Method for determining the purity of {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate

Abstract

To develop new processes for producing Givinostat in accordance with current quality standards and to identify new analytical techniques for determining the purity of Givinostat, such as to allow the identification of the presence of any impurities.SOLUTION: A process for obtaining {6-[(diethylamino)methyl]naphthalen-2-yl}methyl[4-(hydroxycarbamoyl)phenyl]carbamate and / or pharmaceutically acceptable salts thereof having high purity is described. This process makes it possible to obtain a product having an amount of any single unknown impurity equal to or less than 0.10%, as well as a product having a purity greater than 99.5%, preferably equal to or greater than 99.6%. An HPLC method for determining the purity of the product and possible impurities thereof is also described.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The object of the present invention is a method for obtaining {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof having high purity. Such a method makes it possible to obtain a product having an amount of any single unknown impurity of 0.10% or less, and a product having a purity of more than 99.5%, preferably 99.6% or more. A further object of the present invention is an HPLC method for determining the purity of the product and its possible impurities.

Background Art

[0002] Givinostat (registered trademark), also known by the name ITF2357 (IUPAC name {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate), is a hydroxamic acid and is used in the form of its hydrochloride salt, especially the hydrochloride monohydrate. It acts as an inhibitor of histone deacetylase (HDAC) and exerts its action on class I and II enzymes of the same name.

[0003] Givinostat shows a very promising activity profile in both in vitro and in vivo in multiple myeloma and acute myeloid leukemia, and also acts as an anti-inflammatory agent and as an inhibitor of tumor necrosis factor alpha (TNF-α), IL-1, and IL-6 secretion.

[0004] Givinostat is currently used in multiple phase III trials for inflammatory diseases (Duchenne and Becker muscular dystrophy, juvenile arthritis, and polycythemia vera), and in clinical trials for blood cancers (myeloma and lymphoma).

[0005] U.S. Patent No. 6,034,096 (US6,034,096, Patent Document 1) reports the preparation of gibinostat, while U.S. Patent No. 7,329,689 (US7,329,689, Patent Document 2) and International Publication No. 2004 / 065355 (WO2004 / 065355, Patent Document 3) detail the preparation and characterization of one of its polymorphic monohydrates.

[0006] U.S. Patent No. 8,518,988 (US8,518,988, Patent Document 4) describes the preparation and characterization of the anhydrous polymorph of gibinostat.

[0007] The method for preparing gibinostat described in the cited literature, according to Scheme 1 below, first defines the synthesis of acyl chloride (II) from intermediate (I) (Step 1), and this acyl chloride is then added as a solid moistened with THF to an aqueous and THF solution of hydroxylamine to yield the final product (Step 2).

[0008]

Chemical formula

[0009] The literature cited above does not describe any impurities from the synthesis.

[0010] The paper (A. Furlan, V. Monzani, L.L. Reznikov, F. Leoni, G. Fossati. D. Modena, P. Mascagni, C. A. Dinarello, Mol. Med. 2011, 17 (5-6) 353-362 (Non-Patent Document 1)) elucidates that intermediate (I) (ITF2375) and the corresponding amide (ITF2374) are the two main in vivo metabolites of gibinostat (ITF2357), and these are respectively derived from the in vivo conversion of the hydroxamic group to carboxylic acid and amide.

[0011] Amide (ITF2374) has the following formula (Ia).

[0012]

Chemical formula

[0013] Therefore, the presence of intermediate (I) or amide (Ia) in gibinostat in an amount exceeding 0.15% is considered reasonable, and these are regarded as impurities.

[0014] Based on the current guidelines in the pharmaceutical field that provide a detailed description of the impurity profile of an active ingredient intended for human use, the development of a new manufacturing method for gibinostat according to current quality standards such that the presence of any impurity can be identified, and the identification of new analytical techniques for determining the purity of gibinostat are fundamentally important.

Prior art documents

Patent documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-patent documents

[0016]

Non-patent Document 1

Non-patent Document 2

Non-Patent Document 3

Summary of the Invention

[0017] (Definition) Unless otherwise defined, all terms, notations, and other scientific and technical terms used in this specification are intended to have the meanings commonly understood by those skilled in the art to which this disclosure pertains. In some cases, terms having commonly understood meanings are defined herein for the sake of clarity and / or to facilitate reference. Therefore, it should not be understood that including such definitions herein represents a substantial difference beyond what is commonly understood in the art.

[0018] As used herein, the term "physiologically acceptable excipient" refers to a substance that has no significant pharmaceutical effect of its own and does not cause harmful reactions when administered to mammals, preferably humans. Physiologically acceptable excipients are well known in the art and are disclosed, for example, in the Handbook of Pharmaceutical Excipients, sixth edition 2009 (Non-Patent Document 2), which is incorporated herein by reference.

