Polymorphs of the triazole antifungal compound PC945

Abstract

The present invention relates to the crystal polymorphic manifestation of compounds of formula (I), and related aspects.

Description

Field of the Invention

[0001] The present invention relates to the crystal polymorphic manifestation of a compound that can be used to treat fungal diseases, and a composition and use thereof.

[0002] background

[0003] The incidence of fungal infections has increased substantially over the past two decades, with invasive forms being a major cause of morbidity and mortality, particularly in immunocompromised or immunosuppressed patients. Disseminated candidiasis, pulmonary aspergillosis, and emerging opportunistic fungi are the most common agents responsible for these serious fungal diseases. A special feature of fungi is their ability to produce an extracellular matrix (ECM) that holds them together and enables them to adhere to the substrate on which they are attached in vitro or in vivo. These biofilms serve to protect them against the hostile environment of the host immune system and resist the activity of antimicrobial agents (Kaur and Singh, 2013).

[0004] Pulmonary aspergillosis can be classified as either a non-invasive or invasive condition. Additional subclassifications are used to characterize the condition of patients who develop allergic symptoms of aspergillosis (called ABPA; allergic bronchopulmonary aspergillosis) compared to patients who do not develop such conditions. Triggers of pulmonary aspergillosis may be acute, such as exposure to high doses of immunosuppressive drugs or intubation in an intensive care unit. Alternatively, it may be chronic, caused by a previous infection such as tuberculosis (TB) (Denning et al., 2011a). Chronic pulmonary Aspergillus infection can cause extensive and persistent lung damage in patients, requiring lifelong treatment with oral azole drugs (Limper et al., 2011).

[0005] An increasing number of studies suggest that Aspergillus infection may play an important role in clinical asthma (Chishimba et al., 2012; Pasqualotto et al., 2009). In addition, recently published work has associated Aspergillus infection with poor clinical outcomes in COPD patients (Bafadhel et al., 2013). Similarly, representative studies have shown an association between the presence of Aspergillus spp. and Candida spp. in patients' sputum and worsening of their lung function (Chotirmall et al., 2010; Agbetile et al., 2012).

[0006] Invasive aspergillosis (IA) produces high mortality in immunocompromised patients, such as those receiving allogeneic stem cell transplants or solid organ transplants (such as lung transplants). The first case of IA reported in immunocompromised patients occurred in 1953. This event coincided with the introduction of corticosteroids and cytotoxic chemotherapy into treatment regimens (Rankin, 1953). Invasive aspergillosis is a major concern in the treatment of leukemia and other hematological malignancies, given its high morbidity and associated mortality. Despite the availability of oral triazoles (Salmeron et al., 2012), the mortality rate in allogeneic hematopoietic stem cell transplant recipients is also often more than 50% (Lin et al., 2001), and the long-term mortality rate can reach 90%. In patients undergoing solid organ (especially lung) transplants, the use of high doses of steroids makes patients susceptible to infection (Thompson and Patterson, 2008). These diseases also occur in less severely immunocompromised patient populations. These patients include those with COPD or cirrhosis, patients receiving high-dose steroids, and individuals with central venous catheters or supported by mechanical ventilation (Dimopoulos et al., 2012).

[0007] Existing antifungal drugs are mainly administered orally or systemically. Since the drug concentrations achieved at the site of infection tend to be lower than those in unaffected organs, these commonly used delivery routes are insufficient for treating pulmonary airway infections. This is especially true for the liver, which is a major site of toxicity in up to 15% of patients treated with voriconazole or who experience elevated transaminase levels (Levin et al., 2007; Lat and Thompson, 2011). Liver exposure also leads to significant drug interactions caused by inhibition of hepatic P450 enzymes (Jeong, et al., 2009; Wexler et al., 2004).

[0008] Furthermore, the widespread use of triazoles in clinical and agricultural settings has led to the growth and emergence of drug-resistant fungal diseases in some locations (Denning et al., 2011b; Bowyer and Denning, 2014).

[0009] Compounds useful for treating fungal diseases are described in WO2016 / 087878 A1 (Sunose et al., 2016) and WO2016 / 087880 A1 (Colley et al., 2016). Compound I is disclosed in the above patent application publications and is 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide:

[0010]

[0011] Compound I is a potent inhibitor of sterol 14α-demethylase (CYP51A and CYP51B) of Aspergillus fumigatus and has been identified as an effective, long-acting antifungal substance in vitro and in vivo (Colley et al., 2017). In addition, topical treatment with Compound I in combination with known systemic triazole active agents has been disclosed to show synergistic antifungal effects against Aspergillus fumigatus in an in vitro human alveolar bilayer model and in the lungs of neutropenic immunocompromised mice (Colley et al., 2019). Compound I was also found to be a more potent inhibitor than posaconazole, voriconazole, and fluconazole against a global collection of Candida auris isolates (Shivaprakash et al., 2019). Biomarker analysis after intranasal administration of Compound I to immunocompromised mice showed that the antifungal effect was cumulative and persistent after repeated dosing (Kimura et al., 2017).

