Urodesine salts

Abstract

The present disclosure particularly relates to a novel glutarate salt form of the compound known as Urodesine or 7-[(3R,4R)-3-hydroxy-4-hydroxymethyl-pyrrolidin-1-ylmethyl]-3,5-dihydro-pyrrolo[3,2-d]pyrimidin-4-one, and methods for preparing same.

Description

[Technical field]

[0001] The present disclosure particularly relates to a novel glutarate salt form of the compound known as Urodesine or 7-[(3R,4R)-3-hydroxy-4-hydroxymethyl-pyrrolidin-1-ylmethyl]-3,5-dihydro-pyrrolo[3,2-d]pyrimidin-4-one, and methods for preparing same. [Background technology]

[0002] Structurally:

[0003] [ka]

[0004] The pharmaceutical compound known as urodesine or 7-[(3R,4R)-3-hydroxy-4-hydroxymethyl-pyrrolidin-1-ylmethyl]-3,5-dihydro-pyrrolo[3,2-d]pyrimidin-4-one inhibits several relevant enzymes, including purine nucleoside phosphorylase (PNP), that are involved in human diseases.

[0005] Urodesine has been developed for the treatment of several human diseases, including, but not limited to, gout, skin disorders, cancer, B-cell and T-cell mediated diseases, bacterial infections and protozoal infections. The use of Urodesine is also described, for example, in U.S. Patent No. 7,553,839.

[0006] Furthermore, pharmaceutical salts of the compound Urodesine are well known in the literature. This includes, but is not limited to, hydrochloride, dihydrochloride, hydrobromide, hemisulfate, p-tosylate, phosphate, citrate, L-tartrate, L-lactate, stearate, maleate, succinate, fumarate and L-malate. Additionally, hemi- and mono-salts of the compounds with C4 organic diacids can include succinic acid, fumaric acid, L-malic acid, maleic acid, L-tartaric acid, L-aspartic acid, and are exemplified in the art.

[0007] Although several salts of Urodesine have been described, many of its salt forms show properties that are not optimal for the method of production and / or application in medicine.For example, its hydrochloride or other salts have been shown to contain polymorphic variants.It may be desirable to obtain a salt of a pharmaceutical compound that is free of or has a small number of polymorphic variants and is stable in crystalline form.

[0008] Mixed salts may also offer the possibility of different physical properties than those of the unmixed salt alone and may therefore be useful in the manufacture of drug products whose suitability for use depends on the properties of the active pharmaceutical ingredient. Like unmixed salts, mixed salts often exhibit polymorphic forms, some of which are unstable.

[0009] Moreover, there are still considerable technical obstacles associated with the creation of useful urodesine salts in terms of processing.It is worth noting that even replicating free form urodesine is not without its own challenges, as mentioned in Org.Process Research and Dev.2009, 13, 928.Then, the second obstacle is to generate stable salts and provide reliable and robust methods for generating them.Although methods for forming various salts of urodesine have been described, to date, such methods have not identified or generated salts that are candidates for use in pharmaceutical products. Summary of the Invention

[0010] It is therefore desirable to develop stable salts (mixed or unmixed) and processes for the production of stable salts that would be useful in the manufacture of improved Urodesine pharmaceutical products. In a first aspect, the presently disclosed invention comprises at least one salt of urodesine selected from glutarate, malonate and / or oxalate.

[0011] Although such salt forms have not previously been discovered, characterized, or produced in the prior art, the inventors have studied them in detail to generate suitable additional candidates for clinical use, characterizing corresponding selections to determine their suitability for potential pharmaceutical applications. Because of the difficulties in generating these salts in the first instance, such studies went beyond merely the typical studies performed during routine salt selection.

[0012] In an embodiment, the salt comprises a hemi-salt. The stoichiometry of the hemi-salt results in a stable form, which can optionally be converted to an anhydrous form upon heating and drying. However, finding a method to result in a stable hemi-form is a technical challenge that has been overcome by the present inventors.

[0013] In an embodiment, the salt comprises the hemiglutarate salt of urodesine, and in a preferred embodiment, the hemi-L-glutarate crystalline salt of urodesine. Although initially technically difficult to produce, the crystalline form of the hemi-L-glutarate salt was ultimately found to exhibit no polymorphic variants compared to other stable salt forms of Urodesine known in the art. A physically stable salt that does not exhibit polymorphism is a highly desirable property in pharmaceutical manufacturing. Moreover, the hemi-L-glutarate salt has a very good physical stability compared to other salts tested. Moreover, this selected salt compared favorably with the pharma-ceutically acceptable succinate salt and showed surprisingly less degradation than the succinate salt over a period of two weeks under some conditions.

[0014] Previous disclosures have not been useful in identifying the preparation of certain novel salts of Urodesine that fall within the scope of the invention described and claimed herein, or in enabling their characterization. The art generally uses Urodesine free base as starting material to prepare the corresponding salt. Little previous work has been done to ascertain whether this particular form meets the acceptable criteria for salt selection. The absence of literature guidance appears to be related to the issue of recrystallization, and finding a method to make a stable crystalline form is inventive, since there is nothing in the art that teaches or guides how to produce it, and the usual methods did not result in a usable crystalline form. There is no guidance in the prior art that recrystallization from a particular solvent results in that form, or any other particular novel form known to be stable and useful.