[0019] The term "pharmaceutically acceptable salt" refers to those salts which have the biological efficacy and properties of the compound being salted and which do not produce any harmful reactions when administered to mammals, preferably humans. Pharmaceutically acceptable salts can be inorganic or organic salts. Examples of pharmaceutically acceptable salts include, but are not limited to, carbonates, hydrochlorides, bromates, sulfates, bisulfates, citrates, maleates, fumarates, trifluoroacetates, 2-naphthalenesulfonates, and para-toluenesulfonates. More information on pharmaceutically acceptable salts can be found in Handbook of pharmaceutical salts, P. Stahl, C. Wermuth, WILEY-VCH, 127-133, 2008 (Non-Patent Document 3), which is hereby incorporated by reference into this specification.

[0020] The terms "comprising", "having", "including", and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), and it should be considered to provide support for terms such as "consist essentially of", "consisting essentially of", "consist of", or "consisting of".

[0021] The terms "consist essentially of" and "consisting essentially of" should be construed as semi-closed terms, meaning that no other components that significantly affect the basic and novel features of the present invention are included (thus, optional excipients may be included).

[0022] The term "consist of" or "consisting of" should be interpreted as a closed term.

[0023] The term "having a high purity" refers to a purity exceeding 99.5%, preferably 99.6% or more.

[0024] Within the scope of this description, the term Gibinostat or ITF2357 is intended to refer to the hydrochloride salt of {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate, particularly the hydrochloride monohydrate (CAS number 732302-99-7). The hydrochloride salt alternatively has a CAS number of 199657-29-9, and the free base has a CAS number of 497833-27-9.

[0025] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0026] The term "acid-insensitive aprotic solvent" refers to a solvent that does not contain components sensitive to acids. THF is an example of an acid-sensitive solvent. DMSO, acetonitrile, dimethylacetamide, or dimethylformamide are understood to be examples of solvents that are not acid-sensitive.

[0027] The term "unknown impurity" refers to any unknown impurity present in {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or its pharmaceutically acceptable salts.

[0028] (Brief Description of the Invention) Referring to the methods for preparing Gibinostat used in the state of the art, the inventors observed that these methods result in Gibinostat having a single unknown impurity content exceeding 0.10%.

[0029] Accordingly, the applicant of the present application faced the problem of developing a new method for preparing gibinostat having any single unknown impurity of 0.10% or less (area% of HPLC chromatogram). Here, this content is the level expected by the ICH for unknown impurities when the daily dose of the active substance is less than 2 g in the case of gibinostat.

[0030] Surprisingly, in step 1 (Scheme 1), it was found to be important to carry out the halogenation reaction of intermediate (I) in a solvent containing no acid-sensitive components, reduce the equivalent amount of the halogenating agent required to complete the desired reaction, and at the same time obtain intermediate (II) having an impurity amount lower than that obtained by applying known techniques.

[0031] Similarly, surprisingly, the inventors found that in step 2 (Scheme 1), the order of addition of intermediate (II) and hydroxylamine is important for obtaining a final product having a single unknown impurity of 0.10% or less based on the area of the chromatogram of relative purity analysis.

[0032] Accordingly, in a first aspect, the present invention relates to a method for preparing {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof having any unknown single impurity in an amount of 0.10% or less and / or high purity.

[0033] In a preferred embodiment, the method of the present invention enables to obtain {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof having a purity of more than 99.5%, preferably 99.6% or more.

[0034] According to the second aspect, the present invention relates to {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, preferably hydrochloride, more preferably hydrochloride monohydrate, which has any unknown single impurity in an amount of 0.10% or less, or any single impurity other than intermediate (I) or amide (Ia) in an amount of 0.15% or less, preferably 0.10% or less.

[0035] According to the third aspect, the present invention relates to {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, preferably hydrochloride, more preferably hydrochloride monohydrate, which has a purity of more than 99.5%, preferably 99.6% or more.

[0036] According to the fourth aspect, the present invention relates to a new HPLC analysis method for determining the purity of {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof and detecting its impurities.

[0037] According to the fifth aspect, the present invention relates to {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, preferably hydrochloride, more preferably hydrochloride monohydrate, which has an unknown impurity at an RRT of 0.93±0.02 and / or an unknown impurity at an RRT of 1.21±0.02 and / or an unknown impurity at an RRT of 1.51±0.02 and / or an unknown impurity at an RRT of 1.75±0.02 in an amount of 0.10% or less, where the RRT is measured using the HPLC method according to the present invention.

[0038] According to the sixth aspect, the present invention relates to a method for preparing {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, which method comprises an HPLC method for determining the purity according to the present invention.