[0012] Polymorphism is an important consideration in drug development as it affects the physicochemical and biopharmaceutical properties of a compound. This property occurs when an entity (e.g. a small molecule) can adopt different crystal lattices. The resulting changes in solid-state packing can lead to differences in storage stability, solubility properties and density, which can affect formulation and product manufacturing, as well as dissolution characteristics: an important factor in determining in vivo bioavailability.

[0013] There remains a need to provide polymorphic forms of Compound I for use in treating fungal diseases. In particular, there is a need to provide a crystalline form of Compound I that has appropriate physical and chemical stability and other drug-related properties associated with topical administration to the lung, including its susceptibility to micronization, its formulation as an easily dispersible aqueous suspension, and adequate bioavailability and acceptable pharmacokinetic properties. SUMMARY OF THE INVENTION

[0015] The present invention provides a compound of formula (I):

[0016]

[0017] It is a crystalline form of 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide, wherein the crystalline form is polymorph Form 1.

[0018] In addition, the present invention provides compounds of formula (I):

[0019]

[0020] It is a crystalline form of 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide, wherein the crystalline form is polymorph Form 2.

[0021] The skilled artisan will appreciate that, although a specific tautomer is shown above, the compound may exist in multiple tautomeric forms. The present invention includes all such tautomeric forms.

[0022] The compound of formula (I) is referred to herein as Compound I.

[0023] As described in the Examples, crystalline polymorph Form 1 and crystalline polymorph Form 2 have distinctly different crystal structures. In particular, polymorph Forms 1 and 2 of the present invention have the following features and properties that make them particularly suitable for use as therapeutic substances. The polymorphs exhibit high melting points [~202°C (Form 1) and ~183°C (Form 2)]; are physically robust (determined by XRPD, TGA and DSC analysis); have good chemical stability (determined by 1 H NMR spectroscopy and HPLC analysis); easily micronized to a respirable particle size; and bioavailable when administered as an inhalable aqueous suspension. Therefore, Form 1 polymorph and Form 2 polymorph are expected to be useful in the various therapeutic uses described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : High resolution XRPD pattern of a representative sample of polymorph Form 1.

[0026] Figure 2 : High resolution XRPD pattern of a representative sample of polymorph Form 2.

[0027] Figure 3 : Vertical alignment of high resolution XRPD patterns of polymorphic forms 1 and 2.

[0028] Figure 4 : Overlay of high resolution XRPD patterns of polymorphic forms 1 and 2.

[0029] Figure 5 : Calculated XRPD pattern of polymorph Form 1.

[0030] Figure 6 : Calculated XRPD pattern of polymorph Form 2.

[0031] Figure 7 : Comparison of the XRPD pattern of a prior art Form 3 sample with the XRPD patterns of representative samples of Forms 1, 2 and 3.

[0032] Figure 8 : Thermal analysis (DSC and TGA) of the crude input material (polymorph Form 3).

[0033] Fig. 9 : DSC graph of polymorph Form 1 obtained from aqueous acetone (5% H2O).

[0034] Fig.10 : XRPD pattern of polymorph Form 1 obtained from aqueous acetone (5% H2O) after micronization.

[0035] Fig.11 : XRPD pattern of polymorph Form 2 obtained from THF / TBME.

[0036] Fig.12 : DSC graph of polymorph Form 2 obtained from THF / TBME.

[0037] Fig.13 : High resolution XRPD pattern of polymorph Form 2 obtained from THF / TBME after micronization.

[0038] Fig.14 : Crystal structure of polymorph Form 1 viewed along the a-axis.

[0039] Fig.15 : Crystal structure of polymorph Form 2 viewed along the b-axis.

[0040] Fig.16 : Rapid scan DSC pattern of polymorph Form 1 (40°C / min).

[0041] Fig.17 : Rapid scan DSC pattern of polymorph Form 2 (40°C / min).

[0042] Fig.18 : Thermal analysis (DSC and TGA) of polymorph Form 2 obtained from MIBK.

[0043] Fig.19 : Mean plasma drug concentrations of polymorph Form 1 following inhalation administration to male rats at a nominal dose of 2.2 mg / kg.