[0015] The glutarate salt made by the inventors was not an obvious or obvious candidate for selection when compared to other salts available. Usually, salt selection requires safety considerations and may include review of several analytical parameters to determine other beneficial chemical and physical properties, such as clearly sharp diffraction peaks in the graph, review of any clearly amorphous peaks, weight loss of the solvent, and the ability to obtain crystalline forms under various conditions. On initial review, the glutarate salt met safety considerations. For example, Glutar Although the acid appears to be acceptable in terms of safety, it is otherwise not an immediate choice for the salt under standard criteria.For example, initial analysis showed low crystallinity (no sharp diffraction peaks) and some obvious solvent weight loss.However, it was unclear from previous studies in the art whether the hemiglutarate form could form stable crystals, so extensive further experiments and tests were carried out to clarify and determine this.

[0016] Then, the applicant encountered technical problems that needed to be overcome in order to obtain it in a stable crystalline form. For example, the hemiglutarate salt had to be made first, and further recrystallizations in different solvents, in different orders, and under different conditions were required to obtain stable crystals, rather than the crystallization being standardized. The inventors succeeded in determining a stable crystalline form. In an embodiment, it is disclosed that when the salt is a glutarate salt, it can be (a) 50% to 100% crystalline, more particularly at least 50% 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.

[0017] Moreover, useful and stable salt mixtures are made plausible and feasible by the tests herein, where additional salts are already known to be stable, but may benefit from the inclusion therewith of the novel hemi-L-glutarate salt.

[0018] Such combination avoids the problems associated with salt mixtures in the art, such as the problems related to instability and polymorphic behavior.Thus, in one embodiment, the present invention can comprise a composition that comprises at least glutarate (as defined and described herein) in combination with a second additional pharma- ceutically acceptable, physically and chemically stable second salt.In an embodiment, this second salt can comprise hemisuccinate.

[0019] In embodiments, the salts of Urodesine can be selected from differently characterized salts, such as amorphous hemi-salts, such as oxalates and hemi-malonates, etc. The inventors have also been able to make and characterize these alternative salts for the first time, and in so doing provide further alternative options for pharmaceutical manufacturing.

[0020] In embodiments, combinations of two or more of these salts may be selected from, for example, glutarate, hemiglutarate or hemi-L-glutarate, and malonate or oxalate.

[0021] The inventors have been able to determine the good formulation stability in water for the salt of the present invention, and therefore the possibility of scale-up useful for formulation into pharmaceutical products.This study will help support further research on IV formulations for use in, for example, animal and clinical trials.Therefore, the present invention also relates to pharmaceutical compounds comprising the salt form of Urodesine of the present invention, or mixtures of salt compositions as described herein above.

[0022] The present invention also extends to a pharmaceutical compound comprising a therapeutically effective amount of a salt form as described herein or a composition of a salt mixture of the invention as described herein, for use as a medicament, where the pharmaceutical compound may be for use as an inhibitor of PNP.

[0023] In a further aspect, the present disclosure provides a method for preparing urodesine hemiglutarate or hemi-L-glutarate.It has been found that each step of recrystallization is crucial to obtain hemi-salt or hemi-L-salt.In particular, ethanol, in conjunction with each other step, is necessary for the formation of hemiglutarate crystals, otherwise only other salts such as mono-salt (1:1) are obtained, which is less desirable.

[0024] The present disclosure relates to a method for preparing urodesine hemiglutarate or urodesine hemi-L-glutarate, comprising: (a) preparing an aqueous solution of urodesine free base and stirring, optionally at room temperature; (b) adding glutaric acid or L-glutaric acid to the mixture of step (a) and optionally stirring at room temperature for 30 minutes; (c) lyophilizing the solution of step (b) to obtain a white solid product; (d) dissolving the solid product of (c) in water, optionally heating to 75° C., adding ethanol, and optionally stirring at 75° C. for 30 minutes to form a homogenous solution; (e) adding acetonitrile dropwise to the solution of (d), optionally over a period of 60 minutes; (f) stirring the solution of (e) for 60 minutes, optionally at 75° C., and optionally cooling the solution to 0° C. over 60 minutes; (h) filtering and washing with acetonitrile to obtain urodesine hemi-salt glutarate. The present invention provides a method comprising:

[0025] Furthermore, when glutaric acid is added in the above methods, it may be added in an amount of the desired final salt form to aid in the crystallization process. Thus, in some embodiments, the present disclosure provides a method for preparing a glutarate salt of urodesine, particularly the hemiglutarate salt or the hemi-L-glutarate salt.

[0026] The above methods may include a time-holding step after one or more of the disclosed steps. In an embodiment, the method for preparing urodesine hemiglutarate described above comprises the step (a) of preparing an aqueous solution of urodesine free base by reacting the free form of:

[0027] [ka]

[0028] The method may further include requiring the use of Desirably, the method according to the present invention and its embodiments allow for the reliable and consistent production of urodesine hemiglutarate, which was not possible in the prior art.

[0029] Identifying, obtaining and increasing the yield of the newly described salts required for further pharmaceutical and clinical processing in the desired applications has been made possible by significant experimental methods and further technical modifications thereof.Thus, considerable technical challenges associated with the lack of identifiable and reproducible new salt candidates have been overcome, as well as their practical use in formulations and batch production for further applications in medicine and in the treatment of diseases.

[0030] In embodiments, the salt compounds of the present disclosure can be prepared according to specific examples further described in the description below. Terms and Abbreviations: As used herein, the following terms and standard methods have the meanings set forth below.

[0031] The term "API" refers to active pharmaceutical ingredient. The term "mono" refers to a 1:1 ratio of API:acid, respectively, in the crystal structure of the salt of the compound Urodesine.

[0032] The term "hemi" refers to a 2:1 ratio of API:acid, respectively, in the crystal structure of the salt of the compound Urodesine. The term "inert organic solvent" refers to a solvent that does not chemically interfere with the reaction.