Brief Description of the Drawings

[0039]

Figure 1

Embodiments for Carrying Out the Invention

[0040] (Detailed Description of the Invention) The object of the present invention is a method for preparing {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof having any unknown single impurity in an amount of 0.10% or less, said method comprising: i) preparing a solution or suspension of a compound of formula (II) in an organic solvent;

[0041]

Chemical Formula

[0042] (wherein X is a halogen, preferably chlorine.) ii) adding hydroxylamine to the solution or suspension obtained in step i); and the method comprises.

[0043] Within the scope of this description, steps i) and ii) are designated as step 2 according to the method of the present invention.

[0044] In a preferred embodiment, the organic solvent in step i) of step 2 is selected from the group consisting of THF, methyl-THF, dioxane, ethylene glycol dimethyl ether, and bis(2-methoxyethyl) ether.

[0045] Preferably, the organic solvent is used in an amount containing 1 to 100 parts by volume per part by weight of the compound of formula (II).

[0046] Preferably, the organic solvent has a water content of less than 0.5%.

[0047] Preferably, step ii) of step 2 of the method according to the present invention is carried out at room temperature.

[0048] In a preferred embodiment, the method of the present invention (step 2) further comprises step iii) of isolating {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate as the free salt or as a pharmaceutically acceptable salt, preferably the hydrochloride salt, more preferably the hydrochloride salt monohydrate.

[0049] In a more preferred embodiment, the compound of formula (II) is obtained from the corresponding acid of formula (I) by reaction with a halogenating agent, preferably a chlorinating agent (step a) in an aprotic dipolar solvent that is not acid-sensitive.

[0050] Preferably, the compound of formula (II) is isolated from the reaction mixture by precipitation with an organic solvent, preferably subsequent filtration (step b).

[0051] Within the scope of this description, steps a) and b) are designated as step 1 according to the method of the present invention.

[0052] A solvent that is not acid-sensitive is a solvent that does not contain acid-sensitive components (thus, for example, does not contain THF). This characteristic of the solvent makes it possible to use a reduced amount of the halogenating agent as compared to the prior art.

[0053] As an example, THF is a representative of solvents sensitive to acids. This is because under acidic conditions, THF can decompose and generate reactive species that can form unknown by-products in the halogenation reaction.

[0054] Preferably, in step a) of step 1, the aprotic bipolar solvent that is not sensitive to acids is selected from DMSO, acetonitrile, dimethylacetamide, or dimethylformamide, more preferably dimethylformamide.

[0055] Examples of chlorinating agents that can be used in the method of the present invention are thionyl chloride (SOCl2), phosphorus trichloride (PCl3), phosphorus oxychloride (POCl3), or phosphorus pentachloride (PCl5). In addition, the corresponding brominating agents, SOBr2, PBr3, POBr3, or PBr5 can be used.

[0056] In a preferred embodiment of step b) of step 1, the organic solvent used to precipitate compound (II) is selected from aliphatic or aromatic hydrocarbons, ethers, esters, or alcohols, more preferably toluene or THF.

[0057] A further object of the present invention is {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate or a pharmaceutically acceptable salt thereof having an amount of any single unknown impurity of 0.10% or less obtained by applying only step 2 defined above with respect to known techniques.

[0058] In a more preferred embodiment, the present invention relates to {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof having a purity of more than 99.5%, preferably 95.6% or more, by applying both step 1 and step 2 defined above with respect to known techniques.

[0059] A further object of the present invention is {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, more preferably the hydrochloride monohydrate, which has any unknown single impurity in an amount of 0.10% or less, or any single impurity other than intermediate (I) or amide (Ia) in an amount of 0.15% or less, preferably 0.10% or less.

[0060] A further object of the present invention is {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, more preferably the hydrochloride monohydrate, which has a purity of more than 99.5%, preferably 99.6% or more.

[0061] A further object of the present invention is {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, more preferably the hydrochloride monohydrate, which has an unknown impurity at an RRT of 0.93 ± 0.02 and / or an unknown impurity at an RRT of 1.21 ± 0.02 and / or an unknown impurity at an RRT of 1.51 ± 0.02 and / or an unknown impurity at an RRT of 1.75 ± 0.02 in an amount of 0.10% or less, and the RRT is measured using the HPLC method according to the present invention.

[0062] In a preferred form of jealousy, the RRT is measured using the following HPLC method. - Stationary phase: A support based on silica particles containing a C18 alkyl chain and having a carbon load of less than 9 wt%. Mobile phase A: Water buffered to pH 3.7 - 3.8 Mobile phase B: Methanol buffered to pH 3.7 - 3.8 The following gradient elution method is used

[0063] [Table 1]

[0064] Preferably, an ammonium formate-formic acid buffer solution with a pH of 3.7 to 3.8 was used. Preferably, a UV detector with a wavelength of 263 nm was used. Preferably, the column temperature is 25 ± 1 °C. Preferably, the injection volume is 5 μL. Preferably, the flow rate is 0.25 mL / min. Preferably, the product sample is diluted with DMSO.