[0044] Fig. 20 : Mean plasma drug concentrations of polymorph Form 2 following inhalation administration to male rats at a nominal dose of 2.2 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0046] An X-ray powder diffraction (XRPD) pattern consists of a plot of the intensity of a diffracted beam versus the diffraction angle 2θ. The 2θ peak positions of an X-ray powder diffraction pattern are directly related to the unit cell of the crystalline phase, resulting in a unique pattern for each polymorphic form. Thus, the XRPD pattern is characteristic for each polymorph and can be used to distinguish one polymorph from another. X-ray powder diffraction patterns were obtained directly from samples of polymorphic forms 1 and 2 prepared as described herein ( Figure 1 and 2 ). From the visual comparison of the two graphs ( Figure 3 and 4 ) It can be clearly seen that Form 1 and Form 2 of Compound 1 represent two different crystalline states.

[0047] When comparing two (or more) diffraction patterns, a reflection at 2θ is considered unique (and therefore characteristic of a crystalline form) if no reflection is observed within ±0.2° 2θ. Polymorphic Forms 1 and 2 exhibit a number of unique reflections (Table 1).

[0048] Table 1: Unique XRPD reflections of polymorphic forms 1 and 2. Reflection (2θ)

[0049]

[0050] Using synchrotron light source x-ray crystallography, the structural elucidation of polymorphic forms 1 and 2 enabled the XRPD patterns of polymorphic forms 1 and 2 to be calculated from the experimental diffraction values ​​obtained from the single crystal data set. For both polymorphs, the experimental data ( Figure 1 and 2 ) and computational graphs ( Figure 5 and 6 ) have a very close correlation.

[0051] Thus, a crystalline form of polymorph Form 1 of Compound 1 is provided, which has substantially Figure 1 The X-ray powder diffraction pattern is shown.

[0052] Suitably, there is provided a crystalline form of polymorph Form 1 of Compound 1, wherein the crystalline form has an X-ray powder diffraction pattern comprising 3, 4, 5, 6 or 7 peaks selected from (±0.2) 7.0, 7.4, 7.9, 18.2, 19.7, 20.8, 24.7° 2-θ.

[0053] Other peaks of the XRPD pattern of polymorph Form 1 are shown in Table 3. Accordingly, suitably, a crystalline form of polymorph Form 1 of Compound I is provided, wherein the crystalline form has an X-ray powder diffraction pattern comprising the following seven peaks: (± 0.2) 7.0, 7.4, 7.9, 18.2, 19.7, 20.8 and 24.7° 2-θ, and 1, 2, 3, 4 or 5 peaks selected from (± 0.2) 10.2, 16.7, 17.9, 23.3 and 24.2° 2-θ.

[0054] Suitably, there is provided a crystalline form of polymorph Form 1 of Compound 1, wherein the crystalline form has and The unit cell size is , and the α, β and γ angles are 90°.

[0055] Thus, a crystalline form of polymorph Form 2 of Compound 1 is provided, which has substantially Figure 2 The X-ray powder diffraction pattern is shown.

[0056] Provided is a crystalline form of Polymorph Form 2 of Compound 1, wherein the crystalline form has an X-ray powder diffraction pattern comprising 3, 4, 5 or 6 peaks selected from (±0.2) 10.8, 17.0, 20.3, 22.7, 23.9 and 24.3° 2-θ.

[0057] Peaks of other XRPD patterns of polymorph Form 2 are shown in Table 3. Accordingly, suitably, a crystalline form of polymorph Form 2 of Compound I is provided, wherein the crystalline form has an X-ray powder diffraction pattern comprising the following six peaks: (± 0.2) 10.8, 17.0, 20.3, 22.7, 23.9 and 24.3 ° 2-θ, and 1, 2, 3, 4, 5 or 6 peaks selected from (± 0.2) 16.6, 19.5, 20.3, 22.0, 26.6 and 26.9 ° 2-θ.

[0058] Suitably, there is provided a crystalline form of polymorph Form 2 of Compound 1, wherein the crystalline form has and The unit cell dimensions are , as well as an α angle of 90°, a β angle of 101.54° and a γ angle of 90°.

[0059] Form 3 (Prior Art) Polymorph

[0060] Compound I is disclosed in patent application publications WO2016 / 087878 A1 and WO2016 / 087880 A1. In the applications, compound I prepared according to the disclosures thereof is isolated by one of the following methods: (a) adding water to a reaction mixture of compound I in pyridine to obtain a crude solid compound I, which is then purified by flash column chromatography, eluting with 0-3% MeOH in DCM; (b) adding water to a cooled reaction mixture of compound I in DMF, followed by further cooling the reaction mixture and collecting by filtration to obtain a solid compound I, followed by slurrying the filter cake in water and collecting the solid by filtration, or (c) adding water to a reaction mixture of compound I in DMSO, followed by extracting compound I with EtOAc, evaporating the volatiles in vacuo, and purifying the residue by flash column chromatography, eluting with 0-2% MeOH in DCM, and repurifying by flash column chromatography, eluting with 0-50% EtOAc in DCM.

