.2°).

[0053] Thus, for example, in one embodiment, the present invention provides Compound (1) in a substantially crystalline form (Form B) having an X-ray powder diffraction pattern characterized by a major peak at a diffraction angle of 14.0° (±0.2°).

[0054] In another embodiment, the present invention provides Compound (1) in a substantially crystalline form (Form B) having an X-ray diffraction pattern characterized by a major peak at a diffraction angle of 20.6° (±0.2°).

[0055] In another embodiment, the present invention provides Compound (1) in a substantially crystalline form (Form B) having an X-ray powder diffraction pattern characterized by a major peak at a diffraction angle of 24.0° (±0.2°).

[0056] In another embodiment, the present invention provides Compound (1) in a substantially crystalline form (Form B) having an X-ray powder diffraction pattern characterized by a major peak at a diffraction angle of 24.2° (±0.2°).

[0057] In another embodiment, the X-ray powder diffraction pattern of Compound (1) in substantially crystalline form (Form B) is characterized by the presence of major peaks at two or more (e.g., three or more) diffraction angles, particularly at four diffraction angles selected from 14.0°, 20.6°, 24.0° and 24.2° (±0.2°).

[0058] The X-ray powder diffraction pattern of compound (1) in Form B may also have lesser peaks at the diffraction angles given in Table B (i.e., 8.8, 13.0, 13.8, 14.4, 17.3, 19.3, 21.3 and 28.7 (±0.2°)).

[0059]

[0060] Therefore, the present invention also provides a compound (1) in a substantially crystalline form (Form B), whose X-ray powder diffraction pattern is characterized by the appearance of a main peak at a diffraction angle of 14.0° and / or 20.6° and / or 24.0° and / or 24.2° (±0.2°) as specified above, and optionally one or more other peaks at a diffraction angle selected from 8.8°, 13.0°, 13.8°, 14.4°, 17.3°, 19.3°, 21.3° and / or 28.7° (±0.2°).

[0061] In one embodiment, the X-ray powder diffraction pattern of Compound (1) in substantially crystalline form (Form B) is characterized by the presence of a main peak at a diffraction angle of 14.0° and / or 20.6° and / or 24.0° and / or 24.2° (±0.2°); and optionally one or more other peaks at a diffraction angle of 13.8° and / or 19.3° and / or 21.3° (±0.2°).

[0062] In one embodiment, the X-ray powder diffraction pattern of Compound (1) in substantially crystalline form (Form B) is characterized by major peaks occurring at diffraction angles of 14.0°, 20.6°, 24.0°, 24.2°, 13.8°, 19.3° and 21.3° (±0.2°).

[0063] In another specific embodiment, the X-ray powder diffraction pattern of Compound (1) in substantially crystalline form (Form B) is characterized by major peaks occurring at diffraction angles of 14.0°, 20.6°, 24.0°, 24.2°, 8.8°, 13.0°, 13.8°, 14.4°, 17.3°, 19.3°, 21.3° and 28.7° (± 0.2°).

[0064] The X-ray powder diffraction pattern may be further characterized by the presence of additional peaks at the diffraction angles (2θ) given in Table C (±0.2°).

[0065]

[0066] The present invention further provides a substantially crystalline form (Form B) of compound (1) which, when combined with Figure 2The relative intensities of these peaks are preferably Figure 2 The peaks shown are identical.

[0067] In a preferred embodiment, the present invention provides a substantially crystalline form (Form B) of Compound (1) having an X-ray powder diffraction pattern substantially as Figure 2 shown.

[0068] The crystalline forms of the present invention can also be characterized by differential scanning calorimetry (DSC).

[0069] The crystal form B of compound (1) was analyzed by DSC, and the results showed that it had an endothermic phenomenon, as shown in the attached figure. Figure 3 As shown, the onset temperature is 100° C. to 110° C. (more particularly 101° C. to 108° C.), and the peak appears at 110° C. to 125° C. (more particularly 111° C. to 114° C.). This phenomenon is caused by the release of water.

[0070] Therefore, the present invention provides a substantially crystalline form (Form B) of compound (1), which exhibits an endothermic phenomenon in DSC analysis, with an onset temperature of 100°C to 110°C (more particularly 101°C to 108°C). The present invention also provides a substantially crystalline form (Form B) of compound (1), which exhibits an endothermic phenomenon, with a peak appearing at 110°C to 125°C (more particularly 111°C to 113°C).

[0071] The crystalline form (Form B) of the present invention can also be characterized by thermogravimetric analysis (TGA).

[0072] The compound (1) in substantially crystalline form B was analyzed by TGA, and the results showed that the weight loss transition started at 85°C to 95°C, for example, 90.86°C, and ended at 110°C to 130°C (for example, 120°C) (see Figure 4 ). The weight loss corresponds to the release of water.

[0073] Based on DSC and TGA data, it can be seen that the above-mentioned substantially crystalline form B compound (1) is a monohydrate. This is also confirmed by single crystal X-ray diffraction studies (see Example 4E below).

[0074] In the substantially crystalline form B of compound (1), one single crystalline form may predominate, although minor (preferably negligible) amounts of other crystalline forms may be present.

[0075] In a preferred embodiment, the present invention provides Compound (1) in a substantially crystalline form (Form B) comprising a single crystalline form having the XRPD characteristics described above and no more than 5% by weight of any other crystalline form of the compound.

[0076] Preferably, the single crystalline form (Form B) is accompanied by less than 4%, or less than 3%, or less than 2% of other crystalline forms, and particularly comprises less than or equal to about 1% by weight of other crystalline forms. More preferably, the single crystalline form is accompanied by less than 0.9%, or less than 0.8%, or less than 0.7%, or less than 0.6%, or less than 0.5%, or less than 0.4%, or less than 0.3%, or less than 0.2%, or less than 0.1%, or less than 0.05%, or less than 0.01% by weight of other crystalline forms, for example, comprising 0% by weight of other crystalline forms.

[0077] As is apparent from the above, the crystalline form (Form B) of Compound (1) can be characterized by a variety of different physicochemical parameters. Therefore, in one embodiment, the present invention provides a substantially crystalline form (Form B) of Compound (1) characterized by any one or more (in any combination) or all of the following parameters, namely:

[0078] (a) its X-ray powder diffraction pattern is characterized by the presence of a main peak with a diffraction angle (2θ) and an intensity given in Table A and optionally Table B; and further optionally its X-ray powder diffraction pattern is characterized by the presence of a main peak with a diffraction angle (2θ) and an intensity given in Table C; and / or

[0079] (b) Its Figure 2 The X-ray powder diffraction pattern shown in FIG. 1 shows a peak at the same diffraction angle as that shown in FIG. 1 , wherein the relative intensity of the peak is Figure 2 The peaks in are the same; and / or

[0080] (c) Its X-ray powder diffraction pattern is basically as follows Figure 2 as shown; and / or

[0081] (d) when analyzed by DSC, it exhibits an endothermic peak at 100°C to 115°C; and / or

[0082] (e) When thermogravimetric analysis (TGA) is performed, the weight loss occurs at 85°C to 130°C (eg, 90-120°C).

[0083] Process for preparing the free base of compound (1) in crystalline form

[0084] The present invention also provides a process for preparing the free base of Compound (1) in crystalline form.

[0085] Therefore, another aspect of the present invention provides a method for preparing a substantially crystalline free base of compound (1); the method comprising:

[0086] (i) forming an aqueous suspension of an acid addition salt of compound (1), stirring the suspension at a temperature of 25°C to 75°C for a period of time sufficient to disproportionate the acid addition salt to form a crystalline form of the free base of compound (1), and then isolating the crystalline form; or

[0087] (ii) forming an aqueous suspension of an amorphous form of compound (1) free base, wherein the aqueous suspension is unbuffered or buffered to a pH of 1.75 to 7.25, stirring the aqueous suspension at a temperature of 25°C to 55°C for a period of time sufficient to convert the amorphous form of compound (1) free base into a crystalline form of compound (1) free base, and then isolating the crystalline form.

[0088] In process variant (i), the choice of temperature and stirring time will affect whether Form A or Form B is produced. Thus, for example, to obtain Form A, the process may be carried out at a higher temperature, for example, 65-75° C. (more particularly about 70° C.). Conversely, to obtain Form B, the process may be carried out at a lower temperature, for example, 25-55° C. The formation of the crystalline form may be promoted by adding an alcohol (e.g., isopropanol) to the process mixture.

[0089] The starting material for process variant (i) is an acid addition salt of compound (1). The acid addition salt may be amorphous.

[0090] The acid addition salt of the compound (1) may be, for example, an inorganic acid salt, for example, a hydrochloride, a hydrobromide or a sulfate. Methods for preparing the salt are familiar to those skilled in the art. The salt may be prepared by adding the free base compound to a solution of the counter ion in a solvent. The solvent may be a polar protic solvent, for example, 2-propanol or methanol, and may include a polar aprotic cosolvent, for example, dichloromethane.

[0091] Process variant (ii) generally results in Form B.

[0092] In method variation (ii), the aqueous suspension may be buffered or unbuffered. For example, it may be unbuffered or buffered to a pH of 2, 5 or 7. The aqueous suspension is stirred and gently heated, for example, to a temperature of about 25°C to about 35°C, for example, about 30°C. The aqueous suspension is stirred for a period of time sufficient to convert the amorphous form of the compound (1) free base into the crystalline form of the compound (1) free base. The stirring is usually for at least 1 day, more usually for at least 2 days or at least 3 days, and in one embodiment, for 5 days.

[0093] In alternative conditions of process variant (ii), the process can be carried out at a higher temperature (e.g., 45-55° C., in particular about 50° C.) for a shorter time (e.g., at least 12 hours, or at least 15 hours; e.g., 20 hours). In order to assist the formation of Form B, seed crystals or Form A or Form B may be added.

[0094] Amorphous salt of the compound of formula (1)

[0095] A number of amorphous salts of the compound of formula (1) were prepared (see Example 2). Accordingly, another aspect of the present invention provides a salt of the compound of formula (1) in amorphous form.

[0096] The salt may be a hydrochloride, sulfate, naphthalene disulfonate (naphthalene-1,5-disulfonate), ethane disulfonate (ethane disulfonate), toluene sulfonate (p-toluene sulfonate), methane sulfonate (methane sulfonate), naphthalene sulfonate (2-naphthalene sulfonate), benzene sulfonate (phenyl sulfonate), isethionate (2-hydroxyethyl sulfonate), ethane sulfonate (ethyl sulfonate) or hydrobromide of the compound of formula (1).

[0097] One group of salts consists of the hydrochloride, sulfate, hydrobromide or naphthalene disulfonate salts of the amorphous compound of formula (1).

[0098] In other embodiments, the present invention provides:

[0099] Amorphous hydrochloride of the compound of formula (1);

[0100] Amorphous sulfate of the compound of formula (1);

[0101] an amorphous hydrobromide salt of the compound of formula (1); and

[0102] Amorphous naphthalene disulfonate of the compound of formula (1).

[0103] The amorphous salt can be prepared by reacting the free base form of the compound with a suitable acid in an organic solvent, preferably a polar aprotic solvent (e.g., isopropyl acetate), or a mixture of a polar aprotic solvent and a polar protic solvent (e.g., a mixture of isopropyl acetate and 2-propanol).

[0104] The amorphous salt of the present invention may be in the form of particles having a mass median diameter of 1 μm to 100 μm.

[0105] The mass median diameter of the particles can be 2 μm to 50 μm, for example, 2 μm to 25 μm, or 2 μm to 10 μm. The particles can be orally administered (usually an orally adminstrable formulation, optionally containing one or more pharmaceutically acceptable excipients) or administered by other routes, for example, inhalation. When the particles are used for administration by inhalation, the mass median diameter of the particles is typically 1 μm to 10 μm, or 1 μm to 5 μm.

[0106] Particle size can be determined using image analysis, laser diffraction or sieving techniques.

[0107] The particles can be prepared by mechanical micronization or solution-based phase separation. Examples of mechanical micronization methods include grinding techniques.

[0108] Solution-based techniques generally involve the use of liquids, compressed gases, near-critical liquids or supercritical fluids as solvents or cryogenic media for rapid freezing. These techniques involve phase separation of the solvent and the drug compound by evaporation, expansion, freezing or changing the solvent composition.

[0109] The particles can be prepared by freeze drying. Alternatively, the particles can be prepared by spray drying. Therefore, in one embodiment, the amorphous salt of the present invention is spray dried.

[0110] The amorphous salts are useful as therapeutic agents or, as described above, as intermediates for preparing the free base of formula (1) in crystalline form.

[0111] Preparation method of compound of formula (1)

[0112] Compound (1) can be prepared by a series of steps as shown in Scheme 1 below.

[0113]

[0114] Solution 1

[0115] The reaction of intermediate compound (2) with intermediate compound (3) to give compound (1) is described in Example 685 of our earlier application PCT / IB2016 / 001507. The reaction is carried out in dimethylformamide (DMF) and trimethylamine in the presence of an amide bond forming promoter, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU). In PCT / IB2016 / 001507, intermediate compound (2) is prepared by hydrolysis of the corresponding tert-butyl ester, which itself is prepared by reaction of intermediate compound (4) with amine (6).

[0116] The present invention makes many improvements to the method described in PCT / IB2016 / 001507.

[0117] First, the process conditions for the reaction of the intermediate compound (2) with (3) in the final step were improved. Therefore, the coupling agent TBTU was no longer used, but coupling agents such as N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) were used instead. In addition, alternative bases such as diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP), as well as triethylamine, were used.

[0118] Secondly, instead of reacting the intermediate compound (4) with the amine (6) and then hydrolyzing the tert-butyl ester to obtain the compound (2) as described in PCT / IB2016 / 001507, the tert-butyl ester unit in the intermediate compound (4) is first hydrolyzed to obtain the carboxylic acid (5), and then the carboxylic acid (5) is reacted with the amine (6) to obtain the compound (2).

[0119] Another aspect of the present invention provides a method for preparing a compound of formula (1), the method comprising reacting a compound of formula (2) with a compound of formula (3):

[0120]

[0121] The reaction is carried out in an aprotic solvent in the presence of a tertiary amine base and an amide bond promoter, wherein the amide bond promoter is selected from N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

[0122] In a specific embodiment, the amide bond promoter is N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU).

[0123] Examples of tertiary amine bases used in the process are diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP) and triethylamine and mixtures thereof.

[0124] In a specific embodiment, the tertiary amine base is diisopropylethylamine (DIPEA).

[0125] Examples of aprotic solvents are dichloromethane, ethyl acetate and dimethylformamide.

[0126] In a specific embodiment, the aprotic solvent is dichloromethane.

[0127] In a preferred embodiment, a method for preparing a compound of formula (1) is provided, which comprises reacting a compound of formula (2) with a compound of formula (3) in an aprotic solvent of dichloromethane in the presence of a tertiary amine base diisopropylethylamine (DIPEA) and an amide bond promoter N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU).

[0128] The reaction between compound (2) and compound (3) is usually carried out without external heating, for example, at a temperature not exceeding 25° C. Thus, for example, once the reactants are combined to form a reaction mixture, the reaction mixture may be stirred at a temperature of 15-25° C. until the reaction is complete.

[0129] In another aspect, the present invention provides a process for preparing a compound of formula (1) as defined herein, said process comprising:

[0130] a) making a compound of formula (5):

[0131]

[0132] Reaction with a compound of formula (6),

[0133]

[0134] The compound of formula (2) is obtained:

[0135]

[0136] and

[0137] b) reacting a compound of formula (2) with a compound of formula (3):

[0138]

[0139] The compound of formula (1) is obtained and then optionally formed into a salt or crystalline form thereof.

[0140] Step (a) is usually carried out in a polar aprotic solvent such as 1-methyl-2-pyrrolidone (NMP). The reaction is carried out under high temperature conditions, for example, a temperature exceeding 60°C, more usually exceeding 70°C, in particular a temperature of 75-95°C (e.g., 80-95°C).

[0141] Step (a) is carried out in the presence of a base, which may be an inorganic base such as an alkali metal carbonate, for example, potassium carbonate.

[0142] The progress of the reaction between the compound of formula (5) and the compound of formula (6) can be monitored to determine the extent of the reaction. For example, the reaction can be monitored until the residual content of the compound of formula (5) is below the desired level (e.g., below 1 mol% of its original amount). The reaction time of step (a) is generally 1 to 8 hours, for example, 2 to 7 hours, generally 4 to 6 hours.

[0143] Step (b) is carried out under the conditions described above for reacting compound (2) and compound (3) to produce compound (1).

[0144] Another aspect of the present invention provides a method for preparing a compound of formula (2), the method comprising:

[0145] a) making a compound of formula (5):

[0146]

[0147] Reaction with a compound of formula (6),

[0148]

[0149] The compound of formula (2) is obtained:

[0150]

[0151] The reaction is carried out under the conditions described above for step (b).

[0152] Compound (5) can be prepared by hydrolysis of the tert-butyl ester compound of formula (4):

[0153]

[0154] For example, a mineral acid such as concentrated hydrochloric acid is used. The hydrolysis reaction may be assisted by mild heating, for example, at a temperature of 30-45°C, more typically 35-40°C. A hydrocarbon or chlorinated hydrocarbon cosolvent may be used. One such cosolvent is toluene.

[0155] Compound (4) can be prepared by the method described in PCT / IB2016 / 001507; see, for example, Preparation 94 therein.

[0156] Compositions containing compounds of formula (1)

[0157] The solubility of the compound of formula (1) in water is relatively poor. Therefore, the present invention provides a composition of compound (1) comprising a primary vehicle other than water. The composition is suitable for oral administration.

[0158] We have found that compound (1) has good solubility in many non-aqueous solvents. Therefore, the present invention provides a pharmaceutical composition comprising a compound of formula (1) and a medium selected from:

[0159] -C 2-4 alcohol;

[0160] - polyether compounds;

[0161] -C8-C 18 Monoesters of long-chain fatty acids with glycerol or propylene glycol;

[0162] -C8–C 10 di- or triglycerides of long-chain fatty acids;

[0163] and mixtures thereof.

[0164] The pharmaceutical composition may be in the form of a solution of compound (1) in a vehicle.

[0165] Another aspect of the present invention provides a method for preparing a pharmaceutical composition containing a compound of formula (1), the method comprising dispersing the compound of formula (1) in a medium selected from:

[0166] -C 2-4 alcohol;

[0167] - polyether compounds;

[0168] -C8-C 18 Monoesters of long-chain fatty acids with glycerol or propylene glycol;

[0169] -C8–C 10 di- or triglycerides of long-chain fatty acids;

[0170] and mixtures thereof.

[0171] Typically, the compound of formula (1) is dispersed in a medium to form a solution or suspension. In one embodiment, the compound of formula (1) is suspended in the medium. In another embodiment, the compound of formula (1) is dissolved in the medium to form a suspension.

[0172] The medium may include monohydroxy or polyhydroxy C 2-4 Alcohol, preferably C 2-3 Alcohol, for example, ethanol or propylene glycol.

[0173] When the medium comprises a polyether compound, the polyether compound may be polyethylene glycol (PEG). The average molecular weight of the polyethylene glycol may be 200 to 10,000 g / mol, for example, 300 to 8,000 g / mol. In one embodiment, the average molecular weight of the polyethylene glycol is about 200 to 400 g / mol, for example, 300 to 450 g / mol.

[0174] Depending on the nature and relative amounts of the components of the vehicle, the composition may be liquid, semi-solid or solid. For example, when a higher molecular weight PEG is used, the viscosity of the composition may increase to the point where it is considered a "semi-solid" or solid, while when a lower molecular weight PEG is used, a liquid composition may result. In the context of such compositions, reference to a solution includes solid solutions as well as liquid (or semi-solid) solutions.

[0175] Alternatively or additionally, the vehicle may comprise caprylic acid or capric acid and monoesters, diesters and triesters of caprylic acid or capric acid. Examples of these esters include propylene glycol monocaprylate, monocaprylin, dicaprylin, tricaprylin, monocaprin, dicaprin, tricaprin. Caprylocaproylmacrogol-8glycerides ( ALF) is a commercially available vehicle comprising mono-, di- and tri-glycerides of caprylic and capric acid and mono- and diesters of polyethylene glycol having an average molecular weight of 200 to 400 g / mol.

[0176] In another alternative, the vehicle may include monoglycerides of longer chain fatty acids such as linoleic acid or oleic acid. Examples of these vehicles include monolinolein glyceride (Maisine CC TM ) and monoolein (40 type, Peceol TM ).

[0177] In one embodiment, the medium is selected from ethanol, propylene glycol, polyethylene glycol, and mixtures thereof. For example, the medium may include a combination of propylene glycol and ethanol, for example, a combination of propylene glycol and ethanol in a ratio of 50:50 to 90:10% w / w (e.g., a ratio of propylene glycol and ethanol of 75:25 or 85:15% w / w). In one embodiment, the medium includes a combination of propylene glycol and ethanol in a ratio of 75:25 to 90:10% w / w.