[0065] A further object of the present invention is a pharmaceutical composition containing {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0066] In a preferred embodiment of the pharmaceutical composition, the active ingredient is in the form of micronized particles having an average size of less than 200 μm, preferably 100 μm to 1 μm, more preferably 50 μm to 5 μm.

[0067] A further object of the present invention is also a method for determining the purity of the product {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, which comprises eluting the product through an HPLC column having a stationary phase containing an alkyl chain bonded to an inorganic support, such as silica, and subsequently detecting the same product and its impurities using a detector suitable for measuring the amount of the analyte eluting from the column, such as a UV, MS or RID type detector.

[0068] In a preferred embodiment of the method according to the invention, the alkyl chain is of the octadecyl, octyl or butyl (C18, C8 or C4) type, preferably C18. In an even more preferred embodiment, the stationary phase consists of a silica support derivatized with a C18 alkyl chain having a carbon load of less than 9% by weight.

[0069] The carbon load means the carbon content in weight % of the stationary phase bonded to the silica. A high carbon load (15 - 25%) results in a more hydrophobic stationary phase surface and can retain the most hydrophobic impurities, making its correct quantitative evaluation impossible.

[0070] Some columns of this type are commercially available, for example ACE5 C18 - 300, Halo C18 (solid core), YMC - Pack OSD - A, BioBasic - 18 PEEK. In a preferred embodiment, column Halo C18 - 90Å (code 95812 - 902 manufactured by Advanced Materials Technology) is used.

[0071] Different eluents selected from water, polar organic solvents, or mixtures thereof can be used with the combinations of stationary phases and detectors described above. The polar organic solvent is a C1 - C4 alcohol, preferably methanol, or acetonitrile.

[0072] Preferably, in the HPLC method of the present invention, mixtures of water and methanol, water and acetonitrile can be used, optionally with or without a gradient of elution, and optionally with or without a buffer. Preferably, the above mixtures can be used with the addition of a buffer. More preferably, an ammonium formate - formic acid buffer with a pH of 3.7 - 3.8 is used.

[0073] In a preferred embodiment, the execution of the chromatograph according to the following scheme uses the following elution method.

[0074]

Table 2

[0075] In this elution method, eluate A means water and an ammonium formate-formic acid buffer solution with a pH of 3.7 to 3.8, and eluate B means methanol and an ammonium formate-formic acid buffer solution with a pH of 3.7 to 3.8.

[0076] A further object of the present invention is also a method for preparing {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, which includes a method for determining the purity according to the present invention.

[0077] The examples reported in the following examples column should be considered as examples of the methods of the present invention and should not be regarded as limiting the scope of the effectiveness of the present invention itself.

Examples

[0078] (Part of the examples) Example 1 (Comparative example) Synthesis of intermediate (II) according to the prior art 73 g (0.1796 mol) of intermediate (I) and 1.12 L of dry THF are filled into a 2 L reaction vessel that has been pre-dried and is under an inert atmosphere. The resulting suspension is stirred at room temperature for 30 minutes. The internal temperature is lowered to 3 - 5 °C, 64 g of thionyl chloride (0.538 mol) is added over 5 minutes or more, then the reaction mixture is heated to the reflux temperature and held with stirring for about 1 hour. 2 g of dry DMF is added and the mixture is stirred at the same temperature for an additional 4 hours. Next, the reaction mixture is concentrated under reduced pressure until a residual volume of about 0.3 L is reached. Then, 0.35 L of toluene is filled in and the mixture is concentrated again. This operation is repeated once more. After cooling the mixture to room temperature, dry THF (0.7 L) is added and the mixture is stirred for about 30 minutes. Next, the white precipitate of intermediate (II) is filtered and washed with dry THF. The wet product (107 g) is stored at 5 °C and used as it is.

[0079] Example 2 (Comparative Example) Synthesis of Gibinostat by the Method of the Prior Art (Experiment TT180) 125 mL of dry THF and 60 mL of water are pre-dried and charged into a 1 L reaction vessel under an inert atmosphere. Aqueous hydroxylamine (21 g, 50% w / w) is added to this solution, and the resulting mixture is cooled to 5 °C. 19 g of wet Intermediate (II) from Example 1 (corresponding to 13 g of Intermediate (I) of the starting material) is charged all at once, and the internal temperature is gradually raised to 20 ± 3 °C. Progressive dissolution is observed, and the resulting mixture is maintained under stirring for 30 - 40 minutes. Then, water (55 mL) is added, and then 6N HCl aqueous solution (about 60 g) is slowly added until pH < 2 is reached while maintaining the internal temperature at 20 ± 3 °C. Then, THF is removed under reduced pressure until the total volume is approximately halved, and a white precipitate is observed. The mixture is cooled to 10 °C and maintained at this temperature under stirring for 30 minutes. The product is isolated by vacuum filtration and washed with water. 16.4 g of wet crude Gibinostat is obtained.