[0061] For the purposes of the present disclosure, the previously disclosed Compound 1 obtained by the synthetic routes described therein is a poorly crystalline representation of Compound 1, which is referred to herein as Form 3. A common feature of the routes described is that Compound 1 is rapidly generated by the addition of water to a solution of Compound 1 in a miscible organic solvent (e.g., DMF or DMSO), or by evaporation of the solvent mixture after chromatographic purification, thereby producing polymorphic Form 3. As described below, water is a strong anti-solvent for Compound 1, which results in its rapid precipitation in a nearly amorphous state in the described cases. The same principle also applies to samples recovered by chromatography, where rapid evaporation of the eluent containing Compound 1 produces polymorphic Form 3, rather than more ordered crystalline forms such as Form 1 or Form 2.

[0062] Without being limited by theory, experimental data teach that precipitation of Compound 1 in a volatile solvent (e.g., DCM) by rapid evaporation of the solution produces a disordered solid state. The same applies to the (almost instantaneous) precipitation of Compound 1 by the addition of an antisolvent; for example, by quenching a solution of Compound 1 in a miscible organic solvent with water. Both previously disclosed methods do not allow sufficient time for the compound of Formula I to reach a thermodynamically more stable, highly ordered crystalline state, such as exemplified by polymorphic forms 1 and 2 disclosed herein.

[0063] A representative sample of Form 3 was prepared using the following method. To a suspension of 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzoic acid (2.50 g, 4.24 mmol), EDCI (1.63 g, 8.48 mmol) and DMAP (0.03 g, 0.21 mmol) in pyridine (30 mL) was added 4-fluoroaniline (0.41 mL, 4.32 mmol). The reaction mixture was stirred at 60 °C for 2 hours, then cooled to RT, diluted with water (60 mL) and stirred for 5 min. The resulting solid was collected by filtration, washed with water (3x10 mL) and ether (2x15 mL) to give a tan powder. The crude product thus obtained is purified by flash column chromatography (SiO2, 40g, 0-3% MeOH in DCM solution, gradient elution). The resulting yellow solid (2.37g) is suspended in DMSO (6.50mL), and the mixture is heated at 60°C until completely dissolved. The solution is cooled to RT, then in the stirred solution, water (20mL) is added to produce a white solid precipitate. The solid is collected by filtration, washed with water (3x10mL) and ether (3x5mL), then dried under vacuum at 50°C for 3 days, to obtain Compound I (2.25g, 77%) as an off-white solid.

[0064] Figure 7 The XRPD pattern of Polymorph Form 3 of Compound 1 prepared as described above ("Prior Art Sample") is shown, compared to a representative batch of Form 3 samples used in this study ("Form 3 Control"), and compared to the XRPD patterns of Form 1 and Form 2 polymorphs. The XRPD pattern of the Form 3 material showed that it was poorly crystalline and therefore it was not considered to be an easily developable entity. Therefore, no comparative bioavailability data were obtained for this polymorphic form (see Examples).

[0065] Properties of polymorphic forms 1 and 2

[0066] Thermodynamic stability of polymorphic forms 1 and 2 at different temperatures

[0067] It has been determined that the crystalline states referred to herein as Form 1 and Form 2 constitute a bidirectionally transforming polymorphic pair. This property arises from the temperature dependence of their relative thermodynamic stabilities, where one polymorph is the most thermodynamically stable manifestation of the substance up to a characteristic (empirically determined) temperature; above this temperature, the order of their stability is reversed. In the present case, suspensions containing equal amounts of both polymorphs were ripened at 50°C, 70°C, and 135°C in one of six different solvents (Table 6). In all experiments conducted at 50°C and 70°C, the 1:1 mixture was converted to a suspension containing only the Form 2 polymorph. In contrast, all mixtures maintained at a temperature of 135°C were converted to Form 1. These data support the hypothesis that the thermodynamic relationship between Form 1 and Form 2 is bidirectionally transforming and that the temperature of the stability transition (bidirectional transition point) is within the temperature range of 70-135°C.

[0068] Cross-seeding experiments were performed at temperatures below the bidirectional transition point to investigate the propensity to produce Form 1 or Form 2 under different conditions (see Tables 8 to 13 herein). In the single organic solvent system described in Table 8, a mixture containing equal amounts of Form 1 and Form 2 converted to give a suspension containing only the Form 2 polymorph. The exception was the binary mixture of IPA doped with 5% water, which remained a polymorphic mixture (Table 8, entries 10-12). It is possible to interpret this result as arising from two different pathways. The first inference is that the solubility of Compound I in aqueous IPA solution, regardless of the form, is too low for polymorphic interconversion to occur, and therefore kinetic factors determine the results. The second hypothesis is that the presence of water has an effect on the polymorphic mixture in favor of Form 1, although it remains a metastable polymorph within the experimental temperature range.