[0033] The term "isostructural" is used to describe crystalline materials that have the same type of crystal structure, e.g., when a new molecular entity replaces another molecular entity in the crystal structure without significantly disturbing the unit cell.

[0034] The term "pharmacologically acceptable" with respect to pharma- ceutically acceptable carriers, excipients, and the like, means pharmacologically tolerable and substantially non-toxic to a subject to which a particular compound is administered.

[0035] The term "pharmaceutically acceptable salt" refers to a salt that retains the known biological effectiveness and properties of the standard compound, which may be acceptable for pharmaceutical use, and which is non-toxic. Abbreviation: ACN: Acetonitrile DSC: Differential scanning calorimeter DMSO: Dimethyl sulfoxide DVS: Dynamic Vapor Sorption EtOH: Ethanol FaSSIF: Fasted-state simulated intestinal fluid FeSSIF: fed-state simulated intestinal fluid HPLC: High Performance Liquid Chromatograph MeOH: Methanol NMR: nuclear magnetic resonance PLM: Polarized Light Microscopy RT: room temperature RRT: Relative retention time SGF: Simulated gastric fluid TFA: Trifluoroacetic acid TGA: Thermogravimetric analyzer THF: tetrahydrofuran TRS: Total Related Substances XRPD: X-ray powder diffraction method The following figures serve to provide a graphical analysis illustrating the results of various analytical tests showing the physical and chemical properties of the salts in this disclosure. [Brief description of the drawings]

[0036] [Figure 1] 1 shows a labeled XRPD pattern of Urodesine hemi-L-glutarate salt provided and prepared in accordance with the present disclosure. [Diagram 2] 1 shows a DSC and TGA overlay of the same urodesine hemi-L-glutarate salt provided and made in accordance with the present disclosure. [Diagram 3] 1 shows an overlay of XRPD of urodesine hemi-L-glutarate salt before and after DVS according to the present disclosure. [Figure 4] 1 shows an HPLC overlay demonstrating high solubility of a sample of Urodesine hemi-L-glutarate salt provided and made in accordance with the present disclosure. [Diagram 5] 1 shows an XRPD overlay of the hemi-L-glutarate salt provided in accordance with the present disclosure, demonstrating excellent solid-state stability. [Figure 6] 1 shows the labeled pattern of a scaled-up sample of urodesine hemi-L-glutarate salt provided and made in accordance with the present disclosure. [Figure 7] 1 shows a DSC and TGA overlay of a scaled-up sample of urodesine hemi-L-glutarate salt provided and made in accordance with the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Each and every example described herein is intended to be illustrative of the invention, but is not intended to be a complete limitation of the compositions or methods of the disclosure. Identification and preparation of hemi-salts derived from urodesine (free) The structure of the hemi-salt of urodesine under investigation is:

[0038] [ka]

[0039] Method of preparation Free base urodesine, prepared by pre-HPLC from urodesine succinate (A), was mixed with acid in a 2:1 ratio in water and then lyophilized to give four potential urodesine salts: Urodesine hemimalonate (B), Urodesine hemioxalate (C), Urodesine hemiadipate (D), and Urodesine hemi-L-glutarate (E) It was expected that the preferred hemi-salt form of

[0040] In either case, the resulting salt products had to be analyzed and characterized to actually ascertain whether the hemi-salt could be reliably produced, and if so, whether it could be produced in the desired structural form. Crystalline / amorphous salt form: Apart from the reference hemisuccinate salt, which is known to be crystalline, the compounds attempted to be made and described in this disclosure may exist in a crystalline or non-crystalline (e.g., amorphous) state.

[0041] Whether a compound exists in a crystalline state can be easily determined by standard techniques, which are defined herein.Crystals and their crystalline structures are characterized using multiple techniques, including single crystal X-ray crystallography, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and infrared spectroscopy, such as Fourier transform infrared spectroscopy (FTIR).The behavior of crystals under various humidity conditions can also be analyzed by gravimetric vapor sorption tests and by XRPD.These techniques are useful for characterizing the salts produced and identifying whether the product is suitable or not suitable for further investigation.

[0042] In particular, X-ray crystallography involves the analysis and interpretation of the X-ray diffraction of a single crystal. In amorphous solids, the three-dimensional structure normally present in crystalline forms is absent, and the positions of the molecules relative to one another in the amorphous form are essentially random.

[0043] In an attempt to obtain crystals, the hemi-salt under investigation is recrystallized from water and other organic solvents.The present disclosure provides the solvates formed by incorporating non-toxic pharma- ceutically acceptable solvents into the solid-state structure (e.g., crystalline structure) of the compounds provided herein.Examples of such solvents can include water, alcohol (e.g., ethanol, isopropanol, and butanol) and dimethylsulfoxide.

[0044] Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray crystallography can help determine whether solvates are formed in any case. The solvates can be stoichiometric or non-stoichiometric solvates and can include hydrates such as hemihydrates, monohydrates and dihydrates. Alternatively, the resulting compound can be anhydrous (e.g., anhydrous crystalline form).

[0045] Only three crystalline hemi-salts were successfully obtained (Ulodesine hemisuccinate, Urodesine hemiadipate and Urodesine hemi-L-glutarate), the latter being particularly difficult to obtain: of these, only reference salts and reference synthesis methods were known from the art, and it was not possible to achieve the L-glutarate using the reference methods.