[0080] The crude Gibinostat is suspended in 285 mL of an aqueous solution of NaHCO3 (6.5 g) at 20 ± 3 °C, then THF (265 mL) is added, and progressive dissolution is observed. The resulting solution is stirred for 30 minutes, and then ethyl acetate (132 mL) is added. After 15 minutes, stirring is stopped and the two phases are separated. The aqueous phase is discarded, and the organic phase is treated with 37% HCl with vigorous stirring until pH < 2 is reached. The resulting suspension is stirred for 30 minutes, and then the precipitate is isolated by vacuum filtration and washed with THF. 17 g of wet pure Gibinostat is obtained.

[0081] The product is dried in an oven at 30 °C under reduced pressure for 16 hours. 7.2 g of dry pure Gibinostat is obtained. The purity of this Gibinostat is 98.8%, indicating the presence of 0.23% of unknown impurities.

[0082] Example 3 Synthesis of Gibinostat according to the conventional method for Step 1 and according to the method of the present invention (Experiment TT177) for Step 2 Pre-dry and place 29.3 g of the wet intermediate (II) from Example 1 (corresponding to 20 g of the starting material intermediate (I)) in a 1 L reaction vessel under an inert atmosphere, suspend it in 192 mL of dry THF, and adjust the internal temperature to 20 ± 3 °C. Add aqueous hydroxylamine (32 g, 50% w / w) all at once and maintain the mixture under stirring for 30 - 40 minutes. Then add water (176 mL), and a gradual dissolution of the precipitate is observed. Slowly add 6N HCl aqueous solution (about 93 g) while maintaining the internal temperature at 20 ± 3 °C until the pH reaches < 2. Then remove THF under reduced pressure until the total volume is approximately halved, and a white precipitate is observed. Cool the mixture to 10 °C and maintain this temperature for 30 minutes under stirring. Isolate the product by vacuum filtration and wash it with water. 29 g of wet crude gibinostat is obtained.

[0083] Suspend the wet crude gibinostat in an aqueous solution of 408 mL of NaHCO3 (10 g) at 20 ± 3 °C, then add THF (408 mL), and a gradual dissolution is observed. Stir the resulting solution for 30 minutes, then add ethyl acetate (204 mL). After 15 minutes, stop stirring and separate the two phases. Discard the aqueous phase and treat the organic phase with 37% HCl while stirring vigorously until the pH reaches < 2. Stir the resulting suspension for 30 minutes, then isolate the precipitate by vacuum filtration and wash it with THF. 31 g of wet pure gibinostat is obtained.

[0084] Dry the product in an oven at 30 °C under reduced pressure for 16 hours. 15.6 g of dry pure gibinostat is obtained. HPLC purity analysis shows that the product contains no unknown impurities in an amount exceeding 0.10%.

[0085] Example 4 Synthesis of gibinostat following the conventional method for Step 1 and the method of the present invention (Experiment TT183) for Step 2 Pre-dry and place 29.3 g of the wet intermediate (II) from Example 1 (corresponding to 20 g of the starting material intermediate (I)) in a 1 L reaction vessel under an inert atmosphere, suspend it in 192 mL of dry THF, and adjust the internal temperature to 20 ± 3 °C. Add aqueous hydroxylamine (32 g, 50% w / w) by pouring it over a period of more than 15 minutes, and maintain the mixture under stirring for 30 - 40 minutes. Then, add water (176 mL), and a gradual dissolution of the precipitate is observed. Slowly add 6N HCl aqueous solution (about 91 g) while maintaining the internal temperature at 20 ± 3 °C until the pH reaches <2. Then, remove THF under reduced pressure until the total volume is approximately halved, and a white precipitate is observed. Cool the mixture to 10 °C and maintain this temperature for 30 minutes under stirring. Isolate the product by vacuum filtration and wash it with water. 36 g of wet crude gibinostat is obtained.

[0086] Suspend the wet crude gibinostat in 408 mL of an aqueous solution of NaHCO3 (10 g) at 20 ± 3 °C, then add THF (408 mL), and a gradual dissolution is observed. Stir the resulting solution for 30 minutes, then add ethyl acetate (204 mL). After 15 minutes, stop stirring and separate the two phases. Discard the aqueous phase, and treat the organic phase with 37% HCl while stirring vigorously until the pH reaches <2. Stir the resulting suspension for 30 minutes, then isolate the precipitate by vacuum filtration and wash it with THF. 27 g of wet pure gibinostat is obtained.

[0087] Dry the product in an oven at 30 °C under reduced pressure for 16 hours. 15.4 g of dry pure gibinostat is obtained. HPLC purity analysis shows that the product contains no unknown impurities in an amount exceeding 0.10%.