[0069] This hypothesis was investigated in a series of slurry and cross-seeding experiments (Tables 9 to 13), during which it was determined that the presence of water at low levels acted as a co-solvent that favored the dominance of the Form 1 polymorph. This feature was observed when the Form 2 polymorph was slurried in acetone and water alone and in mixtures (Table 9); THF and aqueous THF (Table 10); dioxane / water mixtures (Table 11) and MEK containing increasing proportions of water (Table 12).

[0070] It is evident from the data that it is the presence of water added to these solvents that induces the polymorphic transition. However, except at low levels, water is also an effective antisolvent and significantly inhibits the solubility of both polymorphs. The result of this solubility profile is that the conversion from Form 2 to Form 1 either does not occur or is inhibited by marginal solubility in water alone or in water-rich solvent mixtures (Table 11, entries 3, 6, and 8).

[0071] As expected from the temperature dependence of conversion (according to the Arrhenius equation), polymorph interconversion was observed to occur more readily upon heating (Tables 9, 10 and 12), and presumably due to the increased solubility of the suspended solids. When equilibrated in cooled MEK or THF, the fate of the polymorphs depended not only on the presence of water in the system, but was also influenced by their initial polymorphic composition (Table 13). As expected, a mixture of two polymorphs converted to a suspension of Form 2 in a nominal drying medium, while the single polymorph remained unchanged. In contrast, the presence of water again favored the Form 1 state, and there is further evidence that the initial presence of this polymorph in the starting mixture (as a seed) promotes interconversion.

[0072] The data from the slurry and cross-seeding experiments disclosed herein indicate that the prevalence of polymorphic Forms 1 and 2 when exposed to a solvent system is affected by the choice of organic solvent, its water content, slurry temperature, contact time, and purity (i.e., its polymorph composition). Nevertheless, the studies described herein provide an understanding of these parameters that enables their control to produce Form 1 or Form 2 as desired and to maintain the physical integrity of Form 1 or Form 2. Furthermore, it was determined that while the Form 1 crystalline state is preferred in aqueous organic media, those media in which water is the major component are substantially inert to polymorph interconversion.

[0073] The behavior of these systems and the lessons they provide are important in designing appropriate formulations by which Compound I can be delivered to a patient.

[0074] The Form 1 and Form 2 polymorphs can be micronised easily. This means that they are suitable for use in formulations for delivery to the respiratory tract, such as the lungs.

[0075] The studies described herein surprisingly demonstrate that the Form 2 polymorph is in particular a suitable crystalline form of Compound 1 for aqueous suspension formulations, despite evidence that Form 2 is metastable in the described environment at ambient temperature. The data obtained from the studies disclosed herein demonstrate that the extremely low solubility of Compound 1 in water provides an effective kinetic barrier to the loss of polymorphic integrity in a medium comprising a high proportion of water. The physicochemical properties of Compound 1 (high molecular weight and lipophilicity) ensure that the two major crystalline forms do not readily interconvert under the described conditions, thus retaining their original polymorphic composition. As demonstrated elsewhere herein, when formulated into an aqueous suspension and administered by inhalation, the Form 2 polymorph unexpectedly and advantageously exhibits superior bioavailability to the Form 1 polymorph. Therefore, the Form 2 polymorph is an advantageous form of Compound 1 as a nebulized aqueous suspension inhalation therapeutic.

[0076] The properties of polymorph stability, combined with the lower systemic bioavailability of Form 1 when administered by inhalation, favor other indications and presentations for the polymorph, as discussed below.

[0077] General synthetic method

[0078] Solvate

[0079] In one aspect, a solvated form of Compound I is provided. When Compound I is matured in a variety of aromatic solvents (e.g., anisole, acetophenone, benzyl alcohol, chlorobenzene and isopropylbenzene), a suitable solvate is obtained. Alternatively, when Compound I is matured in THF, a suitable solvate is obtained. Suitably, when analyzed by TGA, the solvate of Compound I thus obtained contains about 13 to 14% by weight of solvent. Suitably, the ratio of Compound I to the solvent is about a stoichiometric ratio (1:1).

[0080] It was found that the identified solvates of Compound I desolvated with heating to generate the crystalline polymorph of Form 1. For example, the anisole solvate exhibited two distinct endothermic peaks using DSC analysis. The first one was at 112-114°C, consistent with the 13.4% weight loss determined from the TGA graph, corresponding to slightly less than one equivalent of solvent, and 1 The H NMR data were consistent. The second endothermic peak peaked at 202 °C, corresponding to the melting point of Form 1.

[0081] Thus, in one aspect, there is provided a method for preparing Compound 1 as polymorphic Form 1, the method comprising heating a solvated form of Compound 1, such as a solvate formed with anisole, acetophenone, benzyl alcohol, chlorobenzene or cumene, to remove the solvent, thereby producing polymorphic Form 1 of Compound 1. Suitably, the solvated form of Compound 1 is an intermediate in the synthesis of polymorphic Form 1.