[0178] In another embodiment, the vehicle is selected from ethanol, polyethylene glycol 400 (PEG 400) and propylene glycol, and mixtures thereof.

[0179] The composition generally allows oral administration of a total daily dose of compound (1) of no more than 1.2 g / day. In the composition of the present invention, the concentration of compound (1) in the medium may be 10 mg / ml-130 mg / ml, for example, 40 mg / ml-125 mg / ml, more particularly 110 mg / ml-125 mg / ml. At a concentration of 120 mg / ml, taking 10 ml of the composition is equivalent to taking 1.2 g of compound (1).

[0180] Alternatively, the composition can be contained in a capsule. Suitable capsules for delivering the composition described herein include hard gelatin capsules or soft gelatin capsules. In one embodiment, the composition is contained in a soft gelatin capsule. The term "gelatin" used herein refers not only to capsules prepared from gelatin, but also to capsules prepared from non-gelatin equivalents (e.g., pullulan or modified cellulose, such as hydroxypropyl methylcellulose).

[0181] The composition may also include one or more surfactants to aid in the solubility of compound (1) in the selected vehicle. The surfactant may also inhibit precipitation of the compound of formula (1) when the composition is diluted in the gastrointestinal tract.

[0182] The surfactant is typically a nonionic surfactant.

[0183] The nonionic surfactant may be, for example, a polyol ester, a polyoxyethylene ester or a poloxamer.

[0184] In one embodiment, the surfactant is tocopherol polyethylene glycol (TPG) having the following structural formula, for example, D-α-tocopherol polyethylene glycol succinate (TPGS):

[0185]

[0186] wherein n has an average value of about 10 to about 30, more typically about 15 to about 27; for example, about 20 to about 25. A specific TPGS is α-tocopheryl polyethylene glycol 1000 succinate (approximate average molecular weight 1513), wherein the polyoxyethylene group [—O—CH2—CH2] n The molecular weight of is about 1000 (eg, 950 to 1050), and the average value of n is about 22. Examples of polyol esters include ethylene glycol esters and glycerol esters and sorbitan derivatives.

[0187] Fatty acid esters of sorbitan (commonly known as span) and their ethoxylated derivatives (commonly known as tweens) include sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan monooleate (Span 80), sorbitan tristearate (Span 65), sorbitan trioleate (Span 8), polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (20) sorbitan monopalmitate (Tween 40), polyoxyethylene (20) sorbitan monostearate (Tween 60), polyoxyethylene (20) sorbitan monooleate (Tween 80). 80), polyoxyethylene (20) sorbitan tristearate (Tween 65) and polyoxyethylene (20) sorbitan trioleate (Tween 85).

[0188] Other specific examples of surfactants include Cremophor RH 40, RH40), Cremophor EL, EL), Polysorbate 80 (Tween80), Gelucire 44 / 14 (Lauroyl Macrogol-32 Glycerate), Solutol HS-15 (Macroglobulin 15 Hydroxystearate) and ALF (Caprylic / capric macrogol glycerides). In one embodiment, the surfactant is Cremophor RH 40.

[0189] In one embodiment, the composition includes a compound of formula (1), ethanol and tocopherol polyethylene glycol (TPG), for example, D-α-tocopherol polyethylene glycol 1000 succinate (TPGS). Ethanol and TPG may be present in the following ratio: 20:80 ethanol:TPG to 60:40 ethanol:TPG, for example, 30:70 ethanol:TPG to 50:50 ethanol:TPG.

[0190] In another embodiment, in addition to the compound of formula (1), the composition further comprises a vehicle comprising:

[0191] (i) propylene glycol;

[0192] (ii) a nonionic surfactant (such as Cremophor RH40 and tocopherol polyethylene glycol); and optionally

[0193] (iii) Ethanol.

[0194] In another embodiment, in addition to the compound of formula (1), the composition further comprises a vehicle comprising:

[0195] (i) propylene glycol;

[0196] (ii) a polyoxyethylene ester nonionic surfactant; and optionally

[0197] (iii) Ethanol.

[0198] The polyoxyethylene ester nonionic surfactant may be, for example, tocopheryl polyethylene glycol (TPG) as defined above, such as D-α-tocopheryl polyethylene glycol succinate (TPGS).

[0199] In one particular embodiment, the composition is free of ethanol (iii).

[0200] In another specific embodiment, the composition comprises ethanol (iii).

[0201] In another specific embodiment, the medium comprises propylene glycol and tocopheryl polyethylene glycol (TPG). In this embodiment, the medium may comprise TPGS, wherein propylene glycol:TPGS (weight ratio) is 1:2 to 10:1, and may optionally further comprise ethanol, wherein the weight ratio of ethanol:propylene glycol (e.g., D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)) is 1:10 to 2:1, and the weight ratio of propylene glycol:tocopheryl polyethylene glycol is 1:2 to 5:1; and optionally further comprises ethanol, the weight ratio of ethanol:propylene glycol is 1:10 to 2:1.

[0202] The composition comprising a compound of formula (1), propylene glycol, a polyoxyethylene ester nonionic surfactant, and optionally ethanol may conveniently be contained in a capsule, for example a hard gelatin capsule or a soft gelatin capsule.

[0203] In step a), the compound of formula (1) may be in crystalline form. In one embodiment, in step a), the compound of formula (1) may be in crystalline form as described herein.

[0204] The low water solubility of a pharmaceutical compound can be improved by reducing the solid particle size of the compound. By reducing the particle size of the pharmaceutical compound, the surface area that can be solvated is increased.

[0205] Therefore, another aspect of the present invention provides a compound of formula (1) in the form of particles having a mass median diameter of from 1 μm to 100 μm.

[0206] The mass median diameter of the particles can be 2 μm to 50 μm, for example, 2 μm to 25 μm, or 2 μm to 10 μm. The particles can be orally administered (usually an oral formulation, optionally containing one or more pharmaceutically acceptable excipients) or administered by other routes, for example, inhalation. When the particles are used for administration by inhalation, the mass median diameter of the particles is generally 1 μm to 10 μm, or 1 μm to 5 μm.

[0207] Particle size can be determined by image analysis, laser diffraction or sieving.

[0208] The particles can be prepared by mechanical micronization or solution-based phase separation. Examples of mechanical micronization methods include grinding techniques.

[0209] Solution-based techniques generally involve the use of liquids, compressed gases, near-critical liquids or supercritical fluids as solvents or cryogenic media for rapid freezing. These techniques involve phase separation of the solvent and the drug compound by evaporation, expansion, freezing or changing the solvent composition.

[0210] The particles can be prepared by freeze drying. Alternatively, the particles can be formed by spray drying.

[0211] definition

[0212] The compound of formula (1) may be referred to in the present application by its chemical name, or, for convenience, referred to as "the compound", "the compound of formula (1)", "compound (1)" or "the compound of the present invention". These nouns all refer to the compound represented by formula (1) above, and its chemical name is (2R)-2-(6-{5-chloro-2-[(oxacyclohexyl-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide.

[0213] The term "mass median diameter" used herein to define particle size is defined as the diameter at which 50% (mass) of the particles have a larger diameter and 50% (mass) of the particles have a smaller diameter. This diameter refers to the equivalent spherical diameter, which, for non-spherical particles, is equal to the diameter of a spherical particle of the same volume as the non-spherical particle.

[0214] ERK1 / 2 refers to either or both of the ERK1 and ERK2 isozymes of the extracellular signal-regulated kinase (ERK).

[0215] "Potency" is a measure of drug activity expressed as the amount required to produce an effect of a given magnitude. Drugs with high potency elicit a greater response at low concentrations. Potency is proportional to affinity and efficacy. Affinity is the ability of a drug to bind to an enzyme. Efficacy is the relationship between target occupancy and the ability to elicit a response at the molecular, cellular, tissue, or system level.

[0216] The term "inhibitor" refers to an enzyme inhibitor, which is a type of ligand or drug that blocks or impedes a biological response mediated by ERK1 / 2. Inhibitors modulate their effects by binding to the active site on the enzyme or to an allosteric site, or they may interact at a unique binding site that is not normally involved in the biological regulation of enzyme activity. Inhibition may be caused directly or indirectly, may be mediated by any mechanism and at any physiological level. Therefore, inhibition caused by a ligand or drug may manifest itself in functionally different ways under different circumstances. Inhibitory activity may be reversible or irreversible depending on the lifetime of the inhibitor-enzyme complex, which in turn depends on the nature of the inhibitor-enzyme binding.

[0217] As used herein, the term "treatment" in the context of treating a condition, i.e., a state, disorder, or disease, generally refers to treatment and therapy, whether directed to humans or animals (e.g., veterinary applications), wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition, including slowing the rate of progression, halting the rate of progression, alleviating the condition, alleviating or relieving at least one symptom associated with or caused by the condition being treated, and curing the condition. For example, treatment may alleviate one or more symptoms of a disorder or completely cure the disorder.

[0218] As used herein, the term "prevention" (i.e., use of a compound as a preventative measure) in the context of treating a condition, i.e., a state, disorder, or disease, generally refers to prevention or prophylaxis, whether in humans or animals (e.g., veterinary applications), wherein some desired prophylactic effect is achieved, e.g., preventing the onset of a disease or preventing the onset of a disease. Prevention includes total and total blocking of all symptoms of a disorder for an indeterminate period of time, merely slowing down the onset of one or more symptoms of a disease, or reducing the likelihood of the onset of a disease, and does not include alleviation of the condition, alleviation or relief of at least one symptom associated with or caused by the condition being treated, and cure of the condition.

[0219] The scope of preventing or treating a disease state or condition, such as cancer, includes alleviating or reducing the incidence of, for example, cancer.

[0220] As used herein, the term "mediate", as used with ERK1 / 2 as described herein (and for, e.g., various physiological processes, diseases, states, conditions, treatments, treatments or interventions), is intended to operate in a restricted manner such that the various processes, diseases, states, conditions, treatments and interventions to which the term is applied are those in which the protein plays a biological role. When the term is applied to a disease, state or condition, the biological role played by the protein may be direct or indirect, and may be necessary and / or sufficient for the manifestation of symptoms of the disease, state or condition (or its etiology or progression). Thus, protein function (particularly abnormal levels of function, e.g., overexpression or underexpression) is not necessarily the proximal cause of the disease, state or condition: rather, we envision that mediated diseases, states or conditions include those with multifactorial etiologies and complex processes, in which only a portion of the protein in question is involved. When the term is applied to treatment, prevention or intervention, the role played by the protein may be direct or indirect, and may be necessary and / or sufficient for the therapeutic operation, prevention or intervention outcome. Thus, protein-mediated disease states or conditions include the development of resistance to any particular cancer drug or treatment.

[0221] The combination of the present invention may produce a therapeutically effective effect compared to the therapeutic effect of the individual compounds / agents when administered separately.

[0222] The term "effective" includes beneficial effects such as additivity, synergy, reduced side effects, reduced toxicity, prolonged disease progression, prolonged survival, sensitization or resensitization of one agent to another, or improved response rate. Advantageously, an effective effect may allow a reduction in the patient dose of each or any component, thereby reducing the toxicity of chemotherapy while achieving and / or maintaining the same therapeutic effect. In the present invention, a "synergistic" effect refers to a therapeutic effect produced by the combination that is greater than the sum of the therapeutic effects of the agents in the combination when administered alone. In the present invention, an "additive" effect refers to a therapeutic effect produced by the combination that is greater than the therapeutic effect of any of the agents in the combination when administered alone. In the case of solid tumors, the term "response rate" used in the present invention refers to the degree of reduction in tumor size at a given time point (e.g., 12 weeks). Thus, for example, a 50% response rate refers to a 50% reduction in tumor size. A "clinical response" herein refers to a response rate equal to or greater than 50%. A "partial response" is defined herein as a response rate less than 50%, provided that the response rate is greater than 0%.

[0223] As used herein, the term "combination", for example, when applied to two or more compounds and / or agents, refers to a material in which the two or more agents are combined. In the present invention, the terms "combined" and "combination" are interpreted accordingly.

[0224] The association of two or more compounds / agents in a combination may be physical or non-physical. Examples of physically associated combination compounds / agents include:

[0225] A composition (e.g., a unitary formulation) comprising two or more compounds / agents in admixture (e.g., in the same unit dose);

[0226] Compositions comprising materials in which two or more compounds / agents are chemically / physicochemically linked (e.g., cross-linked, molecularly aggregated, or bound to a common vehicle moiety);

[0227] A composition comprising a material in which two or more compounds / agents are chemically / physicochemically co-packaged (e.g., disposed on or within a lipid vesicle, particle (such as a microparticle or nanoparticle), or emulsion droplet);

[0228] A pharmaceutical kit, pack or patient pack in which two or more compounds / agents are co-packaged or co-presented (e.g., as part of a series of unit doses);

[0229] Examples of non-physical combined compounds / agents include:

[0230] Materials (e.g., not a single formulation) containing at least one of the two or more compounds / agents and instructions for promptly combining the at least one compound to form a physical combination of the two or more compounds / agents;

[0231] Materials (e.g., not a single formulation) containing at least one of the two or more compounds / agents and instructions for treatment with the combination of the two or more compounds / agents;

[0232] Materials comprising at least one of the two or more compounds / agents and instructions for administration to a patient population to which the other compounds / agents have been (or are being) administered;

[0233] - A material comprising at least one of two or more compounds / agents in an amount or form specifically suitable for use in combination with the other compounds / agents of the two or more compounds / agents.

[0234] The term "combination therapy" as used herein refers to a treatment including a combination (as defined above) of two or more compounds / agents. Therefore, in the present application, "combination therapy", "combination" and the compound / agent using "combination" may refer to a compound / agent administered as part of the same total treatment regimen. Therefore, the dosage of each compound / agent in two or more compounds / agents may be different: each compound / agent may be administered simultaneously or at different times. Therefore, it should be understood that the compound / agent in the combination may be administered in the same pharmaceutical preparation (such as together) or in different pharmaceutical preparations (i.e., separately) in sequence (e.g., before or after) or simultaneously. Simultaneous administration in the same preparation is administered as a single preparation, while simultaneous administration in different pharmaceutical preparations is administered as a non-single preparation. The dosage of each compound / agent in two or more compounds / agents in the combination therapy may also be different relative to the route of administration.

[0235] As used herein, the term "pharmaceutical kit" defines a series of one or more unit doses of a pharmaceutical composition and a means of metering (e.g., a measuring device) and / or a means of administration (e.g., an inhaler or syringe), optionally all contained in the same outer package. In a pharmaceutical kit comprising a combination of two or more compounds / agents, the individual compounds / agents may be single or non-single formulations. The unit doses may be contained in blister packs. The pharmaceutical kit may optionally further include instructions for use.

[0236] As used herein, the term "pharmaceutical package" defines a series of one or more unit doses of a pharmaceutical composition, optionally contained in the same outer packaging. In a pharmaceutical package comprising a combination of two or more compounds / agents, a single compound / agent may be a single or non-single formulation. The unit dose may be contained in a blister pack. The pharmaceutical package may optionally further include instructions for use.

[0237] Salts, solvates, tautomers and isotopes

[0238] Unless the context indicates otherwise, reference to a compound of formula (1) includes ionic forms, salts, solvates, tautomers and isotopic variations thereof.

[0239] Salt

[0240] The compounds of formula (1) may exist in the form of salts, in particular in the form of acid addition salts. Unless the context indicates otherwise, all these salts belong to the scope of the present invention, and the compounds of formula (1) include the salt forms of these compounds.

[0241] Salts of compound (1) can be synthesized from compound (1) using conventional chemical methods, such as the method described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, these salts can be prepared by reacting the free base form of the compound with a suitable acid in water or in an organic solvent or in a mixture of the two; generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is used.

[0242] Acid addition salts (mono or di) can be formed with a wide variety of acids (inorganic and organic). Examples of acid addition salts include mono or di salts formed with acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, horse urine Acid, hydrohalic acid (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, valeric acid, as well as acylated amino acids and cation exchange resins.

[0243] A specific group of salts consists of salts formed from the following acids: acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, acetic acid, propionic acid, butyric acid, malonic acid, glucuronic acid, lactobionic acid. A specific salt is the hydrochloride.

[0244] The salt forms of the compounds of the present invention are generally pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19. However, non-pharmaceutically acceptable salts may also be prepared as intermediate forms, which are then converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salt forms may be used, for example, to purify or isolate the compounds of the present invention and are also part of the present invention.

[0245] Geometric isomers and tautomers

[0246] Unless the context indicates otherwise, the compound of formula (1) may exist in a variety of different tautomeric forms, and all of these forms are included when referring to the compound of formula (1). In order to eliminate doubts, the compound may exist in one or more tautomeric forms, and only one form is specifically described or illustrated, in any case, formula (1) also includes all other forms.

[0247] The customary method of representing stereochemistry using "hashed lines" or "wedges" is used to represent specific stereochemical forms, for example, as shown for the two molecules below.

[0248] (S)-2-(6-bromo-1-oxoisoindolin-2-yl)-3-hydroxypropionic acid methyl ester

[0249] (R)-tert-Butyl 2-(6-bromo-1-oxoisoindolin-2-yl)propanoate.

[0250] Optical isomers can be characterized and identified by their optical activity (i.e., as + and - isomers, or d and l isomers) or they can be characterized by their absolute stereochemistry using the "R and S" nomenclature developed by Cahn, Ingold and Prelog, see Advanced Organic Chemistry by Jerry March, 4 th Edition, John Wiley & Sons, New York, 1992, pp. 109-114, and see Cahn, Ingold & Prelog, Angew. Chem. Int. Ed. Engl., 1966, 5, 385-415.

[0251] Optical isomers may be separated using a variety of techniques including chiral chromatography (chromatography on chiral supports), which techniques are familiar to those skilled in the art.

[0252] As an alternative to chiral chromatography, separation of optical isomers can be accomplished by forming diastereomeric salts with chiral acids (e.g., (+)-tartaric acid, (-)-pyroglutamic acid, (-)-di-toluoyl-L-tartaric acid, (+)-mandelic acid, (-)-malic acid, and (-)-camphorsulfonic acid), separating the diastereoisomers by preferential crystallization, and then dissociating the salts to yield the individual enantiomers of the free base. Similarly, optical isomers of acidic compounds can be separated by forming diastereomeric salts with chiral amines (e.g., brucine, cinchonidine, quinine, etc.).

[0253] Additionally, separation of enantiomers may be achieved by covalently attaching an enantiomerically pure chiral auxiliary to the compound and then performing diastereoisomer separation using conventional methods such as chromatography. The aforementioned covalent attachment is then cleaved to yield the appropriate enantiomerically pure product. For example, optical isomers of a chiral compound containing a free hydroxyl group may be separated by forming a Mosher's acid ester and then separating the resulting diastereomers using chromatography, followed by cleavage of the ester to recover the free hydroxyl group.

[0254] When a specific stereochemical configuration of a compound of the present invention is shown, this may mean that at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound is present in a stereochemical form that is different from other isomeric forms of the compound. In a general embodiment, 99% or more (e.g., substantially all) of the total amount of the compound of formula (1) is present in the depicted stereochemical configuration.

[0255] Isotope variation

[0256] Here, compound (1) includes all pharmaceutically acceptable isotopically labeled variations thereof, wherein one or more atoms are replaced by atoms having the same atomic number but an atomic mass or atomic mass number different from the atomic mass or atomic mass number usually found in nature.

[0257] Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen such as 2 H(D) and 3 H(T), isotopes of carbon such as 11 C. 13 C and 14 C, isotopes of chlorine such as 36 Cl, fluorine isotopes such as 18 F, nitrogen isotopes such as 13 N and 15 N, and oxygen isotopes such as 15 O.17 O and 18 O.

[0258] Certain isotopically labeled compounds of formula (1), for example, those containing radioactive isotopes, are very useful in drug and / or substrate tissue distribution studies. The compounds of formula (1) also have valuable diagnostic properties. They can be used to detect or identify the formation of complexes between the labeled compound and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with labeling agents such as radioactive isotopes, enzymes, fluorescent substances, luminescent substances (for example, luminol, luminol derivatives, fluorescein, aequorin and luciferase). The radioactive isotope tritium (i.e. 3 H(T)) and carbon-14 (i.e. 14 C) is particularly useful for this purpose due to its ease of introduction and detection.

[0259] With heavier isotopes such as deuterium (i.e. 2 H(D)) substitution has certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in certain circumstances. In particular, regardless of whether hydrogen is explicitly defined or its presence is implied to satisfy the valence of the relevant atoms (especially carbon atoms), any reference to hydrogen in this application should be interpreted as including 1 H and 2 H.

[0260] Using positron emitting isotopes such as 11 C. 18 F. 15 O and 13 N substitution is very useful in positron emission tomography (PET) studies for detecting target occupancy.

[0261] Isotopically labeled compounds of formula (1) can generally be prepared by conventional methods familiar to those skilled in the art or by methods analogous to those described in the accompanying examples and preparations, using an appropriate isotopically labeled reagent in place of the non-labeled reagent previously used.