[0046] Product yield, NMR and LC-MS analysis were determined for each with the following parameters: LC-MS method: Mobile phase: A: Water (10 mM NH4HCO3) B: ACN, gradient: 5% to 95% B in 1.3 min, flow rate: 2.0 mL / min, column: C18 4.6*50MM, 3.5 μm, oven temperature: 40° C. 1H solution NMR was collected on a 400 MHz NMR spectrometer using DMSO-d6 as the solvent. Urodesine hemisuccinate (A) To a solution of urodesine (500.00 mg, 1.89 mmol) in water (30 mL), succinic acid (111.70 mg, 0.95 mmol) was added. The mixture was stirred at room temperature for 30 minutes and then freeze-dried to obtain 556.00 mg of a white solid. The yield was 91%. Another batch of hemisuccinate (424.00 mg) was prepared from 400.00 mg of urodesine. 980.00 mg of urodesine hemisuccinate was dissolved in 3 mL of water and heated to 75° C., then 30 mL of acetonitrile was added dropwise over 1 hour. The mixture was then cooled to 0° C. over 1 hour. The mixture was filtered, and the filter cake was washed with acetonitrile and dried to obtain 702 mg of white solid crystals. The recrystallization yield was 71.6%.

[0047] [Table 1]

[0048] Urodesine Hemiadipic Acid Salt (D) To a solution of urodesine (910.00 mg, 3.44 mmol) in water (50 mL), adipic acid (251.60 mg, 1.72 mmol) was added. The mixture was stirred at room temperature for 30 minutes, then freeze-dried to obtain 1056.00 mg of a white solid. The yield was 91%. 1056 mg of urodesine hemiadipate was dissolved in 5 mL of water, heated to 75° C., and then 30 mL of acetonitrile was added dropwise over 1 hour. The mixture was then cooled to 0° C. over 30 minutes. The mixture was filtered, and the filter cake was washed with acetonitrile and dried to obtain 920 mg of a white solid crystal. The yield was 87.1%.

[0049] [Table 2]

[0050] Urodesine hemi-L-glutarate (E) Previous failures: Urodesine hemi-L-glutarate was dissolved in 3 mL of water and heated to 75° C., then 30 mL of acetonitrile was added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0° C. over 1 hour. The mixture was filtered, the filter cake was washed with acetonitrile, and dried to obtain 320 mg of a white solid. 1H NMR showed that it was urodesine mono-L-glutarate. The obtained solid and the mother liquor were combined, concentrated, and dried by oil pump to obtain 1055 mg of a white solid.

[0051] However, this process, which was previously used to produce the other salts listed above (using water and acetonitrile, etc.), produced a white solid mono form. The desired hemi-crystalline form of the glutarate salt could not be obtained by this method.

[0052] New Method "J" Needed: Ultimately, successful production of urodesine hemi-L-glutarate in a useful crystalline form required the investigation of several technical modifications of the crystallization process. Eventually, a new crystallization method ("J") specific for the hemi-glutarate salt form was determined using a mixed solvent process and using several different solvents: A solution of urodesine (1000.00 mg, 3.78 mmol) in water (50 mL) was added to L- Glutar Acid (278.36 mg, 1.89 mmol) was added. The mixture was stirred at room temperature for 30 min and then lyophilized to give 1155.00 mg of a white solid. The yield was 90%.

[0053] 1055mg of urodesine hemi-L-glutarate was dissolved in 3mL of water, then heated to 75°C, 15mL of ethanol was added, and stirred at this temperature for 30 minutes to form a homogeneous solution. Then 30mL of acetonitrile was added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0°C over 1 hour. The mixture was filtered, and the filter cake was washed with acetonitrile and dried to obtain 810mg of urodesine hemi-L-glutarate as a white solid.

[0054] The yield was 76.8%. Analysis (see further below) determined that this solid was crystalline hemi-L-glutarate and confirmed that a novel alternative method involving several steps and the addition of ethanol is necessary for the effective recrystallization of urodesine hemi-L-glutarate.

[0055] Thus, in certain embodiments, the present disclosure relates to urodesine hemi-L-glutarate formed using the recrystallization process described herein.

[0056] [Table 3]

[0057] Amorphous salts: The other two hemi-salts (urodesine hemi-malonate and urodesine hemi-oxalate) were amorphous. Water, acetonitrile, tetrahydrofuran, and ethanol were used to recrystallize these two hemi-salts, but none of the resulting solids were crystalline. Urodesine hemimalonate (B)

[0058] [ka]

[0059] To a solution of urodesine (1000.00 mg, 3.78 mmol) in water (50 mL) was added malonic acid (196.87 mg, 1.89 mmol). The mixture was stirred at room temperature for 30 minutes and then lyophilized to give 1103 mg of a white solid.

[0060] 1103 mg of urodesine hemimalonate was dissolved in 10 mL of water and then heated to 75° C., then 40 mL of acetonitrile was added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0° C. over 1 hour. The mixture was filtered, the filter cake was washed with acetonitrile and dried to give 625 mg of a white solid. The yield was 56.7%. XRPD showed it to be amorphous. The filtrate was concentrated in vacuum and dried by oil pump. The resulting solid and the amorphous form were combined to give 1025 mg of a white solid.

[0061] 1000 mg of urodesine hemimalonate was dissolved in 5 mL of water and heated to 75° C., then 16 mL of ethanol was added and stirred at this temperature for 30 minutes to form a homogeneous solution. 40 mL of acetonitrile was then added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0° C. over 1 hour. The mixture was filtered, the filter cake was washed with acetonitrile and dried to obtain 715 mg of a white solid. The yield was 71.5%. XRPD showed it to be amorphous. The filtrate was concentrated in vacuum and dried by oil pump. The obtained solid and the amorphous form were combined to obtain 980 mg of a white solid.