[0082] Form 1 polymorph

[0083] In one embodiment, a method for preparing polymorph Form 1 of Compound I is provided, comprising the step of crystallizing Compound I into polymorph Form 1 from a solution of Compound I in a solvent, wherein the solvent is selected from toluene, 2-methoxyethanol, aqueous IPA (e.g., containing 3-6% H2O v / v, e.g., 5% H2O v / v) and aqueous acetone (e.g., containing 5-10% H2O v / v).

[0084] Suitably, the solvent is aqueous acetone (eg containing 5% H2O v / v).

[0085] Form 2 polymorph

[0086] In one embodiment, there is provided a method for preparing polymorph Form 2 of Compound 1, comprising the step of crystallizing Compound 1 into polymorph Form 2 from a solution of Compound 1 in a nominally dry organic solvent, wherein the solvent is selected from MIBK, THF, PrOAc, EtOH, DME, MeOH, MeCN, MeNO2, n-heptane, Et2O, IPA, MEK, acetone, TBME, i-BuOH, or a mixture of THF and TBME. Suitably, the method comprises the step of crystallizing Compound 1 into polymorph Form 2 from a solution of a nominally dry organic solvent mixture, wherein the solvent mixture comprises two or more solvents selected from MIBK, THF, PrOAc, EtOH, DME, MeOH, MeCN, MeNO2, n-heptane, Et2O, IPA, MEK, acetone, TBME, and i-BuOH.

[0087] For purposes of the present invention, "nominal dry" means that no water has been added to the solvent. Depending on the hydrophilicity of the solvent chosen, trace amounts of water may be present, although the experimental conditions are designed to exclude trace amounts of water.

[0088] Suitably, the solvent is MIBK. Alternatively, the solvent system is a mixture of THF and toluene, wherein the mixture suitably comprises 2:1 to 1:2 THF:toluene (e.g. 10:5 v / v; 9:5 v / v; 8:4 v / v; 8:16 v / v THF:toluene, respectively). Alternatively, the solvent system is a mixture of THF and TBME, wherein the mixture suitably comprises 4:1 to 1:2 THF:TBME (e.g. 8:2 v / v; 8:2.5 v / v; 8:4 v / v; 8:6 v / v; 8:8 v / v; 8:10 v / v or 8:12 v / v THF:TBME, respectively).

[0089] Pharmaceutical preparations

[0090] In one embodiment, there is provided a pharmaceutical composition comprising a compound of the invention, optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

[0091] Suitably, the compounds of the present invention are topically applied to the lungs or nose, particularly topically applied to the lungs. Therefore, in one embodiment, a pharmaceutical composition comprising a compound of the present invention optionally in combination with one or more locally acceptable diluents or carriers is provided. Suitably, the compounds of the present invention are administered by inhalation.

[0092] Suitably, the pharmaceutically acceptable diluent or carrier is water.

[0093] The composition may conveniently be administered in unit dosage form and may be prepared by any method well known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA., (1985). The composition may also conveniently be administered in multiple unit dosage form.

[0094] Suitable compositions for pulmonary or intranasal administration include powders, liquid solutions, liquid suspensions, aqueous suspensions, nasal drops containing solutions or suspensions, or pressurized or non-pressurized aerosols.

[0095] Topical administration to the nose or lungs can be achieved by using non-pressurized formulations such as aqueous suspensions. The formulations can be administered by spraying, i.e., using a nebulizer that can be hand-held and portable or non-portable and intended for home or hospital use. An exemplary device is the RESPIMAT inhaler.

[0096] Liquid suspensions and aerosol formulations (whether pressurized or not) generally contain the compound of the invention in the form of particles, for example, with a D of 0.5-10 μm. 50 , suitably about 1-5 μm, for example 1-2 μm. Suitably, the compound of the invention in particulate form has a D of 0.2-1 μm. 50 , for example 0.2-0.5 μm. Suitably, the compound of the invention in particle form has a D of 2-6 μm. 90 , for example 3-4 μm. The compound may be in particle form, for example in micronized form. Micronization may be accomplished using a jet mill, such as those produced by Hosokawa Alpine. The resulting particle size distribution may be measured using laser diffraction (e.g. using a Malvern Mastersizer 2000S instrument). D 10 , D 50 and D 90 The particle size distribution is represented by the value. 50 The median is defined as the particle size in micrometers that divides the distribution in half. The results obtained from laser diffraction are more accurately described as volume distributions, and therefore the D obtained using this method is 50 The value is more meaningfully called Dv 50 As used herein, the Dv value refers to the particle size distribution measured using laser diffraction. Similarly, the Dv value used in the context of laser diffraction is 10 and D 90 Values ​​are considered to refer to Dv 10 and Dv 90 and respectively refer to the distribution where 10% is below D 10value of particle size, and 90% of the distribution is below D 90 value.