[0262] Compound

[0263] Formula (1) also includes within its scope complexes of the compounds (e.g., inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals). Inclusion complexes, clathrates and metal complexes can be formed using methods familiar to those skilled in the art.

[0264] Biological properties

[0265] Compound (1) is contemplated for use in medicine or therapy.

[0266] The compounds of the present invention are inhibitors of ERK1 / 2 and will be used to prevent or treat the diseases or conditions described herein in which ERK1 / 2 plays a role, for example, the diseases and conditions discussed below and the diseases and conditions described above in the "Background of the Invention". In addition, the compounds of the present invention will be used to prevent or treat ERK1 / 2-mediated diseases or conditions, for example, diseases or conditions that require ERK1 / 2 activity or that upregulate ERK1 / 2 activity due to activating mutations in upstream components of the MAPK pathway (such as RAS, K-RAS, NRAS and RAF), such as cancer.

[0267] Preventing or preventing or treating a disease state or condition (such as cancer) includes reducing or decreasing the incidence of the disease or condition. Thus, for example, the compounds of the invention are contemplated for use in reducing or decreasing the incidence of cancer.

[0268] The compounds of formula (1) below include crystalline forms of the compounds of formula (1) described herein and compounds of formula (1) prepared according to the methods described herein.

[0269] Therefore, in other embodiments of the present invention, there are provided:

[0270] A compound of formula (1) for pharmaceutical use.

[0271] - A compound of formula (1) for preventing or treating a disease or condition mediated by ERK1 / 2.

[0272] Use of a compound of formula (1) for preparing a medicament for preventing or treating a disease or condition mediated by ERK1 / 2.

[0273] A method for preventing or treating a disease or condition mediated by ERK1 / 2 in a subject (eg, a mammalian subject such as a human in need thereof), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0274] - A compound of formula (1) that decreases or reduces the incidence of a disease or condition mediated by ERK1 / 2.

[0275] Use of a compound of formula (1) for the preparation of a medicament for reducing or decreasing the incidence of a disease or condition mediated by ERK1 / 2.

[0276] A method of reducing or decreasing the incidence of an ERK1 / 2 mediated disease or condition in a subject (eg, a mammalian subject in need thereof, such as a human), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0277] More particularly, compound (1) is an ERK1 / 2 inhibitor. For example, the compounds of the present invention have inhibitory effects on ERK1 or ERK2 and particularly on ERK1 / 2.

[0278] The ERK inhibitor compound of formula (1) can bind to ERK1 / 2 and show efficacy against ERK1 / 2. In one embodiment, the inhibitor compound of formula (1) is selective for ERK1 / 2 (relative to other kinase family members), and relative to other kinase family members, they preferentially bind to ERK1 and / or ERK2 and / or inhibit ERK1 and / or ERK2.

[0279] The function of ERK1 / 2 in controlling cell signaling is also associated with many diseases, including those associated with cell aggregation (such as cancer, autoimmune diseases, inflammation and restenosis), diseases in which excessive apoptosis leads to cell death (such as stroke, heart failure), neurodegeneration (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis), AIDS, local ischemia (stroke, myocardial infarction) and osteoporosis or the treatment of autoimmune diseases such as multiple sclerosis (MS).

[0280] The ERK1 / 2-mediated disease or condition mentioned in any of the preceding embodiments may be any one or more of the above-mentioned diseases or conditions.

[0281] Thus, the compounds of the invention as defined herein are also contemplated to be useful in the treatment of other diseases such as inflammation, hepatitis, ulcerative colitis, gastritis, autoimmune diseases, inflammation, restenosis, stroke, heart failure, neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, myotonic dystrophy and amyotrophic lateral sclerosis), AIDS, ischemia (such as traumatic brain injury, spinal cord injury, cerebral ischemia, cerebral ischemia / reperfusion (I / R) injury, acute and chronic CNS injury ischemia, stroke or myocardial infarction), degenerative diseases of the musculoskeletal system (such as osteoporosis), autoimmune diseases such as multiple sclerosis (MS) and type I diabetes, and eye diseases such as retinal degeneration due to uncontrolled programmed cell death.

[0282] Due to their affinity for ERK1 / 2, the compounds of the invention will be useful in providing a means of controlling cellular signaling. It is therefore expected that the compounds will prove useful in the treatment or prevention of proliferative diseases such as cancer.

[0283] Therefore, in other embodiments, the present invention provides:

[0284] - A compound of formula (1) for use in preventing or treating a proliferative disease such as cancer.

[0285] Use of the compound of formula (1) for preparing a medicament for preventing or treating a proliferative disease (such as cancer).

[0286] A method of preventing or treating a proliferative disease (such as cancer) in a subject (eg, a mammalian subject such as a human in need thereof), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0287] - A compound of formula (1) for use in reducing or decreasing the incidence of a proliferative disease such as cancer.

[0288] Use of the compound of formula (1) for preparing a drug for reducing or lowering the incidence of proliferative diseases (such as cancer).

[0289] A method of reducing or decreasing the incidence of a proliferative disease (such as cancer) in a subject (eg, a mammalian subject such as a human in need thereof), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0290] Examples of cancers (and benign tumors thereof) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other cancers), such as bladder cancer and urethral cancer, breast cancer, gastrointestinal cancer (including esophageal cancer, gastric cancer (stomach cancer), small intestine cancer, colon cancer, rectal cancer, and anal cancer), liver cancer (hepatocellular carcinoma), gallbladder cancer and biliary system cancer, exocrine pancreatic cancer, kidney cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioalveolar carcinoma, and mesothelioma), brain cancer and neck cancer (e.g., tongue cancer, oral cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer, penis cancer, uterine cancer, Cancers of the cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin, and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (i.e., leukemias, lymphomas) and precancerous and borderline malignant conditions, including hematologic malignancies of the lymphoid system and related conditions (e.g., acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas (e.g., diffuse large B-cell lymphoma [DLBCL]), follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphomas, Hodgkin's lymphoma, capillary monoclonal leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and myeloid hematologic malignancies and related disorders (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia, and primary myelofibrosis), myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia); mesenchymal tumors, e.g., sarcomas of soft tissue, bone, or cartilage (e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcomas, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma), gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; neural crest cell-derived tumors, including melanocytic tumors (e.g., malignant melanoma or uveal melanoma), peripheral and cranial nerve tumors, peripheral neuroblastic tumors (e.g., neuroblastoma), CNS embryonal tumors, paragangliomas; central or peripheral nervous system tumors (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary adenomas, adrenal adenomas, pancreatic islet cell tumors, parathyroid adenomas, carcinoids, and medullary thyroid carcinoma); ocular and adnexal tumors (e.g., retinoblastoma);Germ cell and trophoblastic tumors (e.g., teratoma, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); and pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumor); or various syndromes, congenital diseases, or diseases that predispose patients to malignant transformation (e.g., xeroderma pigmentosum). Other examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, testicular tumors and brain tumors (e.g., neuromas).

[0291] Therefore, the pharmaceutical composition, use or method of the present invention is used to treat a disease or condition in which abnormal cell growth (ie, uncontrolled and / or rapid cell growth) occurs. In one embodiment, the disease or condition in which abnormal cell growth occurs is cancer.

[0292] In one embodiment, the hematological malignancy is a leukemia. In another embodiment, the hematological malignancy is a lymphoma. In one embodiment, the compounds of the present invention are used to prevent or treat leukemia, for example, acute or chronic leukemia, particularly acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) or chronic myeloid leukemia (CML). In one embodiment, the compounds of the present invention are used to prevent or treat lymphoma, for example, acute or chronic lymphoma, particularly Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or diffuse large B-cell lymphoma. In one embodiment, the compounds of the present invention are used to prevent or treat acute myeloid leukemia (AML) or acute lymphocytic leukemia (ALL). In one embodiment, the cancer is AML. In another embodiment, the cancer is CLL.

[0293] Many diseases are characterized by persistent and unregulated angiogenesis. Chronic proliferative diseases are often accompanied by profound angiogenesis, which leads to or maintains an inflammatory and / or proliferative state, or leads to tissue destruction through invasive proliferation of blood vessels. It has been found that tumor growth and metastasis are angiogenesis-dependent. Therefore, the compounds of the present invention can be used to prevent and destroy tumor angiogenesis. In particular, the compounds of the present invention can be used to treat metastasis and metastatic cancer.

[0294] Metastasis or metastatic disease is the spread of a disease from one organ or site to another non-adjacent organ or site. Cancers that can be treated by the compounds of the invention include primary tumors (i.e., cancer cells at the point of origin), locally invasive tumors (cancer cells that infiltrate and infiltrate surrounding normal tissues in a local area), and metastatic (or secondary) tumors (i.e., tumors formed by circulating malignant tumor cells that spread through the bloodstream (hematogenous spread) or through lymphatics or body cavities (implantation metastasis) to other parts and tissues of the body).

[0295] In the above examples, specific cancers include hepatocellular carcinoma, melanoma, esophageal cancer, renal cancer, colon cancer, colorectal cancer, lung cancer (eg, mesothelioma or lung adenocarcinoma), breast cancer, bladder cancer, gastrointestinal cancer, ovarian cancer, and prostate cancer.

[0296] Another subset of cancers includes renal cancer, melanoma, colon cancer, lung cancer, breast cancer, ovarian cancer, and prostate cancer.

[0297] Another subgroup of cancers includes pancreatic cancer.

[0298] Another subgroup of cancers includes leukemias, such as acute and chronic leukemias, acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL).

[0299] Another subgroup of cancers includes mesotheliomas, including malignant peritoneal mesothelioma or malignant pleural mesothelioma.

[0300] Certain cancers are resistant to treatment with specific drugs. This can be due to the tumor type (most common epithelial malignancies have inherent chemoresistant (chemoresistant)), or resistance may occur spontaneously with disease progression or treatment. In this regard, mesothelioma includes mesothelioma resistant to topoisomerase poisons, alkylating agents, anti-tubulin, folic acid antagonists, platinum compounds, and radiotherapy, particularly cisplatin-resistant mesothelioma. Similarly, multiple myeloma includes multiple myeloma or refractory multiple myeloma sensitive to bortezomib, and chronic myeloid leukemia includes chronic myeloid leukemia and refractory chronic myeloid leukemia sensitive to imatinib. In this regard, prostate cancer includes prostate cancer resistant to anti-androgen therapy, particularly prostate cancer resistant to abiraterone or enzalutamide or orchiectomy. Melanoma includes melanoma resistant to BRAF and / or MEK inhibitor therapy.

[0301] These cancers may be cancers that are sensitive to ERK1 or ERK2 inhibition or in particular to ERK1 / 2 inhibition.

[0302] It is further envisioned that the compounds of the invention will be particularly useful in treating or preventing cancer types associated with or characterized by elevated Ras, BRAF and / or MEK signaling.

[0303] Elevated levels of Ras, BRAF, or MEK signaling are present in many cancers and are associated with a poor prognosis. In addition, cancers that harbor activating Ras, BRAF, or MEK mutations may also be sensitive to ERK1 / 2 inhibitors. Elevated levels of Ras, BRAF, or MEK signaling and Ras, BRAF, or MEK mutations can be identified using the techniques presented here. Whether a particular cancer is sensitive to ERK1 / 2 inhibition can be determined using the methods presented in the "Diagnostic Methods" section.

[0304] Another subgroup of cancers includes NRas melanoma and NRas AML.

[0305] Another subset of cancers includes KRas lung cancer, KRas pancreatic cancer, and KRas colorectal cancer (CRC).

[0306] Another subgroup of cancers includes BRAF colorectal cancer (CRC), BRAF lung cancer, and BRAF melanoma.

[0307] In other embodiments, the present invention provides:

[0308] - A compound of formula (1) for use in the prevention or treatment of a disease or condition in which mutant Ras, mutant BRAF or mutant MEK is present.

[0309] Use of a compound of formula (1) for the preparation of a medicament for preventing or treating a disease or condition in which mutant Ras, mutant BRAF or mutant MEK is present.

[0310] A method of preventing or treating a disease or condition in which mutant Ras, mutant BRAF or mutant MEK is present in a subject (eg, a mammalian subject such as a human in need thereof), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0311] - A compound of formula (1) for use in reducing or decreasing the incidence of a disease or condition in which mutant Ras, mutant BRAF or mutant MEK is present.

[0312] - Use of a compound of formula (1) for the preparation of a medicament for reducing or decreasing the incidence of a disease or condition in which mutant Ras, mutant BRAF or mutant MEK is present.

[0313] A method of reducing or decreasing the incidence of a disease or condition in which mutant Ras, mutant BRAF or mutant MEK is present in a subject (eg, a mammalian subject such as a human in need thereof), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0314] - A compound of formula (1) for use in treating (or reducing the incidence of) a cancer selected from NRas melanoma and NRas AML.

[0315] - A compound of formula (1) for use in treating (or reducing the incidence of) a cancer selected from KRas lung cancer, KRas pancreatic cancer and Kras colorectal cancer (CRC).

[0316] - A compound of formula (1) for use in treating (or reducing the incidence of) a cancer selected from BRAF colorectal cancer (CRC), BRAF lung cancer and BRAF melanoma.

[0317] - A compound of formula (1) for use in treating the cancer BRAF melanoma (or reducing the incidence of such cancer).

[0318] - Use of a compound of formula (1) for the preparation of a medicament for preventing or treating a cancer as defined herein.

[0319] A method of treating cancer (or reducing the incidence of cancer) in a subject (eg, a mammalian subject, such as a human), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0320] - A compound of formula (1) for use in the treatment of a disease or condition as described herein, particularly cancer.

[0321] - Use of a compound of formula (1) for the preparation of a medicament for the treatment of a disease or condition as described herein, in particular cancer.

[0322] A method of preventing or treating a disease or condition described herein, particularly cancer, in a subject (eg, a mammalian subject such as a human in need thereof), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0323] - A compound of formula (1) for use in reducing or decreasing the incidence of a disease or condition as described herein, particularly cancer.

[0324] - Use of a compound of formula (1) for the preparation of a medicament for reducing or decreasing the incidence of a disease or condition as described herein, particularly cancer.

[0325] A method of reducing or decreasing the incidence of a disease or condition described herein, particularly cancer, in a subject (eg, a mammalian subject such as a human in need thereof), comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0326] The compounds of formula (1) may also be used to treat tumor growth, pathogenesis, address resistance to chemotherapy and radiation therapy by resensitizing cells to chemotherapy, and as antimetastatic agents.

[0327] All types of therapeutic anticancer interventions must increase the stress applied to target tumor cells. To mitigate the adverse effects of this stress, ERK1 / 2 is directly involved in resistance to the effects of cancer drugs and treatment regimens. Therefore, ERK1 / 2 inhibitors represent a class of chemotherapeutics with the potential to: (i) sensitize malignant tumor cells to anticancer drugs and / or treatments; (ii) reduce or reduce the incidence of resistance to anticancer drugs and / or treatments; (iii) reverse resistance to anticancer drugs and / or treatments; (iv) enhance the activity of anticancer drugs and / or treatments; (v) delay or prevent the development of resistance to anticancer drugs and / or treatments.

[0328] Due to the inhibitory properties of the compounds of the present invention on ERK1 / 2, they will be very useful in controlling cell signaling. Therefore, it is also envisioned that the compounds of the present invention can be used to treat other diseases, such as inflammation (such as hepatitis, ulcerative colitis, gastritis); neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, myotonic dystrophy and amyotrophic lateral sclerosis), AIDS, ischemia (such as restenosis, traumatic brain injury, spinal injury, cerebral ischemia, cerebral ischemia / reperfusion (I / R) injury, acute and chronic CNS injury ischemia, stroke or myocardial infarction), musculoskeletal system degenerative diseases (such as osteoporosis), autoimmune diseases (such as multiple sclerosis (MS) and type I diabetes), and eye diseases, such as retinal degeneration.

[0329] The affinity of the compounds of the invention as ERK1 / 2 inhibitors can be determined using the biological and biophysical assays set forth in the Examples herein.

[0330] Diagnostic Methods

[0331] Before administering the compound of formula (1), it is necessary to screen the subject (eg, patient) to determine whether the disease or condition the patient suffers from or may suffer from is sensitive to treatment with a compound that inhibits ERK1 / 2. The term "patient" includes human patients and animal patients.

[0332] For example, a biological sample collected from a patient is analyzed to determine whether the disease or condition, such as cancer, that the patient suffers from or may suffer from is characterized by genetic abnormalities or abnormal protein expression, resulting in upregulated ERK1 / 2 signaling levels, or sensitivity to normal ERK1 / 2 functional pathways, or upregulated biochemical pathways downstream of ERK1 / 2 activation.

[0333] Examples of such abnormalities that lead to activation or sensitization of the ERK1 / 2 pathway include activating Ras isoform mutations (eg, KRAS) or BRAF mutations, as described in the Background section.

[0334] Ras mutations have been detected in cell lines and primary tumors including but not limited to melanoma, colorectal cancer, non-small cell lung cancer, and pancreatic, prostate, thyroid, urethral, ​​and upper respiratory tract cancers (Cancer Res. 2012; 72:2457-2467).

[0335] The term "upregulated" includes high expression or overexpression, including gene amplification (i.e., multiple gene copies), increased expression due to cytogenetic abnormalities and transcriptional effects, or increased signaling due to ERK1 / 2 activation. Therefore, patients can undergo diagnostic testing to detect characteristic markers of ERK1 / 2 upregulation. The term diagnosis includes screening. With respect to markers, we include genetic markers, including, for example, determination of DNA composition to identify the presence of Ras (e.g., KRAS) or BRAF mutations. The term marker also includes markers characterized by upregulation of ERK1 / 2, including protein levels, protein status, and mRNA levels of the aforementioned proteins. Gene amplification includes greater than 7 copies, as well as an increase of 2 to 7 copies.

[0336] Diagnostic assays for detecting KRAS and BRAF mutations are described in Castro et al. Br. J. Cancer. 2012 Jul 10; 107(2): 345-51. doi: 10.1038 / bjc.2012.259. Epub 2012 Jun 19, "A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer species."; and Gonzalez et al., Br J Dermatol. 2013, Apr; 168(4): 700-7. doi: 10.1111 / bjd.12248, "BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel" and references cited therein.

[0337] Several diagnostic tests for BRAF mutations have been approved by the FDA, and details of these tests can be found on the FDA website. Examples of these diagnostic tests are the cobas 4800 BRAF V600 Mutation Test, a companion test for Roche's vemurafenib product, and the THxID BRAF Test – a companion test for Tafinlar (dabrafenib) and Mekinist (trametinib) products.

[0338] Diagnostic tests and screening are typically performed on biological samples (i.e., body tissues or fluids) selected from tumor biopsy samples, blood samples (exfoliated tumor cell isolation and enrichment), cerebrospinal fluid, plasma, serum, saliva, stool biopsy, sputum, chromosome analysis, pleural fluid, peritoneal fluid, oral mucosal smear, skin biopsy or urine.

[0339] Methods for identification and analysis of cytogenetic abnormalities, gene amplifications, mutations, and protein upregulation are familiar to those skilled in the art. Clinical trials for most genetic variants include, but are not limited to, standard methods such as allele-specific polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), traditional Sanger DNA sequence analysis or next-generation sequencing methods, Sanger dideoxy sequencing, pyrosequencing, multiplex ligation-dependent probe amplification (MLPA), or ARMSPCR. Clinical trials for gene copy number and structural genetic variants include, but are not limited to, standard methods such as RNA sequencing (RNAseq), Nanostring hybridization proximity RNA nCounter assays, or in situ hybridization, such as fluorescence in situ hybridization (FISH). Newer next-generation sequencing (NGS) technologies, such as massively parallel sequencing technologies, allow for whole exome sequencing or whole genome sequencing.

[0340] In RT-PCR screening, tumor mRNA levels are assessed by creating cDNA copies of the mRNA and then amplifying the cDNA using PCR. The methods of PCR amplification, the selection of primers, and the conditions for amplification are familiar to those skilled in the art. Nucleic acid manipulation and PCR are performed using standard methods, as described, for example, in Ausubel, FM et al., eds. (2004) Current Protocols in Molecular Biology, John Wiley & Sons Inc. or Innis, MA et al., eds. (1990) PCR Protocols: a guide to methods and applications, Academic Press, San Diego. Reactions and manipulations involving nucleic acids are also described in Sambrook er al., (2001), 3 rdEd, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Alternatively, commercially available RT-PCR kits (e.g., Roche Molecular Biochemicals) can be used, or methods proposed in U.S. Patents 4,666,828, 4,683,202, 4,801,531, 5,192,659, 5,272,057, 5,882,864, and 6,218,529, which are incorporated herein by reference. Examples of in situ hybridization techniques for evaluating mRNA expression include fluorescence in situ hybridization (FISH) (see Angerer (1987) Meth. Enzymol., 152: 649).