[0062] 1000 mg of urodesine hemimalonate was dissolved in 5 mL of water and heated to 75° C., then 25 mL of tetrahydrofuran was added dropwise over 1 h. The mixture was then stirred at this temperature for 1 h. The mixture was cooled to 0° C. over 1 h. No solid appeared. After standing at 0-4° C. for 3 days, no solid was obtained. The mixture was concentrated in vacuum and dried on an oil pump to give 1000 mg of a white solid.

[0063] 1000 mg of urodesine hemimalonate was dissolved in 5 mL of water, then heated to 75° C., and then 25 mL of ethanol was added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0° C. over 1 hour. The mixture was left to stand at room temperature for 1 day. The mixture was filtered, and the filter cake was washed with ethanol and dried to obtain 520 mg of a white solid. The yield was 52.0%. XRPD showed that it was amorphous. The filtrate was concentrated in vacuum and dried by oil pump. The resulting solid and the amorphous form were combined to obtain 969 mg of a white solid.

[0064] [Table 4]

[0065] Urodesine hemioxalate (C)

[0066] [ka]

[0067] To a solution of ulodesine (1000.00 mg, 3.78 mmol) in water (50 mL) was added oxalic acid (170.33 mg, 1.89 mmol). The mixture was stirred at room temperature for 30 minutes and then lyophilized to give 1045 mg of a white solid.

[0068] 1045 mg of urodesine hemioxalate was dissolved in 10 mL of water and heated to 75° C., then 40 mL of acetonitrile was added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0° C. over 1 hour. The mixture was filtered, the filter cake was washed with acetonitrile and dried to obtain 712 mg of a white solid. The yield was 68.0%. XRPD showed that it was amorphous. The filtrate was concentrated in vacuum and dried by oil pump. The obtained solid and amorphous were combined to obtain 915 mg of a white solid.

[0069] 900mg of urodesine hemioxalate was dissolved in 5mL of water and heated to 75°C, then 16mL of ethanol was added and stirred at this temperature for 30 minutes to form a homogeneous solution. 40mL of acetonitrile was then added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0°C over 1 hour. The mixture was filtered, the filter cake was washed with acetonitrile and dried to obtain 705mg of a white solid. The yield was 77.7%. XRPD showed that it was amorphous. The filtrate was concentrated in vacuum and dried by oil pump. The obtained solid and amorphous were combined to obtain 900mg of a white solid.

[0070] 900 mg of urodesine hemioxalate was dissolved in 5 mL of water and heated to 75° C., then 25 mL of tetrahydrofuran was added dropwise over 1 h. The mixture was then stirred at this temperature for 1 h. The mixture was cooled to 0° C. over 1 h. The mixture was filtered, the filter cake was washed with acetonitrile and dried to give 716 mg of a white solid. The yield was 79.7%. XRPD showed it to be amorphous. The filtrate was concentrated in vacuum and dried by oil pump. The resulting solid and the amorphous form were combined to give 880 mg of a white solid.

[0071] [Table 5]

[0072] Salt form characterization studies The new salts made for the first time, especially hemiglutarate, hemiadipate and two other newly formed salts (which were amorphous), were compared with known salts in properties / characteristics. These previously uncharacterized salts of Urodesine (adipate, glutarate, malonate and oxalate) were finally prepared according to the above-mentioned method. An exemplary pharma-ceutically acceptable salt of Urodesine, hemisuccinate, was selected for comparison and made according to the method known in the art.

[0073] The characteristics and properties herein were determined using standard analytical procedures in accordance with the Description section that follows.

[0074] analysis technology The discussion herein is aided by graphical illustrations of various analytical techniques, including polarized light microscopy (PLM), X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).

[0075] Polarized Light Microscopy (PLM) A small amount of sample (<1 mg) is placed on a glass slide, a drop of liquid paraffin is added, and the slide is covered with a slip. The sample, dispersed in oil, is viewed through the microscope eyepieces and camera / computer system. Representative sample images are captured and annotated to measure grain size and crystal habit.

[0076] X-ray powder diffraction method (XRPD) XRPD is a technique used on powdered or microcrystalline samples for the structural characterization of materials.

[0077] X-ray powder diffraction (XRPD) patterns were acquired on a Bruker D8 Advance equipped with a CuK source (1.54056 Å) operated at a minimum of 40 kV and 40 mA. Scan between 4 and 40 degrees 2-theta for each sample. The 2-theta step width was 0.05 and the scan speed was 0.5 s / step.

[0078] Differential Scanning Calorimetry (DSC) DSC is a thermal analysis technique in which the difference in the amount of heat required to raise the temperature of a sample and a reference is measured as a function of temperature.

[0079] Differential scanning calorimetry analysis was performed on a TA Instruments DSC unit (Model DSC25). Samples were placed in non-hermetic aluminum pans and heated at 10° C. / min from ambient temperature to 300° C. with a nitrogen purge at 50 mL / min.

[0080] Thermogravimetric analysis (TGA) TGA is a type of test performed on a sample to determine the change in weight versus a change in temperature.

[0081] Thermogravimetric analysis was performed on a TA Instrument TGA unit (Model: TGA500). Samples were placed in platinum pans and heated at 10° C. / min from ambient temperature to 300° C. with nitrogen purge at 60 mL / min (sample purge) and 40 mL / min (balance purge).

[0082] Dynamic Vapor Sorption (DVS) Moisture sorption profiles were generated using a DVS moisture balance flow system (model Advantage 1.0) at 25 °C with the following conditions: sample size approximately 10 mg, drying at 25 °C for 60 min, adsorption range 0% to 95% RH, desorption range 95% to 0% RH, and step interval 5%. Equilibrium criteria were <0.01% weight change in 5 min for a maximum of 120 min.