[0088] Example 5 Synthesis of gibinostat on an industrial scale, following the conventional method for Step 1 and the method of the present invention for Step 2 Pre-dried, 50.0 kg of wet intermediate (II) (corresponding to 40 kg of intermediate (I) as starting material) is suspended in 400 L of dry THF in a 2000 L reaction vessel under an inert atmosphere, and the internal temperature is set to 20 ± 3 °C. Aqueous hydroxylamine (67.2 kg, 50% w / w) is added by pouring it over a period of 15 minutes or more, and the mixture is maintained under stirring for 30 - 40 minutes. Then, water (447 L) is added, and a gradual dissolution of the precipitate is observed. 6N HCl aqueous solution (about 170 kg) is slowly added while maintaining the internal temperature at 20 ± 3 °C until the pH reaches <2. Then, THF is removed under reduced pressure until the total volume is approximately halved, and a white precipitate is observed. The mixture is cooled to 10 °C and maintained at this temperature for 30 minutes under stirring. The product is isolated by vacuum filtration and washed with water. 60 kg of wet crude gibinostat is obtained.

[0089] The wet crude gibinostat is suspended in a solution consisting of 860 L of an aqueous solution of NaHCO3 (21 kg) and 860 L of THF at 20 ± 3 °C, and a gradual dissolution is observed. The resulting solution is stirred for 30 minutes, and then ethyl acetate (440 L) is added. After 15 minutes, stirring is stopped and the two phases are separated. The aqueous phase is discarded, and the organic phase is treated with 37% HCl while stirring vigorously until the pH reaches <2. The resulting suspension is stirred for 30 minutes, and then the precipitate is isolated by vacuum filtration and washed with THF. 58 kg of wet pure gibinostat is obtained.

[0090] The product is dried in an oven at 30 °C under reduced pressure for 16 hours. 35 kg of dry pure gibinostat is obtained. HPLC purity analysis shows that the product contains no unknown impurities in an amount exceeding 0.10% and has an overall purity exceeding 99.5%.

[0091] Example 6 (Experiment TT259) Synthesis of gibinostat by the method of the present invention (Step 1 + Step 2) Step 1: Synthesis of intermediate (II) 100 g (0.2460 mol) of Intermediate (I) and 300 mL of dimethylformamide are charged into a pre-dried 3 L reaction vessel under an inert atmosphere. The suspension thus obtained is stirred at 20 - 25 °C for 30 minutes. 40 g (0.3362 mol) of thionyl chloride is added, a slight exotherm is observed, and it is held to maintain the reaction mixture at 20 - 25 °C. The resulting suspension is held at 20 - 25 °C for a further 2 hours and then two vacuum cycles are carried out, the pressure is returned to nitrogen to remove the gas generated by the reaction. Next, 2000 mL of THF is added and the mixture is maintained at 20 - 25 °C for 1 hour. The suspension is then filtered on a Buchner and washed with 300 mL of THF. The wet product (133 g) is stored at 5 °C and used as is.

[0092] Step 2: Synthesis of Gibinostat 133 g of Intermediate (II) from Step 1 and 960 mL of THF are charged into a pre-dried 3 L reaction vessel under an inert atmosphere. The resulting suspension is stirred and thermostatted at 12 - 18 °C. 160 g (2.424 mol) of 50% w / w aqueous hydroxylamine solution is added to this suspension (the reaction is exothermic and the temperature of the mixture reaches from 15 °C to 28 °C). Next, it is thermostatted at 17 - 23 °C and held at these conditions for 40 minutes (40’).

[0093] Next, while storing at the same temperature, 1100 mL of deionized water is added and a progressive dissolution of the precipitate is observed. At the end point, 220 g of 15% w / w HCl aqueous solution is added until the pH of the mixture reaches 1.2 - 1.8. The mixture is held at 17 - 23 °C with stirring for 30 minutes and then concentrated under reduced pressure to a residual volume of approximately 1400 mL while maintaining the internal temperature at 25 °C. The pressure is then returned and 1000 mL of deionized water is charged. Next, the mixture is cooled to 7 - 13 °C and held with stirring for 1 hour. The suspension is filtered by washing with 400 mL of deionized water acidified with 1.2 g of 37% HCl.

[0094] Return the wet filtrate to a reaction vessel containing 5 kg of sodium bicarbonate, 1000 mL of deionized water and 1000 mL of THF. Stir the mixture, heat it to 47 - 53 °C and hold the mixture under these conditions for 3 hours. Next, cool the mixture to 17 - 23 °C to allow the phases to settle. Re-extract the separated aqueous phase with 500 mL of ethyl acetate. Next, combine the organic extracts, add 200 mL of 37% HCl with vigorous stirring and observe the precipitation of the product. Hold the mixture under stirring for 30 minutes and then filter it, washing the panel with 400 mL of THF.