[0341] Typically, in situ hybridization includes the following major steps: (1) fixation of the tissue to be analyzed; (2) pre-hybridization treatment of the sample to increase accessibility of the target nucleic acid and reduce nonspecific binding; (3) hybridization of the nucleic acid mixture with nucleic acids in the biological structure or tissue; (4) post-hybridization washing to remove unbound nucleic acid fragments during hybridization; and (5) detection of hybridized nucleic acid fragments. Probes in this application are typically labeled, for example, with radioactive isotopes or fluorescent reporters. Certain probes are long enough, for example, from about 50, 100 or 200 nucleotides to about 1000 or more nucleotides in length, to specifically hybridize to the target nucleic acid under stringent conditions. Standard methods for performing FISH are described in Ausubel, FM et al., eds. (2004) Current Protocols in Molecular Biology, John Wiley & Sons Inc and Fluorescence In Situ Hybridization: Technical Overview by John MS Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd ed.; ISBN: 1-59259-760-2; March 2004, pps. 077-088; Series: Methods in Molecular Medicine.

[0342] Gene expression profiling methods are described in DePrimo et al. (2003), BMC Cancer, 3:3. Briefly, the protocol is as follows: (dT)24 oligomers are used to initiate first-strand cDNA synthesis, such as from polyadenylated mRNA, and then random hexamer primers are used to synthesize second-strand cDNA, thereby synthesizing double-stranded cDNA from total RNA. Double-stranded cDNA is used as a template for in vitro cRNA transcription using biotinylated ribonucleotides. According to the protocol described by Affymetrix (Santa Clara, CA, USA), cRNA is chemically fragmented and then hybridized overnight on a human genome microarray or hybridized overnight with gene-specific oligonucleotide probes on a human genome microarray. Alternatively, single nucleotide polymorphism (SNP) arrays (a DNA microarray) can be used to detect polymorphisms within a population.

[0343] Alternatively, the protein product expressed by the mRNA can be detected by immunohistochemistry or immunofluorescence of tumor samples, microplate solid phase immunoassay, Western blotting, capillary electrophoresis, 2-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry, and other specific protein detection methods familiar to those skilled in the art. Detection methods include the use of site-specific antibodies. Those skilled in the art will recognize that all such well-known methods for detecting ERK1 / 2 upregulation, detecting ERK1 / 2 variants or mutants, or detecting 11q22 amplification can be applied to the present invention.

[0344] Abnormal levels of proteins such as ERK1 / 2 can be determined using standard protein assays, such as those described herein. Tissue samples such as tumor tissues can also be tested for elevated or overexpressed levels of proteins by assays such as those provided by Chemicon International. The target protein will be immunoprecipitated from the sample lysate and its level will then be measured. Detection methods also include the use of markers.

[0345] ERK overexpression can be determined by tumor biopsy. Methods for evaluating gene copy changes include those commonly used in cytogenetic laboratories, such as MLPA (multiplex ligation-dependent probe amplification) - a multiplex PCR method that detects abnormal copy number, or other PCR methods that can detect gene amplification, gain, and deletion.

[0346] If appropriate, Ex-functional assays can also be used, for example, to measure circulating leukemic cells in cancer patients to assess response to inhibitors.

[0347] Therefore, all of these methods can also be used to identify tumors that are particularly suitable for treatment with the compounds of the present invention.

[0348] Therefore, in other embodiments, the present invention provides:

[0349] A compound of formula (1) for use in treating or preventing a disease or condition (or for reducing or lowering the incidence of such disease or condition) in a patient who has been screened and determined to have a disease or condition that is susceptible to treatment with an ERK1 / 2 inhibitory compound (i.e., an ERK1 / 2 inhibitor), or who is at risk of developing such disease or condition.

[0350] Use of a compound of formula (1) for the preparation of a medicament for treating or preventing a disease or condition (or alleviating or reducing the incidence of such disease or condition) in a patient who has been screened and determined to have a disease or condition that is sensitive to treatment with an ERK1 / 2 inhibitory compound (i.e., an ERK1 / 2 inhibitor), or who is at risk of developing such disease or condition.

[0351] A method for treating or preventing a disease or condition (or alleviating or reducing the incidence of such disease or condition) in a patient who has been screened and determined to have a disease or condition that is sensitive to treatment with an ERK1 / 2 inhibitory compound (i.e., an ERK1 / 2 inhibitor), or who is at risk of developing such disease or condition, comprising administering to the patient a therapeutically effective amount of a compound of formula (1).

[0352] Another aspect of the present invention includes compounds of the present invention for the prevention or treatment of cancer in patients selected from subgroups with overexpression or activating mutations of ERK1 / 2 signaling pathways (such as Ras, BRAF or MEK). Therefore, in other embodiments, the present invention provides:

[0353] A compound of formula (1) for use in the treatment or prevention of cancer (or reduction or decrease in cancer incidence) in a patient selected from a subgroup with overexpression or activating mutations in the ERK1 / 2 signaling pathway (such as Ras (such as KRAS), BRAF or MEK).

[0354] Use of a compound of formula (1) for preparing a drug for treating or preventing cancer (or reducing or lowering the incidence of cancer) in a patient selected from a subgroup with overexpression or activating mutations in the ERK1 / 2 signaling pathway (such as Ras (such as KRAS), BRAF or MEK).

[0355] A method for treating or preventing cancer (or reducing or lowering the incidence of cancer) in a patient selected from a subgroup with overexpression or activating mutations of Ras (e.g., KRAS), BRAF or MEK in the ERK1 / 2 signaling pathway, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (1).

[0356] A method for diagnosing and treating a disease or condition mediated by ERK1 / 2, the method comprising (i) screening a patient to determine whether the disease or condition the patient suffers from or may suffer from is susceptible to treatment with a compound having affinity for ERK1 / 2; and (ii) if the patient's disease or condition is determined to be susceptible thereto, then administering to the patient a compound of formula (1).

[0357] Pharmaceutical preparations

[0358] The compound (1) and the new crystalline form of the compound (1) prepared by the novel method of the present invention can be administered to a subject independently, but are more usually administered in the form of a pharmaceutical composition (eg, formulation).

[0359] Therefore, in another embodiment, the present invention provides a pharmaceutical composition comprising the compound of formula (1) and at least one pharmaceutically acceptable excipient and optionally other therapeutic or prophylactic agents as described herein.

[0360] The present invention further provides a method for preparing a pharmaceutical composition comprising combining (eg, mixing) the compound of formula (1), at least one pharmaceutically acceptable excipient, and optionally other therapeutic or prophylactic agents as described herein.

[0361] The pharmaceutically acceptable excipient can be selected from, for example, a carrier (e.g., a solid, liquid or semisolid carrier), an adjuvant, a diluent, a filler or a bulking agent, a granulating agent, a coating agent, a release controlling agent, a binder, a disintegrant, a lubricant, a preservative, an antioxidant, a buffer, a suspending agent, a thickener, a flavoring agent, a sweetener, a taste masking agent, a stabilizer or any other excipient conventionally used in a pharmaceutical composition. Examples of excipients for various pharmaceutical compositions are described in more detail below.

[0362] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human subject) within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reaction or other problems or complications and with a reasonable benefit / risk ratio. Each excipient must also be "acceptable" in terms of compatibility with the other ingredients of the formulation.

[0363] Pharmaceutical compositions containing compound (1) can be formulated using well-known methods, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

[0364] The pharmaceutical composition may be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, sublingual, intraocular, intraaural, rectal, intravaginal or transdermal administration. If the composition is administered parenterally, they may be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or delivered directly to a target organ or tissue by injection, infusion or other delivery means. Delivery may be by bolus, short-term infusion or long-term infusion, and may be delivered passively or by using a suitable infusion pump or injection pusher.

[0365] Pharmaceutical preparations suitable for gastrointestinal administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, cosolvents, surfactants, organic solvent mixtures, cyclodextrin complexing agents, emulsifiers (for forming and stabilizing emulsion preparations), liposome components for forming liposomes, gelling polymers for forming polymer gels, freeze drying protective agents and combinations of various agents, especially for stabilizing soluble active ingredients and imparting isotonicity to the preparations and the blood of designated recipients. Pharmaceutical preparations for parenteral administration can also be in the form of aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents (RG Strickly, Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, Vol 21 (2) 2004, p 201-230).

[0366] These preparations can be provided in unit dose or multi-dose containers, for example, sealed ampoules, vials and pre-filled syringes, which can be stored in freeze-dried (lyophilized) conditions, and only require the addition of a sterile liquid carrier, for example, water for injection, before use. In one embodiment, the preparation is provided as an active pharmaceutical ingredient (e.g., freeze-dried or other subdivided dry form) in a bottle, which can then be reconstituted with a suitable diluent.

[0367] The pharmaceutical preparation can be prepared by freeze-drying the compound of formula (1). Freeze-drying refers to a method of freeze-drying a composition. Therefore, freeze-drying and freeze-drying are synonymous here. Freeze-drying is a dehydration method in which a solvent-containing matrix is ​​frozen and then evacuated to remove the solvent by sublimation, that is, directly converted from a solid frozen state to a gaseous state. Freeze-drying generally includes three steps: a freezing step, a primary drying step, and a secondary drying step. During the freezing step, the matrix is ​​frozen to a temperature far below its melting point. In the subsequent primary drying step, the frozen matrix is ​​evacuated to remove the solvent by sublimation. Most of the solvent in the matrix is ​​removed in this step. However, after the primary drying step is completed, there may still be a small amount of solvent that is bound to the matrix or adsorbed onto the matrix. In order to remove these residual solvents, a vacuum is maintained, but the partially dried matrix is ​​heated to a temperature at which it is no longer frozen. Thereby, the residual solvent is removed by evaporation.

[0368] The freeze drying process can be carried out in a freeze drying apparatus, the structure of which can be a completely conventional structure. The freeze drying apparatus usually has a chamber in which a freeze drying container containing a solution can be placed for freeze drying. The chamber is usually connected to a vacuum source (e.g., a vacuum pump) to reduce the pressure in the chamber. The apparatus may also have a device for freezing or heating the contents in the chamber.

[0369] The pharmaceutical preparation can also be prepared by spray drying the compound of formula (1). Spray drying is a method of producing micron-sized particles, wherein a matrix solution or suspension dissolved in a solvent is sprayed in a fine mist to form a droplet dispersion of the solution or suspension, and then heated and / or partially evacuated to remove the solvent by evaporation. The matrix is ​​first dissolved or suspended in a suitable solvent, and then the solution is passed through an atomizer or nozzle to a drying chamber. A heated gas (e.g., air or nitrogen) can also be injected into the drying chamber to contact the atomized feed, or the drying chamber can be partially evacuated to cause solvent evaporation. When the solvent evaporates, solid particles of the matrix are formed and these particles are recovered. The size of the resulting particles in the powder can be changed by selecting a suitable atomizer or nozzle.

[0370] The spray drying process can be carried out in a spray dryer, the structure of which can be a completely conventional structure. The spray dryer is usually equipped with an atomizer or nozzle that disperses the solution into a fine spray (usually less than 500 μm in diameter). The fine spray is introduced into a drying chamber. The drying chamber usually has an inlet for receiving a heated gas (such as air or nitrogen) and is optionally connected to a vacuum source (such as a vacuum pump) so that the pressure in the chamber can be reduced. The drying chamber may also have an outlet through which solid particles formed by the evaporation of the solvent of the spray droplets can be collected.

[0371] Sterile powders, granules and tablets can be used to prepare extemporaneous injection solutions and suspensions.

[0372] The parenteral injection pharmaceutical composition of the present invention can also include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders (reconstructed into sterile injections or dispersions before use). Examples of suitable aqueous carriers and non-aqueous carriers, diluents, solvents or vehicles include water, alcohol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and their suitable mixtures, vegetable oils (such as olive oil, sunflower seed oil, safflower oil or corn oil) and injectable organic esters, such as ethyl oleate. For example, coating (or thickening) materials (such as lecithin) can be used, in the case of dispersions, by maintaining the required particle size, and by using surfactants, to maintain suitable fluidity.

[0373] The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Protection against the action of microorganisms may be ensured by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents for regulating osmotic pressure may also be included, such as sugars, sodium chloride, etc. Absorption of injectable drug forms may be prolonged by the addition of agents that delay absorption, such as aluminum monostearate and gelatin.

[0374] In one embodiment of the present invention, the pharmaceutical composition is in a form suitable for intravenous administration, such as injection or infusion. When intravenous administration is adopted, the solution can be used directly, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% glucose) before administration.

[0375] In another embodiment, the pharmaceutical composition is in a form suitable for subcutaneous (sc) administration.

[0376] Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches, such as buccal patches.

[0377] Thus, tablet compositions may comprise a unit dose of the active compound with an inert diluent or carrier such as a sugar or sugar alcohol, for example lactose, sucrose, sorbitol or mannitol; and / or a non-sugar diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as microcrystalline cellulose (MCC), methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and starch such as corn starch. Tablets may also contain standard ingredients such as binders and granulating agents, such as polyvinylpyrrolidone, disintegrants such as swellable cross-linked polymers such as cross-linked carboxymethylcellulose, lubricants such as stearates, preservatives such as parabens, antioxidants such as BHT, buffers such as phosphate or citrate buffers, and effervescent agents such as citrate / bicarbonate mixtures. These excipients are well known and need not be discussed in detail herein.

[0378] Tablets may be designed to release the drug when it comes into contact with gastric fluid (immediate-release tablets) or to release the drug in a controlled manner over a longer period of time (controlled-release tablets) or to release the drug in a specific area of ​​the gastrointestinal tract.

[0379] Capsule preparations may be hard gelatin or soft gelatin and may contain active ingredients in solid, semi-solid or liquid form. Gelatin capsules may be formed using animal gelatin or its equivalent products of synthetic or plant origin.

[0380] Solid dosage forms (e.g., tablets, capsules, etc.) may be coated or uncoated. Coatings may be used as protective films (e.g., polymers, waxes, or coatings) or as a means of controlling drug release or for aesthetic or identification purposes. Coatings (e.g., Eudragit™-based polymers) may be designed to release active ingredients at a desired location in the gastrointestinal tract. Thus, coatings may be selected to degrade under certain pH conditions in the gastrointestinal tract, thereby selectively releasing the compound in the stomach or ileum, duodenum, colon, or jejunum.

[0381] Alternatively, or in addition to coating, the drug can be presented in a solid matrix containing a controlled release agent (e.g., a sustained release agent suitable for releasing the compound in a controlled manner in the gastrointestinal tract). Alternatively, the drug is presented in a polymer coating, such as a polymethacrylate polymer coating, which can be suitable for selectively releasing the compound under different acidic or alkaline conditions in the gastrointestinal tract. Alternatively, the matrix material or the release-retarding coating can be in the form of an erodible polymer (e.g., a maleic anhydride polymer), which is substantially continuously eroded when the dosage form passes through the gastrointestinal tract. In another alternative, the coating can be designed to decompose under the action of microorganisms in the intestine. As another alternative, the active compound can be formulated into a delivery system that provides osmotic control of the release of the compound. Osmotic release and other delayed release or sustained release formulations (e.g., formulations based on ion exchange resins) can be formulated using methods familiar to those skilled in the art.

[0382] Formula (1) compound can be formulated with carriers and administered in the form of nanoparticles. Nanoparticles increase the surface area, help compound absorption, and provide the possibility of direct penetration into cells. Nanoparticle delivery systems are described in "Nanoparticle Technology for Drug Delivery", edited by Ram B Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, published on March 13, 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 (1-2), 167-172 and Sinha et al., Mol. Cancer Ther. August 1, (2006) 5, 1909.

[0383] The pharmaceutical composition may comprise from about 1% (w / w) to about 95% active ingredient and from 99% (w / w) to 5% (w / w) pharmaceutically acceptable excipient or excipient combination. In particular, the composition may comprise from about 20% (w / w) to about 90% (w / w) active ingredient and from 80% (w / w) to 10% pharmaceutically acceptable excipient or excipient combination.

[0384] The pharmaceutical compositions of the invention may be, for example, in unit dosage form, such as ampoules, vials, suppositories, dragees, tablets or capsules, or in the form of prefilled syringes.

[0385] The pharmaceutically acceptable excipients can be selected according to the desired physical form of the preparation, and can be selected from, for example, diluents (such as solid diluents, such as fillers or extenders; liquid diluents, such as solvents or co-solvents), disintegrants, buffers, lubricants, glidants, release controlling agents (such as release-retarding or release-delaying polymers or waxes), binders, granulating agents, pigments, plasticizers, antioxidants, preservatives, flavoring agents, taste masking agents, osmotic pressure regulators and coating agents.

[0386] Those skilled in the art have the expertise to select the appropriate amounts of ingredients for the formulation. For example, tablets and capsules typically contain 0-20% disintegrants, 0-5% lubricants, 0-5% glidants and / or 0-99% (w / w) fillers / or bulking agents (depending on the drug dose). They may also contain 0-10% (w / w) polymer binders, 0-5% (w / w) antioxidants, 0-5% (w / w) pigments. In addition, sustained-release tablets also contain 0-99% (w / w) polymers (depending on the dose). The film coating of the tablet or capsule typically contains 0-10% (w / w) controlled-release (e.g., delayed-release) polymers, 0-3% (w / w) pigments and / or 0-2% (w / w) plasticizers.

[0387] Parenteral formulations typically contain 0-20% (w / w) buffer, 0-50% (w / w) cosolvent, and / or 0-99% (w / w) water for injection (WFI) (depending on the dose and whether lyophilized). Intramuscular depot formulations may also contain 0-99% (w / w) oil.

[0388] Oral pharmaceutical compositions can be prepared by combining the active ingredient with a solid carrier, granulating the resulting mixture if necessary, and processing the mixture into tablets, dragee cores or capsules after adding suitable excipients if necessary or desired. It can also be contained in a polymer or wax matrix to allow the active ingredient to diffuse or release in a metered amount.

[0389] The compounds of the present invention can also be formulated as solid dispersions. Solid dispersions are uniform, finely dispersed phases of two or more solids. Solid solutions (molecular dispersions) - a type of solid dispersion, are familiar in the field of pharmaceutical technology (see (Chiou and Riegelman, J. Pharm. Sci., 60, 1281-1300 (1971)) and can be used to increase the dissolution rate and improve the bioavailability of poorly water-soluble drugs.

[0390] The present invention also provides a solid dosage form comprising the above-mentioned solid solution. Solid dosage forms include tablets, capsules and chewable tablets, or dispersible tablets or effervescent tablets. Known excipients can be mixed with solid solutions to obtain the desired dosage form. For example, capsules can contain solid solutions mixed with (a) disintegrants and lubricants, or (b) disintegrants, lubricants and surfactants. In addition, capsules can contain fillers, such as lactose or microcrystalline cellulose. Tablets can contain solid solutions mixed with at least one disintegrant, lubricant, surfactant, filler and glidant. Chewable tablets can contain solid solutions mixed with fillers, lubricants, and, if necessary, additional sweeteners (such as artificial sweeteners), and suitable flavorings. Solid solutions can also be formed by spraying drug solutions and suitable polymers onto the surface of inert carriers such as sugar beads ("non-pareils"). These sugar beads can then be loaded into capsules or pressed into tablets.

[0391] Drug preparations can be provided to patients in a "patient pack" containing the entire course of treatment in a single package (usually a blister pack). The advantage of a patient pack over a traditional prescription where the pharmacist packs the patient's medication from a bulk supply is that the patient always has access to the package insert contained in the patient pack, which is not usually included in the patient's prescription. The inclusion of the package insert has been shown to improve patient compliance.

[0392] Compositions for topical use and intranasal delivery include ointments, creams, sprays, patches, gels, drops and inserts (eg, intraocular inserts). These compositions can be formulated using known methods.

[0393] Examples of formulations for rectal or vaginal administration include pessaries and suppositories, which may be prepared, for example, from shaped moldable or waxy materials containing the active compound. Solutions of the active compound may also be used for rectal administration.

[0394] Compositions for inhalation administration may be in the form of inhalable powder compositions or liquid or powder sprays, and may be administered in standard form using powder inhaler devices or aerosol dispensing devices. These devices are familiar to people. For inhalation administration, powder formulations typically include the active compound and an inert solid powder diluent (such as lactose).

[0395] The compound of formula (1) is generally provided in a unit dosage form and therefore generally contains a sufficient amount of the compound to provide the desired level of biological activity. For example, the formulation may contain 1 nanogram to 2 grams of active ingredient, for example, 1 nanogram to 2 milligrams of active ingredient. Within this range, a specific subrange of the compound is 0.1 milligram to 2 grams of active ingredient (more typically 10 milligrams to 1 gram, for example 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example, 1 microgram to 10 milligrams, for example 0.1 milligram to 2 milligrams of active ingredient).

[0396] For oral compositions, unit dosage forms may contain 1 mg to 2 g, more usually 10 mg to 1 g, for example 50 mg to 1 g, for example 100 mg to 1 g, of active compound.

[0397] The compounds of the invention will be administered to a patient (eg, a human or animal patient) in need thereof in an amount sufficient to achieve the desired therapeutic effect.