[0083] Water content (KF) Three blank samples were measured simultaneously, then approximately 10 mg of sample (two samples were tested simultaneously) was weighed and tested by Karl Fischer titration-volumetric method.

[0084] 1H NMR 1H solution NMR was collected on a Bruker 400 MHz NMR spectrometer using DMSO-d6 as the solvent. The properties of the crystalline and amorphous forms are summarized in the discussion and tables below.

[0085] Limited characterization of amorphous salts Summary table

[0086] [Table 6]

[0087] Urodesine hemimalonate The XPRD patterns of the two batches of urodesine hemimalonate showed that the compound was amorphous with no distinct diffraction peaks, therefore, no further characterization was performed.

[0088] Urodesine hemioxalate XPRD patterns showed that one batch was poorly crystalline, nearly amorphous, with only one small distinct diffraction peak, and the other batch was amorphous, and therefore no further characterization was performed.

[0089] Characterization of crystalline salts Following clear indications of crystalline form for the hemi-succinate, hemi-dipicate and hemi-L-glutarate salts, further characterization studies were performed and then solubility was determined.

[0090] The following summary table shows the overall results of further analytical tests carried out to characterize the three crystalline salts.

[0091] [Table 7-1]

[0092] [Table 7-2]

[0093] Urodesine hemi-L-glutarate was a white solid by visual observation, and birefringent and non-birefringent in the form of particles and irregular clumps under PLM. XPRD patterns showed that the compound was crystalline, as shown in Figure 1, but probably with a low degree of crystallinity, consistent with the PLM results. Information showing distinct diffraction peaks is presented below.

[0094] [Table 8]

[0095] As shown in Figure 2, the DSC trace showed a single melting point at 190.03 °C. A weight loss of 2.637% was observed from room temperature to 190 °C under the TGA curve. This could be due to desolvation of water since the water content determined by Karl Fischer titration was 2.53%. Urodesine hemi-L-glutarate is moderately hygroscopic, and a water uptake of 2.53% was observed in the DVS plot from 0% RH to 80% RH at 25 °C, and the crystal morphology showed no change before and after DVS as shown in the XRPD overlay in Figure 3. Based on the DSC, TGA and water content results, Urodesine hemi-L-glutarate was inferred to be anhydrous.

[0096] Solubility Test Thermodynamic solubility studies were conducted on three crystalline urodesine salts in water, SGF, FaSSIF and FeSSIF at room temperature (21-25°C) for 24 hours. The procedure for the solubility study was as follows: Appropriate amounts of each salt were weighed into 2 mL glass vials, respectively, and then 0.4 mL of the desired vehicle (water, SGF, FaSSIF and FeSSIF) was added to form a suspension. All samples were then vortexed for 30 seconds to ensure homogeneous dispersion and placed in a constant temperature shaking incubator at 37°C and 200 rpm for 24 hours. If the API dissolved, more material was added to maintain the suspension. Concentration, XRPD and pH were tested at each desired time point. A summary of the results is presented below.

[0097] [Table 9]

[0098] Urodesine hemiadipate, urodesine hemisuccinate and urodesine hemi-L-glutarate all showed high solubility (approximately 100 mg / mL) at 24 hours at 37° C. Figure 4 shows the HPLC overlay of samples of urodesine hemi-L-glutarate in the solubility study in four vehicles (water, SGF, FaSSIF, FeSSIF).

[0099] The XRPD patterns of the residual solids of Urodesine hemi-L-glutarate in water, FaSSIF and FeSSIF changed compared to the initial solid form, which was found to be the free base (Ulodesine) based on the 1H NMR results of the residual solids in FaSSIF after the solubility test.

[0100] Solid-State Stability Studies-Hemi-L-Glutarate To investigate the physical and chemical stability of Urodesine hemi-L-glutarate, further studies focusing on potential solid-state stability were carried out under 60°C (closed) and 40°C / 75% RH (open) conditions for 2 weeks, which was also carried out for Urodesine hemi-succinate to provide a means of comparison.

[0101] The procedure for solid-state stability was as follows: 10 mg of Urodesine hemi-succinate and Urodesine hemi-L-glutarate were accurately weighed into 40 mL transparent glass vials, respectively, and then the sample vials were placed in the corresponding conditions. For samples that were opened in humidity conditions, they were opened without caps and covered with aluminum foil with pinholes. For samples that were sealed, all were capped.

[0102] At 0, 1 and 2 weeks, the corresponding samples will be sampled for purity determination by HPLC to assess chemical stability, and another sample will be prepared under corresponding conditions for appearance and XRPD testing to evaluate physical stability.

[0103] The solid state stability test results showed that the two salts showed no change in appearance or XRPD at 60°C and 40°C / 75%RH for 2 weeks. The HPLC test results for the hemi-L-glutarate salt are presented in Figure 5. At 60°C, slight decomposition (0.3%-0.4%) was observed for both salts at 2 weeks. At 40°C / 75%RH, the hemi-L-glutarate salt did not show any increase in related substances, while the hemi-succinate salt decomposed by 0.16-0.3% at 2 weeks.

[0104] Based on the purity results, urodesine hemi-L-glutarate appears to be more chemically stable than urodesine hemi-succinate under 40°C / 75% RH conditions. The results are summarized in the table below.

[0105] [Table 10]

[0106] The hemi-L-glutarate salt has good chemical and physical stability, with no change in appearance or polymorphism at 60°C and 40°C / 75%RH for 2 weeks, which is highly advantageous compared to the hemi-succinate salt. At 60°C for 2 weeks, the two salts showed very little decomposition (0.3%-0.4%) of the same order of magnitude.