[0097] According to a particular aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of the present invention in the form of particles as an aqueous suspension, ie suspended in an aqueous medium.

[0098] Aqueous media typically comprise water and one or more excipients selected from buffers, tonicity adjusters (eg, sodium chloride), pH adjusters, viscosity adjusters, co-solvents (eg, propylene glycol), and surfactants (eg, Lipoid S100).

[0099] Suitably, the aqueous medium comprises at least about 40% water, such as at least about 60% water, at least about 80% water, at least about 95% water or at least about 99% water, such as at least about 99% water.

[0100] Suitably, when Compound I is administered as an aqueous suspension, Compound I is used in crystalline polymorph Form 2. Suitably, the concentration of Compound I in the aqueous suspension formulation is 1-10 mg / mL, for example 4-6 mg / mL.

[0101] Thus, according to one embodiment, there is provided a pharmaceutical composition comprising Compound I used in crystalline polymorph Form 2, particularly in the form of particles suspended in an aqueous medium.

[0102] Local application to the nose or lungs can also be achieved by using a pressurized aerosol formulation. Aerosol formulations typically contain active ingredients suspended or dissolved in suitable aerosol propellants such as chlorofluorocarbons (CFCs) or hydrofluorocarbons (HFCs). Suitable CFC propellants include trichloromonofluoromethane (propellant 11), dichlorotetrafluoromethane (propellant 114) and dichlorodifluoromethane (propellant 12). Suitable HFC propellants include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227). Propellants typically account for 40%-99.5% of the total inhalation composition weight, for example 40%-90%. The preparation may include excipients, including cosolvents (e.g., ethanol) and surfactants (e.g., lecithin, sorbitan trioleate, etc.). Other possible excipients include polyethylene glycol, polyvinyl pyrrolidone, glycerol, etc. Aerosol formulations are packaged in canisters and a suitable dose is provided by a metering valve (for example supplied by Bespak, Valois or 3M, or by Aptar, Coster or Vari).

[0103] Topical administration to the lungs can also be achieved by using a dry powder formulation. The dry powder formulation will contain the compound of the disclosure in the form of particles, typically having an MMAD of 1-10 μm or a D 50, for example about 1-5 μm. Powders of the compounds of the invention in particulate form may be prepared by micronization or similar size reduction methods. Micronization may be performed and tested as described above. The formulations typically contain a topically acceptable diluent, such as lactose, glucose or mannitol (preferably lactose), and typically have a relatively large particle size, such as 50 μm or more, such as 100 μm or more, or D 50 40-150 μm. As used herein, the term "lactose" refers to lactose-containing components, including α-lactose monohydrate, β-lactose monohydrate, anhydrous α-lactose, anhydrous β-lactose and amorphous lactose. The lactose component can be processed by micronization, sieving, grinding, compression, agglomeration or spray drying. It also includes various commercially available forms of lactose, such as (inhalation grade lactose; DFEPharma), (Sieve lactose for dry powder inhalers; Meggle), (DFE Pharma) and (Screening Inhalation Grade Lactose; DFE Pharma) product. In one embodiment, the lactose component is selected from α-lactose monohydrate, anhydrous α-lactose and amorphous lactose. Preferably, the lactose is α-lactose monohydrate.

[0104] The dry powder formulation may also contain other excipients, such as sodium stearate, calcium stearate or magnesium stearate.

[0105] Dry powder preparations are usually delivered using a dry powder inhaler (DPI) device. The example of a dry powder delivery system includes SPINHALER, DISKHALER, TURBOHALER, DISKUS, SKYEHALER, ACCUHALER and CLICKHALER. Other examples of dry powder delivery systems include ECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER, BREEZHALER / NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN, ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIEL dry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN, PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

[0106] In formulations other than aqueous suspensions, such as creams and suppositories, Compound 1 is suitably used in crystalline polymorph Form 1.

[0107] Medical Uses

[0108] Compound I can be used to treat fungal diseases and prevent or treat diseases associated with fungal diseases.

[0109] In one aspect of the present invention, there is provided use of Compound I in the preparation of a medicament for treating fungal diseases and for preventing or treating diseases associated with fungal diseases.

[0110] In another aspect of the present invention, a method of treating a subject suffering from a fungal disease is provided, comprising administering an effective amount of Compound I to the subject.

[0111] In another aspect of the present invention, a method for preventing or treating a disease associated with fungal disease in an individual is provided, comprising administering an effective amount of Compound I to the individual.

[0112] In another aspect of the present invention, there is provided a method for treating an individual suffering from pulmonary or nasal (particularly pulmonary) fungal disease, comprising administering an effective amount of Compound I to the individual by inhalation.