[0398] Treatment

[0399] The compounds of formula (1) can be used to prevent or treat a range of diseases or conditions mediated by ERK1 / 2. Examples of these diseases and conditions are described above.

[0400] The compound is typically administered to a subject in need of such administration (eg, a human or animal patient, particularly a human patient).

[0401] The compound is generally administered in an amount that is therapeutically or prophylactically effective and generally non-toxic. However, in certain circumstances (e.g., in the case of a life-threatening disease), the benefits of administering a compound of formula (1) may outweigh any toxic effects or side effects, in which case it may be contemplated to administer an amount of the compound that would result in a degree of toxicity.

[0402] The compounds may be administered chronically to maintain a beneficial therapeutic effect, or only for a short period of time. Alternatively, administration may be continuous, or in an intermittent manner (eg, pulsed).

[0403] A typical daily dose of a compound of formula (1) may be 100 pg to 100 mg / kg body weight, more typically 5 ng to 25 mg / kg body weight, more typically 10 ng to 15 mg / kg body weight (e.g., 10 ng to 10 mg, more typically 1 μg / kg to 20 mg / kg, e.g., 1 μg to 10 mg / kg), although higher or lower doses may be administered as required. A compound of formula (1) and its subformulae may be administered daily, or repeatedly, for example, every 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days, or 14 days, or 21 days, or 28 days.

[0404] The compound of the present invention can be orally administered within a certain dosage range, for example, 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, such as 2 to 200 mg or 10 to 1000 mg, and specific dosage examples include 10, 20, 50 and 80 mg. In order to achieve the desired therapeutic effect, the compound can be administered once or more per day. The compound can be administered continuously (i.e., administered daily during the treatment period, without interruption). Alternatively, the compound can be administered intermittently (i.e., continuously administered for a period of time, such as one week, and then discontinued for a period of time, such as one week, and then, continuously administered for another period of time, such as one week, etc.) throughout the treatment period. Examples of treatment regimens involving intermittent administration include one week of administration, one week of discontinuation; or two weeks of administration, one week of discontinuation; or three weeks of administration, one week of discontinuation; or two weeks of administration, two weeks of discontinuation; or four weeks of administration, two weeks of discontinuation; or one week of administration, three weeks of discontinuation cycle regimens - one or more cycles can be used, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more cycles. Such discontinuous treatment can also be based on days rather than full weeks. For example, treatment can include daily administration for 1 to 6 days, discontinuation for 1 to 6 days, and this pattern is repeated during the entire treatment regimen. The days (or weeks) in which the compounds of the invention are not administered do not necessarily have to be equal to the days (or weeks) in which the compounds of the invention are administered.

[0405] In one particular administration schedule, the patient will be administered a compound of formula (1) by infusion for 1 hour daily for no more than 10 days, particularly no more than 5 days a week, with the treatment being repeated at required intervals, for example, every two to four weeks, particularly every three weeks.

[0406] The compounds of the present invention can also be administered by push injection or continuous infusion. The compounds of the present invention can be administered daily to once a week, or once every two weeks, or once every three weeks, or once every four weeks during the treatment cycle. If administered daily during the treatment cycle, this daily administration can be interrupted during the treatment cycle of several weeks: for example, administer for one week (or several days), stop administering for one week (or several days), and repeat according to this pattern during the treatment cycle.

[0407] In one dosing schedule, patients may be administered a compound of formula (1) by infusion over a period of one hour per day for five days, with this treatment being repeated every three weeks.

[0408] In another specific dosing schedule, patients are infused for 30 minutes to 1 hour, and then the infusion is maintained for a varying period of time, e.g., 1 to 5 hours, e.g., 3 hours.

[0409] In another specific dosing schedule, the patient is infused continuously for 12 hours to 5 days, and more particularly, is infused continuously for 24 hours to 72 hours.

[0410] However, the ultimate amount of compound administered and the type of composition used will be consistent with the nature of the disease or physiological condition being treated and will be determined by the physician.

[0411] It has been found that ERK1 / 2 inhibitors can be used as single agents or in combination with other anticancer agents. For example, an inhibitor that inhibits ERK signaling is combined with another agent that acts at a different point in the signal transduction cascade or acts through a different mechanism of regulating cell growth, thereby treating two features of cancer development. Combination experiments can be carried out, for example, as described in Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regulat 1984; 22: 27-55.

[0412] The compounds of the invention may be administered as the sole therapeutic agent, or may be administered in combination therapy with one or more other compounds (or treatments) for the treatment of a particular disease state, for example, a neoplastic disease, such as cancer as defined above. For the treatment of the above diseases, the compounds of the invention may advantageously be used in combination with one or more other agents, more specifically, may be used in cancer treatment with other anticancer agents or adjuvants (supportive agents in treatment). Examples of other therapeutic agents or treatments that may be administered with the compounds of formula (1) (whether simultaneously or at different time intervals) include, but are not limited to:

[0413] Topoisomerase I inhibitors

[0414] Antimetabolites

[0415] Tubulin targeting agents

[0416] DNA binding agents and topoisomerase II inhibitors

[0417] Alkylating agent

[0418] Monoclonal antibodies

[0419] Anti-hormones

[0420] Signal transduction inhibitors

[0421] Ubiquitin-proteasome pathway inhibitors

[0422] Immunotherapy

[0423] Cell death regulators

[0424] DNA methyltransferase inhibitors

[0425] Cytokines and retinoids

[0426] Chromatin-targeted therapy

[0427] Radiation therapy, and

[0428] Other therapeutic or prophylactic agents.

[0429] Specific examples of anticancer agents or adjuvants (or their salts) include, but are not limited to, any one or more agents selected from the following groups (i) to (xlviii), and optionally any one or more agents from group (xlix) and / or group (I):

[0430] (i) a platinum compound, for example, cisplatin (optionally in combination with amifostine), carboplatin or oxaliplatin;

[0431] (ii) Taxane compounds, for example, paclitaxel, paclitaxel protein-bound microparticles (Abraxane TM ), docetaxel, cabazitaxel, or larotaxel;

[0432] (iii) topoisomerase I inhibitors, e.g., camptothecin compounds, e.g., camptothecin, irinotecan (CPT11), SN-38, or topotecan;

[0433] (iv) topoisomerase II inhibitors, for example, antitumor epipodophyllotoxin or podophyllotoxin derivatives, for example, etoposide or teniposide;

[0434] (v) Vinca alkaloids, for example vinblastine, vincristine, liposomal vincristine (Onco-TCS), vinorelbine, vindesine, vinflunine or vinvesir;

[0435] (vi) nucleoside derivatives, for example, 5-fluorouracil (5-FU, optionally in combination with leucovorin), gemcitabine, capecitabine, tegafur, UFT, S1, cladribine, cytarabine (Ara-C, cytosine arabinoside), fludarabine, clofarabine or nelarabine;

[0436] (vii) antimetabolites, for example, clofarabine, aminopterin or methotrexate, azacitidine, cytarabine, floxuridine, pentostatin, thioguanine, thiopurine, 6-mercaptopurine or hydroxyurea (hydroxyurea) or trifluridine (optionally in combination with tipiracil);

[0437] (viii) alkylating agents such as nitrogen mustards or nitrosoureas, for example, cyclophosphamide, chlorambucil, carmustine (BCNU), bendamustine, thiotepa, melphalan, threosulfan, lomustine (CCNU), altretamine, busulfan, dacarbazine, estramustine, fotemustine, ifosfamide (optionally in combination with sodium mercaptoethanesulfonate), pipobroman, procarbazine, streptozocin, temozolomide, uracil, mechlorethamine, methylcyclohexylchloroethylnitrosourea or nimustine (ACNU);

[0438] (ix) anthracyclines, anthracenediones and related drugs, for example, daunorubicin, doxorubicin (optionally in combination with dexamethasone), liposomal formulations of doxorubicin (e.g., Caelyx TM 、Myocet TM , Doxil TM ), idarubicin, mitoxantrone, epirubicin, amsacrine, or valrubicin;

[0439] (x) epothilones, such as ixabepilone, patupirone, BMS-310705, KOS-862 and ZK-EPO, epothilone A, epothilone B, desoxyepothilone B (also known as epothilone D or KOS-862), azaepothilone B (also known as BMS-247550), aulimalide, isolaulimalide or luetherobin;

[0440] (xi) DNA methyltransferase inhibitors, for example, temozolomide, azacytidine, decitabine or guadecitabine (SGI-110);

[0441] (xii) folate antagonists, for example, methotrexate, pemetrexed disodium or raltitrexed;

[0442] (xiii) cytotoxic antibiotics, for example antinomycin D, bleomycin, mitomycin C, dactinomycin, carminomycin, daunomycin, levamisole, plicamycin or mithramycin;

[0443] (xiv) tubulin binding agents, for example, combrestatin, colchicine or nocodazole;

[0444] (xv) signal transduction inhibitors, such as kinase inhibitors, for example receptor tyrosine kinase inhibitors (e.g. EGFR (epidermal growth factor receptor) inhibitors, VEGFR (vascular endothelial growth factor receptor) inhibitors, PDGFR (platelet-derived growth factor receptor) inhibitors, Axl inhibitors, MTKI (multi-targeted kinase inhibitors), Raf inhibitors, ROCK inhibitors, mTOR inhibitors, MEK inhibitors or PI3K inhibitors), for example, imatinib mesylate, erlotinib, gefitinib, dasatinib, lapatinib, dovitinib, axitinib, nilotinib, vandetanib, vatalanib, pazopanib, sorafenib, sunitinib, temsirolimus, everolimus (RAD 001), vemurafenib (PLX4032 or RG7204), dabrafenib, encorafenib, selumetinib (AZD6244), trametinib (GSK121120212), dactolisib (BEZ235), buparlisib (BKM-120; NVP-BKM-120), BYL719, cupanisib (BAY-80-6946), ZSTK-474, CUDC-907, apitolisib (GDC-0980; RG-7422), pictilisib (pictrelisib, GDC-0941, RG-7321), GDC-0032, GDC-0068, GSK-2636771, idelalisib (formerly CAL-101, GS 1101, GS-1101), MLN1117(INK1117), MLN0128(INK128), IPI-145(INK1197), LY-3023414, ipatasertib, afuresertib, MK-2206, MK-8156, LY-3023414, LY294002, SF1126 or PI-103, sonolisib (PX-866) or AT13148.

[0445] (xvi) Aurora kinase inhibitors, such as AT9283, barasertib (AZD1152), TAK-901, MK0457 (VX680), cenisertib (R-763), darusetib (PHA-739358), alisertib (MLN-8237), or MP-470;

[0446] (xvii) CDK inhibitors, such as AT7519, roscovitine, seliciclib, alvocidib (flavopiridol), dinaciclib (SCH-727965), 7-hydroxy-staurosporine (UCN-01), JNJ-7706621, BMS-387032 (also known as SNS-032), PHA533533, ZK-304709 or AZD-5438, and including CDK4 inhibitors, such as palbociclib (PD332991) and ribociclib (LEE-011);

[0447] (xviii) PKA / B inhibitors and PKB (akt) pathway inhibitors, such as AT13148, AZD5363, Semaphore, SF1126 and MTOR inhibitors, such as rapamycin analogs, AP23841 and AP23573, calmodulin inhibitors (forkhead translocation inhibitors), API-2 / TCN (triciribine), RX-0201, enzastaurin HCl (LY317615), NL-71-101, SR-13668, PX-316 or KRX-0401 (perifosine / NSC 639966);

[0448] (xix) Hsp90 inhibitors, such as onalespib (AT13387), herbimycin, geldanamycin (GA), 17-allylamino-17-demethoxygeldanamycin (17-AAG), such as NSC-330507, Kos-953 and CNF-1010, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), such as NSC-707545 and Kos-1022, NVP-AUY922 (VER-52296), NVP-BEP800, CNF-2024 (BIIB-021, an oral purine), ganetespib (STA-9090), SNX-5422 (SC-102112) or IPI-504 or TAS-116;

[0449] (xx) monoclonal antibodies (unconjugated or conjugated to radioisotopes, toxins or other agents), antibody derivatives and related agents, such as anti-CD, anti-VEGFR, anti-HER2 or anti-EGFR antibodies, for example, rituximab (CD20), ofatumumab (CD20), ibritumomab tiuxetan (CD20), GA101 (CD20), tositumomab (CD20), epratuzumab (CD22), lintuzumab (CD33), gemtuzumab ozogamicin (CD33), alemtuzumab (CD52), galiximab (CD80), trastuzumab (HER2 antibody), pertuzumab (HER2), trastuzumab-DM1 (HER2), ertuinomab (HER2 and CD3), cetuximab (EGFR), panitumumab (EGFR), nexitozumab MAB (EGFR), nimotuzumab (EGFR), bevacizumab (VEGF), catumaxomab (EpCAM and CD3), abavolumab (CA125), farrelizumab (folate receptor), elotuzumab (CS1), denosumab (RANK ligand), fentozumab (IGF1R), CP751,871 (IGF1R), mapatumumab (TRAIL receptor), metMAB (met), mitumomab (GD3 ganglioside), naptumomab (tamiflu) estafenatox) (5T4) or sildenafil (IL6) or immunomodulators, such as CTLA-4 blocking antibodies and / or anti-PD-1 and PD-L1 and / or PD-L2 antibodies, for example, ipilimumab (CTLA4), MK-3475 (pembrolizumab, formerly lamblizumab, anti-PD-1), nivolumab (anti-PD-1), BMS-936559 (anti-PD-L1), MPDL320A, AMP-514 or MEDI4736 (anti-PD-L1) or tremelimumab (formerly tesimumab, CP-675,206, anti-CTLA-4);

[0450] (xxi) estrogen receptor antagonists or selective estrogen receptor modulators (SERMs) or estrogen synthesis inhibitors, for example, tamoxifen, fulvestrant, toremifene, droloxifene, faloxifen, or raloxifene;

[0451] (xxii) aromatase inhibitors and related drugs, such as exemestane, anastrozole, letrozole, testosterone aminoglutethimide, mitotane, or vorozole;

[0452] (xxiii) antiandrogens (i.e., androgen receptor antagonists) and related agents, such as bicalutamide, nilutamide, flutamide, cyproterone acetate, or ketoconazole;

[0453] (xxiv) hormones and their analogs, for example, medroxyprogesterone, diethylstilbestrol (also known as diethylstilbestrol) or octreotide;

[0454] (xxv) steroids, for example, drostanolone propionate, megestrol acetate, nandrolone (decanoate, phenylpropionate), fluoxymesterone or gossypol;

[0455] (xxvi) steroidal cytochrome P450 17α-hydroxylase-17,20-lyase inhibitors (CYP17), such as abiraterone;

[0456] (xxvii) gonadotropin-releasing hormone agonists or antagonists (GnRA), such as abarelix, goserelin acetate, histrelin acetate, leuprolide acetate, triptorelin, buserelin, or deserorelin;

[0457] (xxviii) glucocorticoids, e.g., prednisone, prednisolone, dexamethasone;

[0458] (xxix) Differentiating agents such as retinoids, retinoids, vitamin D or retinoic acid and retinoic acid metabolism blockers (RAMBAs), for example, accutane, alitretinoin, bexarotene or tretinoin;

[0459] (xxx) Farnesyl transferase inhibitors, for example, tipifarnib;

[0460] (xxxi) chromatin-targeted therapies, such as histone deacetylase (HDAC) inhibitors, for example, sodium butyrate, suberoylanilide hydroxamide acid (SAHA), depsipeptide (FR 901228), dacillast (NVP-LAQ824), R306465 / JNJ-16241199, JNJ-26481585, trichostatin A, vorinostat, chlamydocin, A-173, JNJ-MGCD-0103, PXD-101 or apicidin;

[0461] (xxxii) drugs targeting the ubiquitin-proteasome pathway, including proteasome inhibitors, for example, bortezomib, carfilzomib, CEP-18770, MLN-9708, or ONX-0912; NEDD8 inhibitors; HDM2 antagonists and deubiquitinating enzymes (DUBs);

[0462] (xxxiii) photodynamic drugs, for example, porfimer sodium or temoporphine;

[0463] (xxxiv) Marine bio-derived anticancer agents, such as trabectedin;

[0464] (xxxv) radiolabeled drugs for radioimmunotherapy, e.g., beta particle emitting isotopes (e.g., iodine-131, yttrium-90) or alpha particle emitting isotopes (e.g., bismuth-213 or actinium-225), such as ibritumomab tiuxetan, r-iodotositumomab, or alpha radium;

[0465] (xxxvi) telomerase inhibitors, for example, telomestatin;

[0466] (xxxvii) Matrix metalloproteinase inhibitors, for example, batimastat, marimastat, prinostat or metamastat;

[0467] (xxxviii) recombinant interferons (such as interferon-γ and interferon α) and interleukins (such as interleukin 2), for example, aldesleukin, denileukin diftitox, interferon α2a, interferon α2b or pegylated interferon α2b;

[0468] (xxxix) selective immune response modifiers, for example, thalidomide or lenalidomide;

[0469] (xl) Therapeutic vaccines, such as sipuleucel-T (Provenge) or OncoVex;

[0470] (xli) cytokine activators include lysostigmine, romotide, sizoran, virulin or thymosin;

[0471] (xlii) arsenic trioxide;

[0472] (xliii) G protein-coupled receptor (GPCR) inhibitors, for example, atrasentan;

[0473] (xliv) enzymes such as L-asparaginase, pegaspargase, rasburicase or pegamidase;

[0474] (xlv) DNA repair inhibitors, such as PARP inhibitors, for example, olaparib, velaparib, iniparib, INO-1001, AG-014699 or ONO-2231;

[0475] (xlvi) death receptor (e.g., TNF-related apoptosis-inducing ligand (TRAIL) receptor) agonists, such as mapatumumab (formerly HGS-ETR1), canatumumab (formerly AMG 655), PRO95780, lexalimumab, dulalemin, CS-1008, apolumab, or recombinant TRAIL ligands, such as recombinant human TRAIL / Apo2 ligand;

[0476] (xlvii) Immunotherapy, such as immune checkpoint inhibitors, cancer vaccines, and CAR-T cell therapy;

[0477] (xlviii) cell death (apoptosis) modulators, including Bcl-2 (B-cell lymphoma 2) antagonists, such as venetoclax (ABT-199 or GDC-0199), ABT-737, ABT-263, TW-37, sabutoclax, obaclax, and MIM1 and IAP antagonists, including LCL-161 (Novartis), Debio-1143 (Debiopharma / Ascenta), AZD5582, Birinapant / TL-32711 (TetraLogic), CUDC-427 / GDC-0917 / RG-7459 (Genentech), JP1201 (Joyant), T-3256336 (Takeda), GDC-0152 (Genentech), HGS-1029 / AEG-40826 (HGS / Aegera), or ASTX-660;

[0478] (xlix) Prophylactic (adjuvant) agents; that is, agents that reduce or alleviate the side effects associated with chemotherapeutic agents, e.g.

[0479] - Antiemetics;

[0480] -Agents that prevent or reduce the duration of chemotherapy-related neutropenia and prevent complications caused by decreased platelet, red blood cell, or white blood cell levels, such as interleukin-11 (e.g., oprelleukin), erythropoietin (EPO) and its analogs (e.g., darbepoetin alfa), colony stimulating factor analogs, such as granulocyte macrophage colony stimulating factor (GM-CSF) (e.g., sargramostim), and granulocyte colony stimulating factor (G-CSF) and its analogs (e.g., filgrastim, pegfilgrastim);

[0481] -Agents that inhibit bone resorption, such as denosumab or bisphosphonates, such as zoledronate, zoledronic acid, pamidronate disodium, and ibandronate;

[0482] -Agents that suppress the inflammatory response, such as dexamethasone, prednisone, and prednisolone;

[0483] -Agents used to reduce blood levels of growth hormone and IGF-I (and other hormones) in patients with acromegaly or other rare hormone-producing tumors, such as synthetic forms of the hormone somatostatin, such as octreotide acetate;

[0484] -Antidates for drugs that lower folate levels, such as leucovorin or folinic acid;

[0485] -Agents used for pain, for example, opiates such as morphine, diacetylmorphine, and fentanyl;

[0486] - Nonsteroidal anti-inflammatory drugs (NSAIDs), such as COX-2 inhibitors, for example, celecoxib, etoricoxib, and lumiracoxib;

[0487] -Mucositis agents, such as palifermin;

[0488] -Agents to treat side effects (including loss of appetite, cachexia, edema, or thromboembolic episodes), such as megestrol acetate; and

[0489] (I) Radiation therapy for curative, palliative, or preventive (or adjuvant or neoadjuvant) purposes.

[0490] Each compound in the combination of the present invention can be administered according to independent different dosage arrangements and administered by different routes. Therefore, in two or more agents, the dosage of each agent may be different: each agent can be administered simultaneously or at different times. Those skilled in the art can understand the dosage regimen and combination therapy used by their general common knowledge. For example, the compounds of the present invention can be used in combination with one or more other agents, which are administered according to their existing combination regimens. Examples of standard combination regimens are provided below.