[0107] In summary, applicants have determined that ulodesine hemi-L-glutarate is generally more chemically stable than ulodesine hemi-succinate. Thus, Applicants have concluded that, despite initial challenges, they have been able to successfully create a crystalline form of the hemi-glutarate salt, which exhibits favorable physical and chemical properties compared to available salts of Urodesine.

[0108] Therefore, it may be a useful candidate in pharmaceutical formulation processing. Further stability was investigated in potential example formulation types, such as aqueous, with good consistent yield potential for scale-up production for batch trials.

[0109] Hemi-L-glutarate - Stability of aqueous formulations Further studies focusing on the newly characterized salts were conducted to provide stability data supporting an aqueous intravenous (IV) formulation.

[0110] The formulation contained urodesine hemi-L-glutarate in purified water at a solution concentration of 0.1 mg / ml. Sample conditions were maintained at 21-25°C and protected from light. The test procedure was as follows: 6.4mg of compound Urodesine hemi-L-glutarate was weighed into a 50mL volumetric flask, sonicated until dissolved, and diluted to volume. The target concentration was 0.10mg / ml (calculated as free base). Duplicate samples were prepared by transferring 4ml of solution into an 8mL glass bottle, and all samples were placed in the dark at room temperature. At the desired time points of 0, 3, 7, 10 and 14 days, the sample concentrations were analyzed by HPLC and pH values ​​were measured.

[0111] The results showed that no significant changes were observed in the appearance, concentration and purity of an aqueous solution sample of 0.1 mg / ml (calculated on the free base) of urodesine hemi-L-glutarate at room temperature for 14 days. The formulation was physically and chemically stable for 14 days.

[0112] In summary, it was determined that Urodesine hemi-L-glutarate at 0.1 mg / mL (calculated as free base) in water exhibits good formulation stability at room temperature for 14 days with potential for scale-up, which may be useful to support animal studies, especially in an intravenous (IV) formulation.

[0113] Repeat the salt formation process (to scale up to 1g) Initially, the process of crystalline salt production of the hemi-glutarate salt form was repeated to determine whether providing salt from the free form using this newly established method was reliable and consistent to yield enough precisely identified pure product that would be useful for scale-up. The initial goal was to obtain 1 g of the desired product and determine consistency with the analytical data of the product described above.

[0114] A solution of 1 g of urodesine (1000.00 mg, 3.78 mmol) in water (50 mL) was added to L- GlutarAcid (278.36 mg, 1.89 mmol) was added. The mixture was stirred at room temperature for 30 minutes and then lyophilized to obtain 1170.00 mg of a white solid. 1170 mg of urodesine hemi-L-glutarate was dissolved in 5 mL of water, then heated to 75° C., 16 mL of ethanol was added, and stirred at this temperature for 30 minutes to form a homogeneous solution. 40 mL of acetonitrile was then added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0° C. over 1 hour. The mixture was filtered, and the filter cake was washed with acetonitrile and dried to obtain 1010 mg (1 g) of a white solid. The yield was 79.0%. The final material was first confirmed by LC-MS and NMR as follows:

[0115] [Table 11]

[0116] It was then characterized by PLM, XRPD, DSC and TGA as before. Urodesine hemi-L-glutarate was a white solid by visual observation and was birefringent in the form of particles and irregular clumps under PLM.

[0117] The XPRD pattern shown in Figure 6 indicated that the final compound was crystalline with well-defined diffraction peaks, and the polymorphism of this scale-up (1g) hemi-L-glutarate salt was the same as that of the hemi-L-glutarate salt obtained in the preliminary studies mentioned in the above tables. Information on the well-defined diffraction peaks can be found in the following table.

[0118] [Table 12]

[0119] As seen in FIG. 7, the DSC trace showed a single melting point at 203.41° C. Only a 0.971% weight loss was observed in the TGA curve from room temperature to 195° C. The 1 g hemi-L-glutarate product was determined to be likely anhydrous. Furthermore, its crystallinity was a significant improvement from the initial crystallization, further proving that this salt would be a useful candidate in further formulation testing. Following the successful characterization and selection of the novel salt (hemi-glutarate salt derived from the free form of Urodesine) and validation that the recrystallization method used yielded this product with the same consistent physical and chemical solid state stability, a complete manufacturing method was investigated.

[0120] Complete method for the preparation of urodesine hemi-glutarate Following the complete Urodesine production process, including recrystallization of the free form product, to produce the desired salt was necessary to ensure that the complete manufacturing method disclosed (rather than starting from simple Urodesine free base) resulted in the same useful salt product.

[0121] In particular, it was important that the final hemi-glutarate salt was reliably obtained by such a process, in view of the fact that this final process could be used to obtain larger scale demonstration batches (suitable for pharmacological processing).

[0122] Of particular note, in light of past technical challenges reported in the art, it will be important to establish that the free form of urodesine can be reliably produced, as this is essential for efficient and reliable recrystallization of the newly disclosed hemi-glutarate salt.

[0123] First, the identity of the complete process was established. Below is a scheme showing the overall process steps of the complete synthetic production of the present invention urodesine hemi-glutarate CG689J from the basic components. Synthesis Route:

[0124] [ka]

[0125] Applicant first followed each of the steps above (and described herein above) in accordance with the above scheme conditions.

[0126] Step J-1 Hydroxybenzylation was carried out according to known methods. Polymers of CG689-SM2 (see above) were observed in all results and the main reason for the yield loss. The applicant carried out preliminary tests to optimize the base and replace BnOH as the solvent, because polymer formation occurred during the prolonged solvent removal process. However, in the end, no better solvent could be identified.