[0095] Return the wet filtrate to a reaction vessel containing 5 kg of sodium bicarbonate, 1000 mL of deionized water and 1000 mL of THF. Stir the mixture at 17 - 23 °C and hold it under these conditions for 30 minutes. Next, add 500 mL of ethyl acetate to the mixture at 17 - 23 °C and stir for 15 minutes. Allow the phases to settle and filter the separated organic phase through a 10 - micron microfilter. Then, wash the reaction vessel and lines with a mixture of 120 mL of THF and 60 mL of ethyl acetate, add 200 mL of 37% HCl to the combined organic phase and observe the precipitation of the product. Hold the mixture under stirring for 30 minutes and then filter it, washing the panel with 400 mL of THF. Remove the product (157 g) and dry it under vacuum (<50 mbar) at 25 - 35 °C for 15 hours. Obtain 107 g of the final product.

[0096] Example 7 (Experiment TT267) Synthesis of Gibinostat by the method of the present invention (Step 1 + Step 2) The preparation of intermediate (II) and then its conversion to Gibinostat was repeated as described in Example 6. 105 g of product was obtained at the end point of this method.

[0097] Example 8 (Experiment TT287) Synthesis of Gibinostat by the method of the present invention (Step 1 + Step 2) 100 g (0.2460 mol) of intermediate (I) and 300 mL of dimethylformamide are charged into a pre-dried 3 L reaction vessel under an inert atmosphere. The resulting suspension is stirred at 20 - 25 °C for 30 minutes. Then, 40 g (0.3362 mol) of thionyl chloride is added, and a slight exotherm is observed. This is held to maintain the reaction mixture at 20 - 25 °C. The resulting suspension is held at 20 - 25 °C for a further 2 hours, then two vacuum cycles are performed, the pressure is returned to nitrogen, and the gas generated by the reaction is removed. Next, 2000 mL of toluene is added, and the mixture is maintained at 20 - 25 °C for 1 hour. The suspension is then filtered on a Buchner and washed with 300 mL of toluene. The wet product (133 g) is stored at 5 °C and used as is.

[0098] 133 g of intermediate (II) from the described preparation and 960 mL of THF are charged into a pre-dried 3 L reaction vessel under an inert atmosphere. The resulting suspension is stirred and thermostatted at 12 - 18 °C. 160 g (2.424 mol) of 50% w / w aqueous hydroxylamine solution is added to this suspension (the reaction is exothermic and the temperature of the mixture reaches from 15 °C to 28 °C). Next, this is thermostatted at 17 - 23 °C and held at these conditions for 40 minutes (40’).

[0099] Next, it is held at the same temperature, and 1100 mL of deionized water is added while maintaining the temperature at 17 - 23 °C, and a progressive dissolution of the precipitate is observed. At the end point, 220 g of 15% w / w HCl aqueous solution is added until the pH of the mixture reaches 1.2 - 1.8. The mixture is held at 17 - 23 °C with stirring for 30 minutes, then the mixture is concentrated under reduced pressure while maintaining the internal temperature at 25 °C until it reaches a residual volume of approximately 1400 mL. Then, the pressure is returned and 1000 mL of deionized water is charged. Next, the mixture is cooled to 7 - 13 °C and held with stirring for 1 hour. The suspension is filtered by washing with 400 mL of deionized water acidified with 1.2 g of 37% HCl.

[0100] Return the wet filtrate to a reaction vessel containing 5 kg of sodium bicarbonate, 1000 mL of deionized water and 1000 mL of THF. Stir the mixture, heat it to 47 - 53 °C and maintain these conditions for 3 hours. Next, cool the mixture to 17 - 23 °C and allow the phases to settle. Re-extract the separated aqueous phase with 500 mL of ethyl acetate. Next, combine the organic extracts, add 200 mL of 37% HCl to the extract under vigorous stirring and observe the precipitation of the product. Hold the mixture under stirring for 30 minutes, then filter it and wash the panel with 400 mL of THF.

[0101] Return the wet filtrate to a reaction vessel containing 5 kg of sodium bicarbonate, 1000 mL of deionized water and 1000 mL of THF. Stir the mixture at 17 - 23 °C and maintain these conditions for 30 minutes. Next, add 500 mL of ethyl acetate to the mixture at 17 - 23 °C and stir for 15 minutes. Allow the phases to settle and filter the separated organic phase through a 10 - micron microfilter. Then, wash the reaction vessel and lines with a mixture of 120 mL of THF and 60 mL of ethyl acetate, add 200 mL of 37% HCl to the combined organic phase and observe the precipitation of the product. Hold the mixture under stirring for 30 minutes, then filter the panel and wash it with 400 mL of THF. Remove the product (157 g) and dry it under vacuum (<50 mbar) at 25 - 35 °C for 15 hours. Obtain 107 g of the final product.