[0491] The dose of the taxane compound administered per course of treatment is advantageously 50-400 mg per square meter of body surface area (mg / m 2 ), for example, 75-250 mg / m 2 , especially paclitaxel, is administered at a dose of approximately 175-250 mg / m 2 The dosage of docetaxel is about 75 to 150 mg / m 2 .

[0492] The dosage of the camptothecin compound administered per course of treatment is advantageously 0.1-400 mg per square meter of body surface area (mg / m 2 ), for example, 1-300 mg / m 2 In particular, irinotecan is administered at a dose of about 100-350 mg / m 2The dosage of topotecan is about 1 to 2 mg / m 2 .

[0493] The dosage of the antitumor podophyllotoxin derivative administered per course of treatment is advantageously 30-300 mg per square meter of body surface area (mg / m 2 ), for example, 50-250 mg / m 2 In particular, etoposide is administered at a dose of about 35-100 mg / m 2 The dosage of teniposide is about 50 to 250 mg / m 2 .

[0494] The dosage of antitumor vinca alkaloid administered per course of treatment is advantageously 2-30 mg per square meter of body surface area (mg / m 2 ), especially vinblastine, is administered at a dose of about 3-12 mg / m 2 The dosage of vincristine is about 1 to 2 mg / m 2 The dosage of vinorelbine is about 10 to 30 mg / m 2 .

[0495] The dosage of the antitumor nucleoside derivative administered per course of treatment is advantageously 200-2500 mg per square meter of body surface area (mg / m 2 ), for example, 700-1500 mg / m 2 , especially 5-FU is administered at a dose of about 200-500 mg / m 2 The dosage of gemcitabine is about 800 to 1200 mg / m 2 The dose of capecitabine is about 1000-2500 mg / m 2 .

[0496] The alkylating agent, such as nitrogen mustard or nitrosourea, is advantageously administered in a dose of 100-500 mg per square meter of body surface area (mg / m 2 ), for example, 120-200 mg / m 2 , especially cyclophosphamide, is administered at a dose of about 100-500 mg / m 2 The dosage of chlorambucil is about 0.1 to 0.2 mg / kg, and the dosage of carmustine is about 150-200 mg / m 2 The dosage of chlorambucil is about 100-150 mg / m 2 .

[0497] The dosage of the antitumor anthracycline derivative administered per course of treatment is advantageously 10-75 mg per square meter of body surface area (mg / m 2), for example, 15-60 mg / m 2 In particular, the dosage of doxorubicin is about 40-75 mg / m 2 The dosage of daunorubicin is approximately 25 to 45 mg / m 2 The dose of idarubicin is about 10-15 mg / m 2 .

[0498] The dosage of anti-estrogen preparation is advantageously about 1 to 100mg every day, depending on the disease of specific reagent and treatment. The oral dosage of tamoxifen is advantageously about 5 to 50mg, particularly 10 to 20mg, twice a day, continuing treatment for a long enough time, to achieve and maintain therapeutic effect. The oral dosage of tamoxifen is advantageously about 60mg, once a day, continuing treatment for a long enough time, thereby achieving and maintaining therapeutic effect. The oral dosage of anastrozole is advantageously about 1mg, once a day. The oral dosage of droloxifene is advantageously about 20-100mg, once a day. The oral dosage of raloxifene is advantageously about 60mg, once a day. The oral dosage of exemestane is advantageously about 25mg, once a day.

[0499] The antibody is advantageously administered at a dose of about 1 to 5 mg per square meter of body surface area (mg / m 2 ), or, if different, at doses known in the art. The dosage of trastuzumab administered per course of treatment is advantageously about 1-5 mg per square meter of body surface area (mg / m 2 ), especially 2-4 mg / m 2 .

[0500] If the compound of formula (1) is used in combination therapy with one, two, three, four or more other therapeutic agents (particularly one or two, more particularly one), the compound can be used simultaneously with them or in sequence. In the latter case, two or more compounds will be used over a period of time, and the amount and mode of administration are sufficient to ensure that a beneficial or synergistic effect is achieved. When used in sequence, they can be used at very close intervals (e.g., 5-10 minutes) or at longer intervals (e.g., separated by 1, 2, 3, 4 or more hours, if necessary, or even at longer intervals), and the precise dosage regimen is matched with the properties of the therapeutic agent. These doses can, for example, be used once, twice or more times per course of treatment, and can, for example, be repeated every 7 days, 14 days, 21 days or 28 days.

[0501] It should be understood that the specific method and order of administration of each component in the combination and the respective dosage and regimen will depend on the specific other medicaments and compounds of the present invention to be administered, their routes of administration, the specific tumors to be treated, and the specific hosts to be treated. The optimal method and order of administration and the dosage and regimen of administration can be easily determined by those skilled in the art using conventional methods and considering the information provided herein.

[0502] When used in combination, the weight ratio of the compounds of the present invention to one or more other anticancer agents can be determined by those skilled in the art. The ratio, exact dosage and frequency of administration will depend on the specific compound of the present invention and the other anticancer agent used, the specific disease treated, the severity of the disease treated, the age, weight, sex, diet, time of administration and general physical condition of the particular patient, the mode of administration and other drugs administered to the individual, all of which are familiar to those skilled in the art. In addition, it is clear that the effective daily amount can be reduced or increased based on the response of the patient treated and / or based on the evaluation of the doctor prescribing the compounds of the present invention. The specific weight ratio of the compound of formula (1) of the present invention to another anticancer agent can be 1 / 10-10 / 1, more particularly 1 / 5-5 / 1, and even more particularly 1 / 3-3 / 1.

[0503] The compounds of the invention may also be administered with non-chemotherapeutic treatments such as radiation therapy, photodynamic therapy, gene therapy, surgery and dietary manipulation.

[0504] The compounds of the present invention also have therapeutic applications for sensitizing tumor cells to radiotherapy and chemotherapy. Therefore, the compounds of the present invention can be used as "radiosensitizers" and / or "chemosensitizers" or can be used in combination with another "radiosensitizer" and / or "chemosensitizer". In one embodiment, the compounds of the present invention are used as chemosensitizers.

[0505] The term "radiosensitizer" is defined as a molecule that is administered to a patient in a therapeutically effective amount to increase the sensitivity of cells to ionizing radiation and / or to facilitate the treatment of a disease treatable by ionizing radiation.

[0506] The term "chemosensitizer" is defined as a molecule that is administered to a patient in a therapeutically effective amount to increase the sensitivity of cells to chemotherapy and / or to facilitate the treatment of a chemotherapeutic-treatable disease.

[0507] Many cancer treatment regimens currently utilize radiosensitizers in conjunction with x-ray radiation. Examples of x-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, demethyl misonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives thereof.

[0508] Photodynamic therapy (PDT) of cancer uses visible light as a radioactive agent for sensitizers. Examples of photodynamic radiosensitizers include, but are not limited to, the following: hematoporphyrin derivatives, photoporphyrins, benzoporphyrin derivatives, protoporphyrin tin, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and their therapeutically effective analogs and derivatives.

[0509] The radiosensitizer may be administered with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds that promote the incorporation of the radiosensitizer into target cells; compounds that control the flow of therapeutic agents, nutrients and / or oxygen to target cells; chemotherapeutic agents that act on tumors with or without the application of additional radiation; or other therapeutically effective compounds for the treatment of cancer or other diseases.

[0510] Chemosensitizers can be administered with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds that promote the incorporation of chemosensitizers into target cells; compounds that control the flow of therapeutic agents, nutrients and / or oxygen to target cells; chemotherapeutic agents that act on tumors or other therapeutically effective compounds for the treatment of cancer or other diseases. Calcium antagonists such as verapamil have been found to be useful with anti-tumor agents to establish chemosensitivity in tumor cells that are resistant to accepted chemotherapeutic agents, as well as to enhance the efficacy of these compounds in drug-sensitive malignancies.

[0511] For use with another chemotherapeutic agent in combination therapy, the compound of formula (1) can be formulated together with one, two, three, four or more other therapeutic agents, for example, into a dosage form that contains two, three, four or more therapeutic agents, i.e., formulated as a unit pharmaceutical composition containing all components. Alternatively, each therapeutic agent can be formulated separately and put together in the form of a kit, optionally including its instructions for use.

[0512] It will be appreciated from the foregoing that, in other embodiments, the invention provides a combination of a compound of formula (1) and another therapeutic agent (eg, another therapeutic agent as defined above).

[0513] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of formula (1), a pharmaceutically acceptable carrier and one or more therapeutic agents as defined above.

[0514] In other embodiments, the present invention provides:

[0515] - A combination as defined herein, for use in treating (or in alleviating or reducing the incidence of) a disease or condition as described herein, particularly cancer.

[0516] - Use of a combination as defined herein for the manufacture of a medicament for treating (or reducing the incidence of) a disease or condition as defined herein, particularly cancer.

[0517] A method of preventing or treating (for reducing or decreasing the incidence of) a disease or condition as described herein, particularly cancer, in a subject (e.g., a mammalian subject such as a human in need thereof), the method comprising administering to the subject a therapeutically effective amount of a combination as defined herein.

[0518] - A combination as defined herein for use in inhibiting the growth of tumour cells (eg in a patient).

[0519] - Use of a combination as defined herein for the preparation of a medicament for inhibiting the growth of tumour cells in a patient.

[0520] - A method of inhibiting the growth of tumour cells (eg in a patient), said method comprising contacting the tumour cells with a compound or combination of formula (1) as defined herein.

[0521] In each of the foregoing embodiments, the compound of formula (1) and one or more other therapeutic agents (at least one of which is an anticancer agent) may be administered simultaneously, separately or sequentially in treating a cancer patient.

[0522] In another embodiment, the present invention provides a method for preventing or treating cancer (or reducing or lowering the incidence of cancer), the method comprising administering radiation therapy or chemotherapy and a compound of formula (1) to a patient.

[0523] In another embodiment, the present invention provides a compound of formula (1) for use in combination with radiotherapy or chemotherapy to prevent or treat cancer (or to reduce or decrease the incidence of cancer).

[0524] Examples

[0525] Now, the present invention will be described, however, the present invention is not limited to the specific embodiments described in the following examples. The compounds are named using an automatic naming package such as AutoNom (MDL) or ChemAxon Structure to Name, or are named according to the names provided by the chemical supplier. In these examples, the following abbreviations are used.

[0526] The following compounds were prepared by methods analogous and / or similar to the following general methods.

[0527] The synthetic methods described below are provided solely for the purpose of illustrating such methods; for a given preparation or procedure, the precursor employed need not necessarily be derived from a single batch synthesized according to the described procedure.

[0528] When a compound is described as a mixture of two diastereomers / epimers, the configuration of the stereocenter is not specified and is represented by a straight line.

[0529] As will be appreciated by those skilled in the art, the compounds synthesized using the schemes shown may exist as solvates such as hydrates and / or contain residual solvents or small amounts of impurities. Compounds isolated as salts may be integer stoichiometric, i.e., mono- or di-salts, or intermediate stoichiometric.

[0530] Some of the compounds below are isolated as salts, e.g., depending on the acid used in the purification process. Some of the compounds are isolated as free bases.

[0531] Abbreviations

[0532] ca. Approximately

[0533] conc. concentrated

[0534] Corr. Corrected

[0535] DCM Dichloromethane

[0536] DIPEA N,N-Diisopropylethylamine

[0537] 1,4-DMB 1,4-dimethoxybenzene

[0538] DMF N,N-Dimethylformamide

[0539] d6-DMSO Deuterated dimethyl sulfoxide

[0540] eq. equivalent

[0541] EtOAc Ethyl acetate

[0542] FaSSGF Fasting state simulated gastric fluid

[0543] FaSSIF Fasting State Simulated Intestinal Fluid

[0544] g

[0545] GF / F Glass Microfiber Filter Grade

[0546] h hour

[0547] 1 H Proton

[0548] 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]

[0549] HATU Pyridinium 3-oxide Hexafluorophosphate

[0550] HPLC High Performance Liquid Chromatography

[0551] Kg Kilogram

[0552] L Liter

[0553] LC-MS Liquid chromatography–mass spectrometry

[0554] M Moore

[0555] MeCN Acetonitrile

[0556] MeOH Methanol

[0557] 2-MeTHF 2-Methyltetrahydrofuran

[0558] mg milligram

[0559] Mins

[0560] mL milliliters

[0561] mol mole

[0562] MW Molecular weight

[0563] NMP 1-Methyl-2-pyrrolidone

[0564] NMR Nuclear Magnetic Resonance

[0565] TFA Trifluoroacetic acid

[0566] th Theory

[0567] TLC Thin Layer Chromatography

[0568] TPGS D-α-Tocopheryl polyethylene glycol 1000 succinate

[0569] Uncorr. Uncorrected

[0570] UV

[0571] vol volume

[0572] w / v weight / volume

[0573] w / w weight / weight

[0574] wrt relative to

[0575] wt weight

[0576] BINAP: 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl; CDI: 1,1'-carbonyldiimidazole; DCE: 1,2-dichloroethane; DCM: dichloromethane; DIPEA: diisopropylethylamine; DMSO: dimethyl sulfoxide; DMF: N,N-dimethylformamide; DMAP: -(dimethylamino)pyridine; EtOAc: ethyl acetate; h: hour; HATU: N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate; HBTU: 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate hexafluorophosphate); HCl: hydrochloric acid; HPLC: high pressure liquid chromatography; LC-MS: liquid chromatography-mass spectrometry; LiHMDS: lithium bis(trimethylsilyl)amide; mins.: minutes; MeCN: acetonitrile; MS: mass spectrometry; NBS: N-bromosuccinimide; NMR: nuclear magnetic resonance; PdCl2(dppf)2: (1,1'-bis(diphenylphosphino)-ferrocene) dichloropalladium(II); Pd2(dba)3: tris(dibenzylideneacetone)palladium(0); Petrol: petroleum ether fraction with a boiling point of 40–60°C; PyBOP: (benzotriazol-1-yl-oxy) tris(dimethylamino)phosphine hexafluorophosphate; RT: room temperature; Sat.: saturated; SCX: solid phase cation exchange resin; SPhos: 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl; S-Phos Pd G3: (2-dicyclohexylphospho-2',6'-dimethoxybiphenyl)[2-(2'-amino-1,1'-biphenyl)]methanesulfonate palladium(II); TBDMSCl: tert-butyldimethylsilyl chloride; TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate; TFA: trifluoroacetic acid; THF: tetrahydrofuran; XPhos: 2-dicyclohexylphospho-2',4',6'-triisopropylbiphenyl; XantPhos: 4,5-bis(biphenylphospho)-9,9-dimethylxanthene.

[0577] When the amount is given in weight equivalents (wt) and volume equivalents (vol), 1 vol is equal to 1 milliliter per gram of starting material (defined as having a weight equivalent value of 1 wt). For example, when 50 mg (0.05 g) of starting material (defined as having a weight equivalent of 1 wt) is used, then 20 vol is equal to 1 mL (0.05 x 20 = 1).

[0578] Synthesis method

[0579] All starting materials and solvents were purchased commercially or prepared according to the literature. Unless otherwise stated, all reactions were stirred. Organic solutions were routinely dried over anhydrous magnesium sulfate. Hydrogenation was performed on a Parr hydrogenator, which is a Thales H-cube flow reactor, under the conditions stated or under hydrogen balloon conditions. Microwave reactions were performed in CEMDiscover and Smithcreator microwave reactors, heated to a constant temperature using variable power microwave irradiation. Normal phase column chromatography was routinely performed using an automated flash chromatography system, such as a CombiFlash Companion or CombiFlash RF system, and a pre-filled silica gel (230-400 mesh, 40-63 μm) filter cartridge. SCX was purchased from Supelco and treated with 1M hydrochloric acid before use. Unless otherwise stated, the reaction mixture to be purified was first diluted with MeOH and made acidic by adding a few drops of AcOH. This solution was loaded directly onto the SCX and washed with MeOH. Then, 1% NH3 in MeOH was used to rinse and elute the desired material.

[0580] When a compound is described as a mixture of two diastereomers / epimers, the conformation of the stereocenter is not specified and is represented by a straight line.

[0581] NMR data

[0582] The spectrometer was used to obtain the data at 400 MHz. 1 H NMR spectra. The central peak of chloroform-d, dimethyl sulfoxide-d6, or tetramethylsilane internal standards was used as reference. For NMR data, when the number of assigned protons is less than the theoretical number of protons for the molecule, it is assumed that the apparently missing signals are masked by solvent and / or water peaks. In addition, if proton NMR solvents are used to obtain the spectrum, exchange of NH and / or OH protons with the solvent occurs, and therefore, these signals are usually not observed. BRIEF DESCRIPTION OF THE DRAWINGS

[0583] Figure 1is an X-ray powder diffraction pattern of a crystalline form (“Form A”) of (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide.

[0584] Figure 2 is an X-ray powder diffraction pattern of another crystalline form (“Form B”) of (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide.

[0585] Figure 3 It is a differential scanning calorimetry (DSC) scan of crystalline Form B of (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide.

[0586] Figure 4 The weight loss chart of crystalline form B of (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide obtained by thermogravimetric analysis.

[0587] Figure 5 The weight curve of the crystalline form B of (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide under different humidity conditions was obtained by dynamic vapor sorption analysis.

[0588] Figure 6 The hydrochloride of the amorphous compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0589] Figure 7 The sulfate of the amorphous compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0590] Figure 8The napadisilate salt of the amorphous compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0591] Fig. 9 The amorphous edisulphonate of the compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0592] Fig.10 The toluenesulfonate salt of the amorphous compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0593] Fig.11 The amorphous methanesulfonate of the compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0594] Fig.12 The amorphous naphthalenesulfonate of the compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0595] Fig.13 The amorphous benzenesulfonate of the compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0596] Fig.14 The isethionate salt of the amorphous compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0597] Fig.15 The ethanesulfonate of the amorphous compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0598] Fig.16 The hydrobromide salt of the amorphous compound of formula (1) obtained in Example 2 1 H-NMR spectrum.

[0599] Example 1

[0600] Amorphous (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3- Synthesis of dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide

[0601] Step 1: Methyl 5-bromo-2-(bromomethyl)benzoate

[0602]

[0603] Methyl-5-bromo-2-methylbenzoate (500.0 g, 2.18 mol, 1.0 eq.) and N-bromosuccinimide (Fluorochem, 388.5 g, 2.18 mol, 1.0 eq.) were added to a stirred solution of 1,2-dichloroethane (1.9 L) in a 10 L five-necked flask equipped with a condenser, a stir bar, a N2 inlet, and a bubbler. The mixture was heated to 90°C (oil bath). Azobisisobutyronitrile (5.0 g, 0.03 mol, 0.014 eq.) was dissolved in DCE (100 mL) and 20 mL of this solution was added to the dropping funnel. When the reaction mixture reached 85°C, this solution was added slowly. When the vigorous reflux and foaming stopped, the remaining 80 mL was added to the reaction mixture in one portion and stirred at 90°C for 1 hr. NMR results showed that at the end of the reaction, about 11% of the starting material remained. Then, the reaction mixture was cooled to room temperature by adding dry ice in an oil bath, and once the internal temperature dropped to ~30 ℃, the reaction mixture was quenched with water (2.0 L). After 5 minutes, two identical reaction mixtures were combined and transferred into a separatory funnel to collect the organic layer. The aqueous layer was extracted again with DCM (2x 2.0 L). All organic layers were combined and washed with water (2 L) and brine (2 L), dried with MgSO4, filtered, and concentrated in vacuo to give an orange liquid (1.397 kg, 104%, obtained from 2x 500 g operation).

[0604] Step 2-(2R)-tert-butyl 2-(6-bromo-1-oxo-2,3-dihydro-1H-isoindol-2-yl)propanoate

[0605]

[0606] To the THF stirring solution (5.0 L) in a 10 L five-necked flask equipped with a condenser, a stirrer, a N2 inlet and a bubbler, 5-bromo-2-(bromomethyl)benzoic acid methyl ester (from step 1) (660 g, 2.14 mol, 1.0 eq), (2R)-2-aminopropionic acid tert-butyl ester.HCl (467 g, 2.57 mol, 1.2 eq) and diisopropylethylamine (1.0 L, 6.42 mol, 3.0 eq., d = 0.742) were added. The mixture was heated to 80 ° C (heated overnight in an oil bath). Then, the reaction mixture was cooled to room temperature with dry ice, and then, two identical reaction mixtures were poured into a large separatory funnel. NaHCO3 saturated aqueous solution (5.6 L) was added and the resulting mixture was stirred for 5 minutes. The organic layer was collected and the aqueous phase was extracted with ethyl acetate (2 x 4 L). The organic phases were pooled, washed with brine (5.6 L), stirred for 3 minutes, dried over MgSO4, filtered, and concentrated in vacuo to give an orange-brown residual sticky solid, which was placed in a vacuum oven at 40° C. overnight. The solid (1.4 kg) was slurried in petroleum ether 40-60 (2.5 L) and stirred vigorously overnight, then filtered and washed with petroleum ether (2×300 mL) to give a yellow solid (480 g).