[0127] The polymeric impurities were removed by hot filtration of CG689G to give the desired intermediate in approximately 40% yield.

[0128] Step J-2 The HCl salt of compound 3 was used and the Mannich reaction was carried out in the presence of K2CO3.

[0129] Step J-3 After 72 hours under hydrogenolysis conditions, the benzyl protecting group of CG689H was successfully removed. However, the challenge remained that urodesine was still obtained as the hydrochloride salt, rather than the preferred free base (which would have been desirable for generating the novel salt directly), despite the addition of a basic additive (ammonium hydroxide solution).

[0130] After treatment with ion exchange resin, the free form of urodesine was obtained in approximately 33% yield (over two steps) with high HPLC and chiral purity (98.3%, 99.7%, respectively).

[0131] Step J-4 The final step was performed according to the recrystallization process of the hemi-glutarate salt formation described above. However, although urodesine hemi-glutarate salt was generally obtained, incomplete dissociation of the hydrochloride salt resulted in a mixture of salt forms. When tested with DSC calorimeter and XRPD, the results were not consistent with the findings of the hemi-glutarate salt produced in the analytical testing of a selection of salts described above. It was concluded that in order to obtain the correct salt form with comparable properties, alternative steps would be essential in the preparation before recrystallization.

[0132] Further technical investigation of the method for the preparation of Urodesine according to the above scheme was carried out. The applicant realized that Urodesine hydrochloride compound 3 is a key component in the above preparation of Urodesine CG689I free form. The successful acquisition of Urodesine free base was identified as a key factor.

[0133] [ka]

[0134] After experimental work, applicants determined that when the free base of compound 3 (rather than HCL) was used as the reaction partner in step J-2, the subsequent addition of base in the reaction was not necessary. Conversion of starting material CG689G was determined to be >95% by LC-MS. The reaction was more efficient and productive than the previously used mechanism. Importantly, this change also avoided the dissociation of the hydrochloride salt previously seen in step J-4 (above).

[0135] Using this method, the free form of Urodesine CG689I could be obtained with high HPLC and chiral purity (99.1% and 98.6%, respectively). Analytical studies confirmed that by modifying this step above, reliable and consistent production of the correct hemi-glutarate salt CG689J from Urodesine was again possible from the complete method of Urodesine production.

[0136] A verified batch of Urodesine Hemi-glutarate (35g) It was then desirable to produce batch quantities (35 g) of glutarate product by this novel method in order to demonstrate its feasibility for use in pharmaceutical processing and biological testing.

[0137] To that end, an alternative production route (using the free form of compound 3 as the reaction partner in the Urodesine process) was used to produce the free base of Urodesine, CG689I, as described in the modified process. A newly defined crystallization process (herein above "J") was then utilized to produce a final 35 g demonstration batch of the desired hemi-glutarate salt, CG689J.

[0138] Demonstration batch data

[0139] [Table 13]

[0140] Analysis of Proof Batch 1 (35g) CP-0031535-13:

[0141] [Table 14]

[0142] In summary, the desired product Urodesine hemi-glutarate was obtained in good yield in both batches. The product obtained was proven to be chemically pure, and the salt properties were consistent with those reported in the salt selection studies described above. Therefore, the newly identified and characterized Urodesine salt and the additional novel production method used to make it are believed to be a highly useful solution for providing Urodesine pharmaceutical products and the treatment of pathologies or diseases using the same.

[0143] The above examples are presented for the purpose of illustrating the present invention and should not be construed as imposing limitations on the scope of the present invention. It will be readily apparent that many variations and modifications may be made to the specific embodiments of the present invention described above and in the examples without departing from the principles underlying the invention. All such variations and modifications are intended to be encompassed by this application.

Claims

1. A urodexine compound: 【Chemical 1】 A pharmaceutically acceptable salt thereof, comprising a glutaric acid crystal salt.

2. The salt according to claim 1, comprising a hemiglutaric acid crystal salt.

3. A pharmaceutically acceptable composition comprising the urodexine salt compound according to claim 1 or 2 and a pharmaceutically acceptable excipient.

4. The composition according to claim 3, for use as a medicament.

5. A method for preparing a urodexine hemiglutaric acid crystal salt compound, comprising: (a) preparing an aqueous solution of urodexine free base; (b) adding glutaric acid to the mixture of step (a); (c) lyophilizing the solution of step (b) to obtain a white solid product; (d) dissolving the solid product of (c) in water, adding ethanol to form a homogeneous solution; (e) dropwise adding acetonitrile to the solution of (d); (f) stirring the solution of (e) for 60 minutes; (g) filtering and washing with acetonitrile to obtain a urodexine hemiglutaric acid crystal salt compound. A method for preparing a urodexine hemiglutaric acid crystal salt compound, comprising the above steps.

6. A method for preparing a urodexine hemiglutaric acid crystal salt compound according to claim 5, wherein: the step (a) further comprises stirring the aqueous solution of urodexine free base at room temperature, or the step (b) further comprises stirring the solution with added glutaric acid at room temperature for 30 minutes, or the step (d) further comprises heating the water in which the solid product is dissolved to 75°C, and / or stirring at 75°C for 30 minutes after adding ethanol, or in the step (e), the dropwise addition of acetonitrile to the solution of (d) is carried out over 60 minutes, or in the step (f), the stirring of the solution of (e) is carried out at 75°C for 60 minutes, and / or the solution is cooled to 0°C over 60 minutes.

7. The method for preparing a urodexine hemiglutaric acid crystal salt compound according to claim 5 or 6, wherein the step (a) requires the use of a reactant in free form: 【Chemical 2】 in the preparation of urodexine free base in water.

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