[0102] Example 9 HPLC method for determining the purity and impurities of gibinostat

[0103] [Table 3]

[0104] [Table 4]

[0105] Sample preparation Accurately weigh about 20 mg of the ITF2357 sample, transfer this to a 100 mL flask, and fill to volume with DMSO (concentration 0.20 mg / mL).

[0106] Figure 1 shows a typical chromatogram obtained by the HPLC method according to the present invention for a specially prepared mixture containing all typical impurities of the product.

[0107] Example 11 Purity analysis of gibinostat obtained by the method of the state of the art (Example 2), gibinostat obtained by a conventionally known method for step 1 and by the method of the present invention for step 2 (Examples 3 and 4), and gibinostat obtained by the method of the present invention for both step 1 and step 2

[0108] [Table 5]

[0109] Table 1 shows the relative retention times of gibinostat (RRT1), amide metabolite (1a) (RRT1.08), intermediate (I) (RRT1.27) and unknown impurities, evaluated in particular by the HPLC analysis described in Example 9.

[0110] Experiment TT180 (Example 2) relates to the repetition of the method used in the state of the art, while TT177 (Example 3) and TT183 (Example 4) are experiments in which the method of the present invention was applied with respect to step 2, and relate to the experiment of adding 50% aqueous hydroxylamine to a solution of intermediate (II) prepared according to known techniques dissolved in THF.

[0111] Compared to the standard order (adding intermediate (II) to hydroxylamine), the reverse addition order (adding hydroxylamine to intermediate (II)) makes it possible to obtain a reduction of all unknown impurities of less than 0.10% based on the final reaction product. In contrast, using the standard method results in one impurity well exceeding the limit of 0.10%.

[0112] An even more important aspect of limiting the formation of impurities is the amount of water contained in the mixture containing intermediate (II) to which hydroxylamine is added, which must be limited to within 0.5% based on the weight of the mixture. On the other hand, the addition time of hydroxylamine is not restricted, but unlike the methods of the state of the art, the method of the present invention can be operated at room temperature rather than at temperatures above 5 °C.

[0113] In Examples 6, 7 and 8, the novel method of the present invention is applied to both Step 1 and Step 2. It will be apparent from the values reported in the table that gibinostat having a purity exceeding 99.5%, preferably a purity of 99.6% or more, and no unknown impurities being detectable at all (detection limit of the method 0.02%) is obtained.

Claims

1. A method for determining the purity of the product {6 - [(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, comprising eluting the product through an HPLC column having a stationary phase containing an alkyl chain, and subsequently detecting the product itself and its impurities by a detector.

2. The method according to claim 1, wherein the detector is of the UV, MS or RID type.

3. The method according to claim 1, wherein the alkyl chain is of the octadecyl, octyl, or butyl (C18, C8 or C4) type.

4. The method according to claim 3, wherein the alkyl chain is C18.

5. The method according to claim 1 or 3, wherein the stationary phase consists of particles of a support material containing an alkyl chain.

6. The method according to claim 5, wherein the alkyl chain is C18, C8 or C4.

7. The method according to claim 5, wherein the particles of the support material are silica.

8. The method according to any one of claims 1 to 7, wherein the carbon loading of the stationary phase is less than 9% by weight.

9. The method according to any one of claims 1 to 8, wherein the eluent phase consists of an aqueous solution, a polar organic solvent, or a mixture thereof.

10. The method according to claim 9, wherein the eluent phase is added with a buffer.

11. The polar organic solvent is C 1 -C 4 The method according to claim 9, characterized in that it is alcohol or acetonitrile.

12. The method according to claim 11, wherein the C1-C4 alcohol is methanol.

13. The method has the following operating conditions: - Stationary phase: a support based on silica particles containing a C18 alkyl chain and having a carbon loading of less than 9% by weight; Mobile phase A: water buffered to pH 3.7 - 3.8 Mobile phase B: methanol buffered to pH 3.7 - 3.8 and is carried out using the following gradient elution method 【Table 1】 The method according to claim 11, characterized in that it is carried out according to.

14. {6-[(Diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof, having an unknown impurity at an RRT of 0.93 ± 0.02 and / or an unknown impurity at an RRT of 1.21 ± 0.02 and / or an unknown impurity at an RRT of 1.51 ± 0.02 and / or an unknown impurity at an RRT of 1.75 ± 0.02 in an amount of 0.10% or less, wherein the RRT is measured using the HPLC method according to claim 13.

15. The {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof according to claim 14, wherein the pharmaceutically acceptable salt is a hydrochloride.

16. The {6-[(diethylamino)methyl]naphthalen-2-yl}methyl [4-(hydroxycarbamoyl)phenyl]carbamate and / or a pharmaceutically acceptable salt thereof according to claim 14, wherein the pharmaceutically acceptable salt is a hydrochloride monohydrate.

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