[0607] Step 3: (2R)-2-[1-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2, tert-Butyl 3-dihydro-1H-isoindol-2-yl]propanoate

[0608]

[0609] To a stirred solution of dioxane (4 L) in a 10 L 5-necked flask equipped with an overhead stirrer, condenser, temperature probe, N2 inlet and bubbler was added tert-butyl (2R)-2-(6-bromo-1-oxo-2,3-dihydro-1H-isoindol-2-yl)propanoate (from step 2) (500 g, 1.469 mol, 1.0 eq), bis(pinacolato)diboron (446.3 g, 1.764 mol, 1.2 eq) and anhydrous potassium acetate (433.9 g, 4.409 mol, 3 eq). Then, N2 was used for degassing for 30 minutes, after which Pd(dppf)Cl2 (21.5 g, 0.029 mol, 0.02 eq) was added, and the reaction mixture was further degassed for 10 minutes and then heated to 90°C in an oil bath. It was stirred overnight (Note: After stirring at 90°C for 2-3 hours, the reaction exothermed, rising from 88°C to 104°C, with a lot of reflux, and the solution turned from an orange-red solution to a dark brown color, maintained for ~30 min, and then cooled to 90°C). The reaction was cooled to room temperature with dry ice, then combined and filtered through a celite pad (4L sinter, ~2 inch thick pad). The celite pad was washed with dioxane (1 L) until all color was washed off.

[0610] Step 4: (2R)-2-[6-(2,5-dichloropyrimidin-4-yl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]propane Tert-butyl ester

[0611]

[0612] The solution obtained in step 3 was then divided into two halves, and each portion was added to a five-necked flask with the same configuration as in the previous step. 2,4,5-trichloropyrimidine (404.4 g, 252.8 mL, 2.205 mol, 1.5 eq, D = 1.6) and potassium carbonate (609.4 g, 4.409 mol, 3 eq) were added to the five-necked flask, and then degassed with N2 for 30 minutes, after which Pd(dppf)Cl2 (21.5 g, 0.029 mol, 0.02 eq) was added and further degassed for 10 minutes. The reaction mixture was heated to 65°C in an oil bath, and then water (500 mL) was added through a dropping funnel within 5 minutes (Note: After a few minutes of the initial addition of water, the temperature rose from 62°C to 72°C due to exotherm). It was allowed to react overnight. The reaction was cooled to room temperature with dry ice and filtered through a pad of celite (4 L fritted funnel, ~3 inch thick celite pad) and the celite pad was washed with DCM (2 L). The filtrate was then concentrated to nearly dryness to give a black-brown tarry crude material (1909 g).

[0613] The crude material was divided into two portions and dry loaded onto a silica gel column, eluting with 6 x 4 L 40% ethyl acetate / petrol. (Note: all fractions were combined, pooled, and concentrated to almost dryness - product was purified by TLC with 30% ethyl acetate / petrol Rf = ~0.5). The residual brown mixture was cooled to room temperature, petrol (4 L) was added, and the product was crystallized by rotation for 2 hours. The product was filtered and the filter cake was washed with petrol (2 x 2 L) to give a white solid (548 g).

[0614] Alternatively, tert-butyl (2R)-2-[6-(2,5-dichloropyrimidin-4-yl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]propanoate (intermediate compound (4)) can be prepared according to PCT / IB2016 / 001507 (International Publication No. WO2017 / 068412) - see Preparations 76, 89 and 94 therein.

[0615] Amorphous (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide (Compound 1) was synthesized using tert-butyl (2R)-2-[6-(2,5-dichloropyrimidin-4-yl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]propanoate (from Step 4) according to the following synthetic scheme:

[0616] Step 5: (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro- Hydrogen-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide

[0617]

[0618] Stage 1: Boc-deprotection of (4)

[0619]

[0620] To a 15-25°C solution of (4) (supplied by Manchester Organics) in toluene (13 vol) was added concentrated hydrochloric acid (1 vol) followed by a line rinse with toluene (1 vol). The mixture was heated to 35-40°C and stirred at this temperature until the reaction was complete (qualification criteria: ≤ 2.0% (4) by area, expected reaction time 16-24 hours). The suspension was concentrated under reduced pressure at no more than 50°C until a wet solid was obtained. Toluene (3 x 10 vol) was charged and concentrated under reduced pressure at no more than 50°C to obtain a wet solid after each successive addition. Toluene (4 vol) was added and the suspension was rotated on a rotary evaporator at atmospheric pressure and no more than 50°C for 10 to 20 minutes. The flask was removed from the rotary evaporator and the contents were aged at 15-25°C for at least 1 hour. The solid was collected by filtration, washed with toluene (2 x 2 vol) and pulled dry under nitrogen until toluene content ≤ 6.0% w / w, water content ≤ 2.0% w / w. Intermediate (5) was isolated as an off-white to light beige solid (74-95% th, 64-82% w / w).

[0621] Stage 2: Coupling of intermediate (5) with 4-aminotetrahydropyran

[0622]

[0623] To a 15-25°C solution of (5) (1.0 wt correction, 1.0 mol eq) in 1-methyl-2-pyrrolidone (NMP) (9.5 vol) was added potassium carbonate (0.86 wt, 2.2 mol eq), followed by 15-40°C 4-aminotetrahydropyran (6) (0.4 vol, 1.3 mol eq) and a line rinse with NMP (0.5 vol). The mixture was heated to 80-95°C and stirred at this temperature until the reaction was complete (qualification criteria: ≤1.0 mol% intermediate (5), reaction time 4-6 hours). The mixture was cooled to 15-25°C and 3M hydrochloric acid (10 vol) was added, the temperature being maintained at 15-30°C. Dichloromethane (DCM) (10 vol) was added and the phases were separated. The acidic aqueous phase was back-extracted with DCM (5 vol) and the combined organic phases were washed with purified water (8 x 10 vol) until the NMP content was controlled to ≤ 15.0% w / w. The organic phase was washed with 13% w / w NaCl solution (10 vol), treated with activated carbon (0.3 wt) and dried with magnesium sulfate (1.0 wt). The mixture was filtered to remove the desiccant, washed with DCM (2 x 2 vol) and the combined filtrates were concentrated under reduced pressure at no more than 35° C. to give (2) (74-90% th, 88-107% w / w) as a light brown foam.

[0624] Stage 3: Preparation of Compound 1

[0625]

[0626] To a solution of (2) (1.0 wt, 1.0 mol eq) in DCM (12.5 vol) was added amine (3) (0.68 wt, 1.27 mol eq). The suspension was cooled to 10-15°C and DIPEA (1.67 vol, 4.0 mol eq) was added. The resulting solution was stirred for 5-10 minutes and then HATU (1.15 wt, 1.27 mol eq) was added portion wise, keeping the reaction temperature at <25°C. The mixture was stirred at 15-25°C until HPLC confirmed the completion of the reaction (<0.5% area of ​​(2), typically 1 hour). After the reaction was completed, the reaction was concentrated under reduced pressure at no more than 38°C to give a mobile orange viscous oil. The residue was dissolved in EtOAc (10 vol) and washed with purified water (10 vol), 25% w / w ammonium chloride solution (2 x 10 vol), 8% w / w NaHCO3 solution (2 x 10 vol) and 13% w / w NaCl solution (6 x 10 vol), and then dried over MgSO4 (1.0 wt). The solid was removed by filtration, and the filter cake was washed with ethyl acetate (2 x 2 vol). The filtrate was concentrated on a rotary evaporator at no more than 40° C. to give crude compound 1 as a light yellow foam. Crude compound 1 was purified by dry flash chromatography.

[0627] Under the above conditions, but using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) as coupling agent instead of HATU, 4-dimethylaminopyridine (4-DMAP) as base instead of DIPEA, and dimethylformamide (DMF) as solvent instead of DCM, it was also observed that (2) was fully converted into compound 1.

[0628] Chromatographic analysis procedure:

[0629] Silica gel (20 wts) was loaded into a quick dry column and washed and filled with ethyl acetate (usually 2 x 20 vol). Crude compound 1 (1 wt uncorrected) was dissolved in DCM (4 vol) and carefully loaded onto the column. Then, the chromatographic column was eluted according to the following scheme:

[0630] Pure EtOAc 40x 20vol F1-240

[0631] 1% MeOH in EtOAc 10x 20 vol F41-50

[0632] 5-10% MeOH in EtOAc 3x 20 vol F51-53 column flush

[0633] All fractions collected are analyzed by HPLC, and the product is usually eluted in fractions 12 to 45. In the absence of HPLC data, only those fractions (e.g., fractions 15-25 of the product elution example range above) that TLC shows to be the cleanest and most intense are combined. The external products containing each fraction are analyzed by HPLC to determine whether these fractions have appropriate purity and are suitable for combining with the main product fraction. Once all these fractions are combined and concentrated into foam or lower volume, the product is diluted with ethyl acetate (e.g., 10 vol), stirred, and a solution is obtained, which is then clarified by glass fiber filter paper. Then, the filtrate is concentrated to obtain amorphous compound 1 (65-85% th, 91-119% w / w) in a beige to light yellow foam.

[0634] Example 2

[0635] Preparation of amorphous salt of compound 1

[0636] Prepare 2-propanol stock solutions of various counterions. Add the relevant stock solution (500 μl, 10 vol) to a solution of compound 1 (prepared according to Example 1) in isopropyl acetate (2.5 ml, 50 vol) and stir. Cool the uncrystallized or slightly precipitated solid, evaporate and concentrate or further treat with tert-butyl methyl ether (TBME).

[0637] The solid was separated by filtration and dried with a stream of nitrogen for 96 hours, removed, and subjected to 1 H NMR and XRPD analysis (see table below).

[0638]

[0639]

[0640] Example 3

[0641] Preparation of crystalline form of compound 1

[0642] Example 3A - Preparation of Crystalline Form A

[0643] The hydrochloride salt of (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide (prepared as in Example 2) was suspended in purified water (35 volumes) at 70° C. for 96 hours. The solid was isolated by filtration and dried under a stream of nitrogen for 96 hours, removed, and subjected to XRPD analysis (see Figure 1 ).

[0644] Example 3B - Preparation of Crystalline Form B (Method I)

[0645] The hydrochloride salt of (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide (prepared as in Example 2) was suspended in purified water (2 ml, 20 vol) at 18-23°C. The solution was stirred at 45-50°C for 20 hours. Then, the temperature was lowered to 30-35°C and the solution was further stirred for 96 hours. The XRPD results of the obtained solid were consistent with those of Figure 2 XRPD Figure 1 To.

[0646] Example 3C - Preparation of Crystalline Form B (Method II)

[0647] The hydrochloride salt of (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide (prepared as in Example 2) was suspended in purified water (20 vol) and the mixture was stirred at 40°C under nitrogen for 20 hours. 2-Propanol (7.6 vol) was added and the mixture was stirred at 40°C for 20 hours. The progress of the conversion was monitored by XRPD. The XRPD results of the obtained solid were consistent with those of Figure 2 XRPD Figure 1 To.

[0648] Example 3D - Preparation of Crystalline Form B (Method III)

[0649] (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide (prepared as in Example 1) (100 mg, 1.0 wt.) was added to a container, followed by ethyl acetate or toluene (15 vol) and tert-butyl methyl ether (15 vol). The suspension was stirred at 30°C for 7 days. The product was then isolated by filtration, washed with recovered maturation solvent, and dried at 18-23°C under a stream of nitrogen, providing evidence of crystallization by XRPD analysis. The XRPD pattern of the resulting solid was consistent with that of Figure 2 XRPD Figure 1 To.

[0650] Example 4

[0651] Further characterization of crystalline form B of compound 1

[0652] The crystalline Form B of Compound 1 obtained in Examples 3B, 3C and 3D was studied by X-ray powder diffraction, differential scanning calorimetry, thermogravimetric analysis and dynamic vapor sorption.

[0653] Example 4A—X-ray Powder Diffraction

[0654] Crystals of crystalline form B compound 1 were prepared according to the method of Example 3. X-ray powder diffraction (XRPD) analysis was performed using a Bruker D2Phaser powder diffractometer equipped with a LynxEye detector. Minimal sample processing was performed, but if necessary, the sample could be slightly ground with a mortar and pestle before obtaining the sample. The specimen was placed in a 5 mm bag in the center of the silica gel sample holder (approximately 5-10 mg). The sample was rotated continuously during data acquisition, and the scanning conditions were: step size 0.02°2θ, range 4°-40°2θ, step time 34.5 seconds. The data was processed using Bruker Diffrac.Suite. The X-ray powder diffraction pattern of the crystalline form B of the compound is shown in Figure 2 The 2θ diffraction angle and intensity of each peak are given in the table below.

[0655] Diffraction angle (°) Relative Strength 8.75 23 12.00 13 13.03 23 13.82 39 14.05 100 14.43 20 16.89 12 17.31 22 19.34 36 20.56 85 21.25 45 23.52 18 23.97 74 24.15 71 26.18 18 28.74 22 29.84 13

[0656] Example 4B - Differential Scanning Calorimetry (DSC)

[0657] Crystals of crystalline form B of compound 1 were prepared according to the method of Example 3. Thermal analysis was performed using a Mettler Toledo DSC 821 operated by STARe™ software. The analysis was performed under nitrogen conditions in a 40 μL open aluminum pan with a sample size of 1-10 mg. Typically, the temperature range of the analysis was 20° to 250° with a temperature gradient of 10°C / min. The DSC scan results of the crystalline compound are shown in FIG. Figure 3 shown.

[0658] Example 4C - Thermogravimetric Analysis (TGA)

[0659] Crystals of Compound 1 in Crystalline Form B were prepared according to the method of Example 3. Approximately 7 mg of the sample was placed in a platinum HT TGA pan that had been cleaned with a butane torch and tare using the instrument's automatic tare function. The sample was heated from 25°C to 800°C at 10°C / min under nitrogen. The weight loss curve of the crystalline compound is shown in Figure 4 shown.

[0660] Example 4D - Dynamic Vapor Sorption Analysis

[0661] Crystals of crystalline form B compound 1 were prepared according to the method of Example 3. Approximately 20 mg of the sample was weighed into an aluminum pan and placed in a DVS Intrinsic instrument maintained at 25°C. The sample was allowed to equilibrate at 0% RH for 3 hours, and then the humidity was increased from 0 to 30% RH in 5% increments. Then, the humidity was increased to 90% RH in 10% increments. A similar rate reduction was used in the desorption stage. In each step, a rate of change of 0.002% / min mass / time unit (dt) was set as the equilibrium parameter. The vapor adsorption / desorption curve of the compound is shown in FIG. Figure 5 shown.

[0662] Example 4E - Single Crystal X-ray Diffraction Studies

[0663] The single crystal X-ray structure of crystalline form B of the compound of formula (1) was determined at 100K using crystals obtained by slow evaporation of isopropyl acetate.

[0664] Data were collected using a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero, Atlas CCD diffractometer equipped with an Oxford Cryosystems Cobra cooling device. Data were collected using Cu Kα. The structure was resolved and refined using the Bruker AXS SHELXTL package. All details are shown in the table below. Unless otherwise stated, hydrogen atoms attached to carbon are arranged geometrically and refined using riding isotropic displacement parameters. Hydrogen atoms attached to heteroatoms are located within the difference Fourier synthesis and can be arbitrarily refined using isotropic displacement parameters. Reference diffraction patterns for crystal structures were generated using Mercury (CFaMacrae, "Mercury: visualization and analysis of crystal structures," J. Appl. Cryst., vol. 39, pp. 453-457, 2006).

[0665] Data acquisition and structure refinement

[0666] Diffractometer SuperNova, Dual, Cu at zero, Atlas Radiation Source SuperNova(Cu)X-ray Source,CuKα Data collection methods scanning Theta range for data collection 3.460 to 66.589° Indicator range -14≤h≤15, -10≤k≤9, -14≤ / ≤15 Diffraction points collected 24752 Independent diffraction points 4834[R(int)=0.0371] Range of independent diffraction points 97.4％ Absorption correction Semi-empirical from equivalent values Maximum and minimum transmission 1.00000 and 0.80872 Structural analysis methods Direct Method Structural Elucidation / Refinition Program SHELXTL (Sheldrick, 2013) Refinement method Full Matrix Least Squares Method Based on F2

[0667] Crystallization data

[0668]

[0669]

[0670] The crystal is monoclinic, space group P21, and finally R1=[I>2s(I)]=2.93%.

[0671] The absolute stereochemistry of the compound has been determined using diffraction data and it has been demonstrated that the Flack parameter = -0.004 (7) as depicted herein in formula (1).

[0672] In the asymmetric unit, there is one molecule of the compound of formula (1) and one water molecule, both of which are completely ordered, confirming that crystalline form B is a monohydrate of the compound of formula (1).

[0673] Example 5

[0674] Solubility determination of crystalline form B of compound (1) in aqueous solvents

[0675] The solubility of the crystalline form B and the amorphous form of the compound of formula (1) in purified water and various buffers of equal concentration was determined with stirring times not exceeding 24 hours at temperatures between 18 and 23° C. The pH and temperature of the solution were monitored to confirm that the pH had not changed by the end of the experiment.

[0676] General Procedure

[0677] The sample (50 mg) of the compound of formula (1) was suspended in purified water (2 mL) or a suitable buffer solution and stirred at room temperature (18-23 ° C) for 20-24 hours. The slurry was then centrifuged and diluted with HPLC sample diluent (acetonitrile / water, 1 / 1, v / v) if appropriate, and analyzed using HPLC peak area. The measured value was compared with the relevant calibration curve of the compound of formula (1). Approximate solubility was calculated, and a compensation factor was applied for appropriate dilution factor and %w / w analysis. The dilution factor ensured that the peak area value fell within the desired calibration curve range.

[0678] The solids remaining in the centrifuge tube were dried in a vacuum oven and analyzed by XRPD. If the sample showed any evidence of crystallinity that was different from the expected diffraction pattern of the crystalline form of compound (1), no further analysis was performed. 1 H NMR analysis was performed to confirm its chemical identity.

[0679] The preparation instructions for the buffer used in this study are as follows:

[0680] pH 1.2 – Mix 50 mL of 0.2 M potassium chloride solution with 85 mL of 0.2 M hydrochloric acid solution.

[0681] pH 2 – Mix 50 mL of 0.2 M potassium chloride solution with 13 mL of 0.2 M hydrochloric acid solution.

[0682] pH 3-Mix 100 mL of 0.1 M potassium hydrogen phthalate with 44.6 mL of 0.1 M hydrochloric acid solution.

[0683] pH 4-Mix 100 mL of 0.1 M potassium hydrogen phthalate with 0.2 mL of 0.1 M hydrochloric acid solution.

[0684] pH 5-Mix 100 mL of 0.1 M potassium hydrogen phthalate with 45.2 mL of 0.1 M sodium hydroxide solution.

[0685] pH 6-Mix 100 mL of 0.1 M potassium dihydrogen orthophosphate with 11.2 mL of 0.1 M sodium hydroxide solution.

[0686] pH 7 – Mix 100 mL of 0.1 M potassium dihydrogen orthophosphate with 58.2 mL of 0.1 M sodium hydroxide solution.

[0687] pH 8 – Mix 100 mL of 0.1 M potassium dihydrogen orthophosphate with 93.4 mL of 0.1 M sodium hydroxide solution.

[0688] result

[0689]

[0690]

[0691] Conclusions and Observations

[0692] The crystalline and amorphous forms of the compound of formula (1) are practically insoluble in buffers at pH 1.2-8.0 (approximately <0.1 mg / ml).

[0693] Solubility determination of crystalline form B of compound (1) in non-aqueous solvents

[0694] program

[0695] Form B of compound (1) prepared according to Example 3 was added to a suitable solvent (2 ml) at approximately 1 hour intervals. The solution was purposely saturated and stirred at 18-23°C for 72 hours. The suspension was then centrifuged and the supernatant was sampled and diluted accordingly, and the peak areas of the corresponding analytes were determined by HPLC and compared with the peak areas of standard calibration materials. Approximate solubility was calculated and the solubility was physically checked on the basis of anhydrous solvent to confirm the consistency of the results.

[0696] Observations and Results

[0697]

[0698] The saturated solution was centrifuged and 100 μl of the clear supernatant was added to a 250 ml (ethanol) or 100 ml (residue) volumetric flask and diluted to volume with sample diluent. The diluted samples were then analyzed by HPLC peak area and the estimated maximum solubility was calculated and reported in the table below.

[0699] Solvents Estimated maximum solubility (mg / ml), 18-23℃ Ethanol 337 glycerin 5 PEG 400 229 Propylene glycol 289

[0700] To confirm that the estimated order of magnitude of the solubility was correct, a solution was prepared so that the concentration did not exceed the estimated maximum assay concentration, and the mixture was stirred overnight under the same conditions to confirm dissolution.

[0701] in conclusion

[0702] The results show that the solubility of the compound of formula (1) in ethanol, PEG 400 and propylene glycol is significantly greater than its solubility in water.

[0703] Example 6 Liquid Formulation-I

[0704] Solvents

[0705] a. Propylene glycol - P4347SIGMA-ALDRICH (in accordance with USP test specifications)

[0706] b. Ethanol - 29221 SIGMA-ALDRICH (tested according to European Pharmacopoeia requirements)

[0707] c.Super Refined TM PEG 400-Croda (JP, USP-NF, PhEur)

[0708] d. Propylene glycol and ethanol 75:25 (% w / w) combination

[0709] e. Propylene glycol and ethanol 85:15 (% w / w) combination

[0710] plan

[0711] The compound of formula (1) was incubated in each solvent at 500 mg / mL in a glass vial (using RS9000 heating block, 25°C) with continuous stirring. T = 1, 3, 7, 24 and 48 hours.

[0712] The resulting suspension is ultracentrifuged at 13,000 rpm (i.e., 500-1000 μl sample volume) for 15 minutes and visually inspected to confirm clarity. If the clarity is not satisfactory, repeat the centrifugation procedure to precipitate the remaining undissolved compounds. The supernatant is sampled (e.g., 100 μl-1000 μl), appropriately diluted, and analyzed by HPLC-UV to determine the actual concentration.

[0713] Example 7

[0714] Liquid Formulations – II

[0715] The following liquid formulations were prepared.

[0716] 7-1. The compound of formula (1) was dissolved in 5 g of TPSG and 5 g of propylene glycol.

[0717] 7-2. The compound of formula (1) was dissolved in 6 g of TPSG and 6 g of propylene glycol.

[0718] 7-3. The compound of formula (1) was dissolved in 2.5 g of TPSG and 7.5 g of propylene glycol.

[0719] 7-4. The compound of formula (1) dissolved in TPGS (20% w / w), ethanol (15% w / w) and propylene glycol (65% w / w).

[0720] 7-5. The compound of formula (1) dissolved in propylene glycol (100% w / w).

[0721] 7-6. The compound of formula (1) dissolved in TPGS (10% w / w), ethanol (10% w / w) and propylene glycol (80% w / w).

[0722] The concentration of the compound of formula (1) in each preparation was 100 mg / mL.

[0723] For each formulation, dilution studies were performed in simulated gastric and simulated intestinal fluids as described below. In these experiments, the concentration of the compound of formula (1) was determined using the HPLC-UV method described below.

[0724] LC method parameters

[0725] Column: Halo C18 150x 4.6mm; 2.7μm

[0726] Injection volume: 5 μL

[0727] Detection: UV@221nm

[0728] Mobile phase A: water / acetonitrile / TFA (5 / 95 / 0.05 v / v / v)

[0729] Mobile phase B: water / acetonitrile / TFA (95 / 5 / 0.5 v / v / v)

[0730] Time (minutes) ％A ％B 0.0 100 0 2.0 100 0 24.0 60 40 28.0 50 50 33.0 0 100 36.0 0 100 36.1 100 0 40.0 100 0

[0731] Flow rate: 1.0mL / min

[0732] Column temperature: 40°C

[0733] Running time: 40 minutes

[0734] Integration time: 36 minutes

[0735] Wash bottle: Sample diluent

[0736] Dilution studies

[0737] Each preparation was added to 50 mL of FaSSGF ( http: / / biorelevant.com / fassif-fessif- fassgf / how-to-make / Description Samples were taken at three time points (T = 5 min, 15 min and 30 min) within 1 h (prepared by the method described above), and at least 2 mL was taken for each sample.

[0738] Then, the mixture was mixed with 44 mL of fasting state simulated intestinal fluid FaSSIF (according to http: / / biorelevant.com / fassif-fessif-fassgf / how-to-make / The mixture was further diluted and sampled at 8 time points (T = 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 5 hours and 7 hours). The concentration of the compound of formula (1) in the samples was analyzed by HPLC-UV.

[0739] For formulations 7-1 and 7-2, approximately 65% ​​of the compound of formula (1) remained dissolved after dilution in FaSSGF for 30 minutes. In the next dilution step, when transferred to FaSSIF, the amount of dissolved compound was still approximately 50% at 3 hours and then gradually declined. Extrapolating the results of this dilution study to the "human dilution scenario", it is expected that in formulation 7-1 or formulation 7-2, >60% (i.e., approximately 600 mg) of a 1000 mg dose of the compound of formula (1) will dissolve and remain in solution when entering the stomach.

[0740] For Formulation 7-3, approximately 33% of the drug remained dissolved 30 minutes after dilution in FaSSGF. In the next dilution step, when transferred to FaSSIF, the amount of dissolved drug was still approximately 27% at 3 hours and then gradually declined. Extrapolating the results of this dilution study to the "human dilution scenario", it is expected that in Formulation 7-3, >30% (i.e., approximately 300 mg) of the 1000 mg dose will be dissolved and remain in solution when entering the stomach.

[0741] For Formulation 7-4, approximately 30% of the drug remained dissolved 30 minutes after dilution in FaSSGF. In the next dilution step, when transferred to FaSSIF, the amount of dissolved drug was still approximately 24% at 3 hours and then gradually declined. Extrapolating the results of this dilution study to the "human dilution scenario", it is expected that in Formulation 7-4, >30% (i.e., approximately 300 mg) of the 1000 mg dose will be dissolved and remain in solution when entering the stomach.

[0742] For formulation 7-5, approximately 2% of the drug remained dissolved 30 minutes after dilution in FaSSGF. In the next dilution step, when transferred to FaSSIF, the amount of dissolved drug remained approximately 2%.

[0743] For formulation 7-6, approximately 17% of the drug remained dissolved 30 minutes after dilution in FaSSGF. In the next dilution step, when transferred to FaSSIF, the amount of dissolved drug remained approximately 11%.

[0744] We found that formulation 7-6 was particularly advantageous. A placebo solution of this formulation (i.e., using only the vehicle without any compound of formula (1)) initially formed a transparent liquid containing some precipitates, but these precipitates redissolved after heating to 50°C, and the liquid remained transparent without any precipitates or turbidity after 24 hours and 96 hours. The active solution of formulation 7-6 containing 100 mg / ml compound of formula (1) formed a transparent solution, and after standing at room temperature for 24 hours and 192 hours, the solution remained transparent without any precipitates or turbidity.

[0745] Example 8

[0746] Biological Activity

[0747] Example 8A-ERK2 In vitro Inhibition Assay

[0748] The inhibitory activity of the compounds of the invention was determined using the following protocol.

[0749] The activity of the ERK2 enzyme (Life Technologies) was determined by measuring the phosphorylation of truncated activating transcription factor 2 labeled with green fluorescent protein (ATF2-GFP) (Life Technologies) in a time-resolved fluorescence format. The assay reaction contained 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 μM ATF2-GFP, 20 μM ATP, and 0.25 nM ERK2 in the presence of the compound and was carried out at room temperature for 30 minutes. The reaction was then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA, and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After incubation for at least 30 min, the fluorescence readings were taken on a Pherastar microplate reader (Lanthascreen optical module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio of the A and B readings was used to calculate the signal. The IC was calculated using a sigmoidal dose-response equation (Prism GraphPad software, LaJolla, CA, USA). 50 value.

[0750] In the assay using ERK2, the IC of the compound of formula (1) was 50 The value is 0.0027 μM.

[0751] Example 8B - Antiproliferative Activity

[0752] The antiproliferative activity of the compounds of the invention was determined by measuring the ability of the compounds of formula (1) to inhibit the growth of the human melanoma cell line A375.

[0753] Cell proliferation was determined by measuring the conversion of resazurin (alamar blue) to resorufin in response to mitochondrial activity (Nociari, MM, Shalev, A., Benias, P., Russo, C. Journal of Immunological Methods 1998, 213, 157-167). A375 cells (American Type Culture Collection, Teddington, UK) were cultured in Dulbecco's Modified Eagle Medium + 10% FBS. One day before compound treatment, 2 x 10 3Cells were seeded in 200 μl of complete medium per well of a black 96-well flat-bottom plate. The cells were incubated with a 0.1% (v / v) dimethyl sulfoxide (DMSO) solution of the compound for 4 days, followed by the addition of 20 μl of Alamar Blue. After an additional 6 h of incubation at 37°C, the plates were read on a Spectramax Gemini microplate reader (Molecular Devices; excitation 535 nm, emission 590 nm). The GI was calculated using a sigmoidal dose-response equation (Prism GraphPad software, La Jolla, CA, USA). 50 value.

[0754] In the test using A375 cells, the GI of the compound of formula (1) was 50 The value is 0.0034 μM.

[0755] Combination regimen for cell proliferation

[0756] The following technique was used to evaluate the effect of the combination of the compound of formula (1) (Compound I) and the anticancer agent (Compound II). 3 -4x10 3 Human cancer cell lines (e.g., A375) were seeded in 96-well tissue culture plates at a concentration of 10 cells / well. The cells were allowed to recover for 16-24 hours before addition of compound or DMSO control (0.1-0.5% DMSO). The cells were incubated with a 0.1%-0.5% (v / v) solution of compound in dimethyl sulfoxide (DMSO) for 72-96 hours before addition of 20 μl of Alamar Blue. After an additional 6 hours of incubation at 37°C, the plates were read on a Spectramax Gemini microplate reader (Molecular Devices; excitation 535 nm, emission 590 nm). The GI was calculated using a sigmoidal dose-response equation (Prism GraphPad software, La Jolla, CA, USA). 50 The GI of compound II in the presence of different doses of compound I was determined. 50 When the content of compound I is lower than the effective dose, GI 50 When the effect of compound II and compound I combined is equivalent to the sum of the effects of the two compounds alone, it indicates that they are additive. Antagonistic effect is defined as causing GI 50 An ascending effect, where the effect of two compounds in combination is less than the sum of the effects of the two compounds alone.

[0757] Example 9

[0758] Pharmaceutical preparations

[0759] (i) Tablets

[0760] Tablet compositions comprising a compound of formula (1) are prepared by mixing an appropriate amount of the compound (e.g., 50-250 mg) with a suitable diluent, disintegrant, compressing agent, and / or glidant. One possible tablet comprises 50 mg of the compound, 197 mg of lactose (BP) diluent, and 3 mg of magnesium stearate lubricant, and is compressed into tablets in a manner familiar to people. Such compression tablets are optionally coated with a film.

[0761] (ii) Capsules

[0762] Capsules are prepared by mixing 100-250 mg (e.g. 100 mg) of the compound of formula (1) with an equivalent amount of lactose (e.g. 100 mg) and filling the resulting mixture into standard opaque hard gelatin capsules. Appropriate amounts of suitable disintegrants and / or glidants may be included as required.

[0763] (iii) Injectable Formulation I

[0764] Parenteral compositions for injection can be prepared by dissolving the compound of formula (1) (e.g., in salt form) in water containing 10% propylene glycol to obtain a concentration of 1.5% by weight of the active compound. The solution is then sterilized by filtration and then filled into an ampoule and sealed. Optionally, the solution is first formulated as an isotonic solution before sterilization.

[0765] (iv) Injectable preparation II

[0766] The parenteral composition for injection can be prepared by dissolving the compound of formula (1) (eg, salt form) (2 mg / ml) and mannitol (50 mg / ml) in water, sterilizing the solution by filtration and filling into a sealable 1 ml vial or ampoule or prefilled syringe.

[0767] (v) Injectable preparations III

[0768] The preparation for intravenous injection or infusion can be prepared by dissolving the compound of formula (1) (e.g., in salt form) in water at a concentration of 20 mg / ml and then optionally adjusting the isotonicity. The vial is then sealed and sterilized by autoclave. Alternatively, it can be loaded into an ampoule or vial or pre-filled syringe, sterilized by filtration, and sealed.

[0769] (vi) Injectable preparations IV

[0770] The preparation for intravenous injection or infusion can be prepared by dissolving the compound of formula (1) (e.g., in salt form) in water containing a buffer (e.g., 0.2M acetate solution at pH 4.6) at a concentration of 20 mg / ml. The vial is then sealed and sterilized by autoclave. Alternatively, the pre-filled syringe is then sealed by autoclave or by filtration sterilization and sealed.

[0771] (vii) Subcutaneous or intramuscular injection preparations

[0772] The composition for subcutaneous (intramuscular) administration is prepared by mixing the compound of formula (1) with pharmaceutical grade corn oil to a concentration of 5-50 mg / ml (eg 5 mg / ml). The composition is sterilized and filled into a suitable container.

[0773] (viii) Lyophilized preparations

[0774] Aliquots of the formulated compound of formula (1) were placed in 50 ml vials and lyophilized. During lyophilization, the composition was frozen using a (-45°C) one-step freezing protocol. The temperature was raised to -10°C for annealing, then lowered to -45°C for freezing, then primary dried at +25°C for approximately 3400 minutes, and then a temperature increase step was used to raise the temperature to 50°C for secondary drying. The pressure during primary and secondary drying was set to 80 mTorr.

[0775] (ix) Lyophilized preparation II

[0776] The prepared aliquots of the compound of formula (1) or its salt are placed in 50 mL vials and lyophilized. During lyophilization, the composition is frozen using a (-45°C) one-step freezing scheme. The temperature is raised to -10°C for annealing, then lowered to -45°C for freezing, then primary drying at +25°C for approximately 3400 minutes, and then the temperature is raised to 50°C using a temperature increase step for secondary drying. The pressure during primary and secondary drying is set to 80 mTorr.

[0777] (x) Lyophilized preparation for intravenous administration III

[0778] The aqueous buffer is prepared by dissolving the compound of formula (1) in a buffer. The buffered solution (filtered to remove particulate matter) is placed in a container (e.g., a Class 1 glass bottle), and the bottle is then partially sealed (e.g., covered with a Fluorotec stopper). If the compound and formulation are sufficiently stable, the formulation is sterilized by autoclaving at 121°C for a suitable time. If the formulation is unstable to autoclaving, it can be sterilized using a suitable filter and placed in a sterile bottle under aseptic conditions. The solution is freeze-dried using a suitable cycle. After the freeze-drying cycle is completed, the bottle is backfilled with nitrogen to atmospheric pressure, and the stopper is covered and secured (e.g., aluminum jaws). For intravenous injection, the lyophilized solid can be reconstituted with a pharmaceutically acceptable diluent (e.g., 0.9% saline or 5% glucose). The solution can be used directly or further diluted into an infusion bag (containing a pharmaceutically acceptable diluent, such as 0.9% saline or 5% glucose) before use.

[0779] (xii) Powder in bottle

[0780] The oral composition is prepared by placing the compound of formula (1) into a bottle or vial. It is then reconstituted with a suitable diluent, for example, water, juice, or a commercially available vehicle (such as OraSweet or Syrspend). The reconstituted solution can be dispensed into a measuring cup or oral syringe for administration.

Claims

1. (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide, having formula (1): or its tautomeric form, 50% to 100% in crystalline form.

2. (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 55% crystalline.

3. (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 60% crystalline.

4. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 70% crystalline.

5. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 80% crystalline.

6. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 90% crystalline.

7. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 95% crystalline.

8. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 98% crystalline.

9. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 99% crystalline.

10. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 99.5% crystalline.

11. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is at least 99.9% crystalline.

12. (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide according to claim 1, which is a hydrate.

13. (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 1, which is a monohydrate.

14. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide according to claim 13, wherein the X-ray powder diffraction pattern thereof is characterized in that main peaks appear at diffraction angles of 14.0°±0.2°, 20.6°±0.2°, 24.0°±0.2° and 24.2°±0.2°.

15. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide according to claim 13, wherein the X-ray powder diffraction pattern thereof is characterized in that the main peak diffraction angle and relative intensity are as shown in Table A:

16. (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 15, wherein the X-ray powder diffraction pattern is further characterized by the presence of one or more additional peaks at the diffraction angles given in Table B, and the relative intensities given in Table B:

17. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide according to claim 13, which has a peak at a diffraction angle substantially the same as the diffraction angle in the X-ray powder diffraction pattern shown in Figure 2 of the appended drawings of the specification.

18. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide according to claim 13, whose X-ray powder diffraction pattern is substantially as shown in Figure 2 of the accompanying drawing of the specification.

19. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 13, which exhibits an endothermic phenomenon when analyzed by differential scanning calorimetry, with an initial temperature of 100°C to 110°C.

20. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 19, which exhibits an endothermic phenomenon when analyzed by differential scanning calorimetry, with an initial temperature of 101°C to 108°C.

21. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 13, which exhibits an endothermic phenomenon when analyzed by differential scanning calorimetry and has a peak at a temperature between 110°C and 125°C.

22. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 21, which exhibits an endothermic phenomenon when analyzed by differential scanning calorimetry and has a peak at a temperature between 111°C and 113°C.

23. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 13, which loses weight at 85°C to 130°C during thermogravimetric analysis.

24. The (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to claim 23, which loses weight at 90°C-120°C during thermogravimetric analysis.

25. A method for preparing (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide as claimed in claim 1, the method comprising: (i) forming an aqueous suspension of the acid addition salt of (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide and stirring the suspension at a temperature of 25°C to 75°C for a period of time sufficient to disproportionate the acid addition salt to form a crystalline form of (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide and isolating the crystalline form; or (ii) forming an aqueous suspension of an amorphous form of (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide, wherein the aqueous suspension is unbuffered or buffered to a pH of 1.75 to 7.25, and stirring the aqueous suspension at a temperature of 25° C. to 55° C. for a period of time sufficient to allow the amorphous form of (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide to form an aqueous suspension of an amorphous form of (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)- -2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide is converted into a crystalline form of (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide, and then the crystalline form is isolated.

26. An amorphous (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide hydrochloride, sulfate, naphthalenedisulfonate, edisylate, toluenesulfonate, methanesulfonate, naphthalenesulfonate, benzenesulfonate, isethionate, ethanesulfonate or hydrobromide salt.

27. An amorphous (2R)-2-(6-{5-chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide hydrochloride, sulfate, hydrobromide or naphthalene disulfonate.

28. A method for preparing (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide, the method comprising reacting a compound of formula (2) with a compound of formula (3), The reaction is carried out in an aprotic solvent in the presence of a tertiary amine base and an amide bond promoter, wherein the amide bond promoter is selected from N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

29. The method of claim 28, wherein the tertiary amine base is diisopropylethylamine (DIPEA).

30. A method for preparing (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propionamide having the structure of formula (1), The method comprises: a) making a compound of formula (5) Reaction with a compound of formula (6), To obtain a compound of formula (2), and b) reacting the compound of formula (2) with the compound of formula (3), The compound of formula (1) is obtained and then optionally formed into a salt or crystalline form of the compound of formula (1).

31. A pharmaceutical composition comprising (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide and a medium selected from: -C 2-4 alcohol; - polyether compounds; -C8-C 18 Monoesters of long-chain fatty acids with glycerol or propylene glycol; -C8-C 18 di- or triglycerides of long-chain fatty acids; and mixtures thereof; and optionally a nonionic surfactant.

32. (2R)-2-(6-{5-chloro-2-[(oxacyclohexan-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide in particulate form having a mass median diameter of 1 μm to 100 μm.

33. Use of (2R)-2-(6-{5-chloro-2-[(oxacyclohexane-4-yl)amino]pyrimidin-4-yl}-1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl]propanamide according to any one of claims 1 to 24, an amorphous salt according to claim 26 or 27, a pharmaceutical composition according to claim 31, or a particle form according to claim 32 in the manufacture of a medicament for preventing or treating a disease state or condition requiring or upregulating ERK1 / 2 activity.

34. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from a cancer disease state.

35. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from the group consisting of hepatocellular carcinoma, melanoma, esophageal cancer, renal cancer, colorectal cancer, lung cancer, breast cancer, bladder cancer, gastrointestinal cancer, ovarian cancer, prostate cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer, cervical cancer, myometrial cancer, and endometrial cancer.

36. The use according to claim 35, wherein The colorectal cancer is selected from colon cancer, and / or the lung cancer is selected from mesothelioma and lung adenocarcinoma.

37. The use according to claim 34, wherein the compound or composition is used in combination with one or more other compounds or treatment methods.

38. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from hematological malignancies.

39. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from leukemia and lymphoma.

40. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from hematological malignancies and lymphatic system diseases.

41. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma and post-transplant lymphoproliferative disorder.

42. The use according to claim 41, wherein The B cell lymphoma is selected from diffuse large B cell lymphoma [DLBCL].

43. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from hematological malignancies and myeloid diseases.

44. The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disease, myeloproliferative syndrome, myelodysplastic syndrome and promyelocytic leukemia.

45. The use according to claim 44, wherein The myeloproliferative disorder is selected from the group consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis.

46. ​​The use according to claim 33, wherein The disease state or condition requiring or upregulating ERK1 / 2 activity is selected from adenoma and carcinoma.

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