[0113] In another aspect of the present invention, there is provided a method for preventing or treating a disease associated with pulmonary or nasal (particularly pulmonary) fungal disease in an individual, comprising administering an effective amount of Compound I to the individual by inhalation.

[0114] In particular, the fungal disease may be caused by Aspergillus spp., such as Aspergillus fumigatus or Aspergillus pullulans, especially Aspergillus fumigatus. Fungal diseases can also be caused by Candida spp., such as Candida albicans or Candida glabrata; Rhizopus spp., such as Rhizopus oryzae; Cryptococcus spp., such as Cryptococcus neoformans; Chaetomium spp., such as Chaetomium globosum; Penicillium spp., such as Penicillium chrysogenum, and Trichophyton spp., such as Trichophyton rubrum.

[0115] Diseases associated with fungal diseases are, for example, pulmonary aspergillosis.

[0116] Suitably, there is provided Compound I in Form 2 polymorphic form for use in treating fungal diseases and diseases associated with fungal diseases.

[0117] Alternatively, provided is Compound I in Form 1 polymorphic form for use in treating fungal diseases and diseases associated with fungal diseases.

[0118] Compound I can be used in a preventive treatment by administering Compound I prior to the development of a fungal disease. For example, Compound I can be administered prophylactically to individuals at risk for developing a fungal disease, such as premature infants, children with congenital defects of the lungs or heart, immunocompromised individuals (e.g., individuals with HIV infection), asthmatics, individuals with cystic fibrosis, elderly individuals, and individuals with chronic health conditions that affect the heart or lungs (e.g., congestive heart failure or chronic obstructive pulmonary disease).

[0119] Suitably, there is provided Compound I in Form 1 polymorphic form for use in preventing fungal diseases and diseases associated with fungal diseases.

[0120] Alternatively, provided is Compound I in Form 2 polymorphic form for use in preventing fungal diseases and diseases associated with fungal diseases.

[0121] Individuals include human and animal individuals, particularly human individuals.

[0122] The compounds of the invention are particularly useful for treating fungal diseases such as Aspergillus fumigatus infection and for preventing or treating diseases associated with fungal diseases such as Aspergillus fumigatus infection in at-risk individuals. At-risk individuals are as defined above.

[0123] The compounds of the invention may also be used to treat azole-resistant fungal diseases, for example azole-resistant Aspergillus fumigatus infections, particularly in combination with posaconazole.

[0124] The compounds of the invention may be administered in combination with a second or further active ingredient. The second or further active ingredient may, for example, be selected from other antifungal agents (such as voriconazole or posaconazole), amphotericin B, echinocandins (such as caspofungin) and 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (such as lovastatin, pravastatin or fluvastatin).

[0125] Second or additional active ingredients include active ingredients useful in treating or preventing fungal diseases, such as Aspergillus fumigatus infection, or diseases associated with fungal diseases, such as Aspergillus fumigatus infection, or conditions complicated by fungal diseases, such as Aspergillus fumigatus infection.

[0126] The compounds of the present invention may be co-formulated with the second or additional active ingredients, or the second or additional active ingredients may be formulated and administered separately by the same or different routes.

[0127] For example, the compounds of the invention may be administered to a patient already undergoing systemic treatment with an antifungal agent such as voriconazole or posaconazole.

[0128] For example, the compounds of the invention may be co-administered, e.g. co-formulated, with one or more active agents selected from amphotericin B, an echinocandin (such as caspofungin) and a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor (such as lovastatin, pravastatin or fluvastatin).

[0129] The compounds of the invention may alternatively (or additionally) be co-administered, e.g. co-formulated, with one or more active agents selected from candicidin, filipin, hamycin, natamycin, nystatin, cytomycin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, conazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, propiconazole, ravuconazole, terconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, micafungin, benzoic acid, ciclopirox, flucytosine (5-fluorocytosine), griseofulvin, tolnaftate, and undecylenic acid.

[0130] Preferred combination partners include itraconazole, voriconazole, caspofungin and posaconazole.

[0131] According to one aspect of the present invention, a kit of parts is provided, comprising: (a) a pharmaceutical composition comprising a compound of the present invention and optionally one or more diluents or carriers; (b) a pharmaceutical composition comprising a second active ingredient and optionally one or more diluents or carriers; (c) optionally one or more other pharmaceutical compositions, each of which comprises a third or other active ingredient and optionally one or more diluents or carriers; and (d) instructions for administering the pharmaceutical composition to an individual in need thereof. The individual in need thereof may suffer from or be susceptible to a fungal disease, such as an Aspergillus fumigatus infection.

[0132] The compounds of the invention may be administered at suitable intervals, for example once a week, once every two days, once a day, twice a day, three times a day or four times a day.

[0133] A suitable dosage for a human of average weight (50-70 kg) is expected to be about 50 μg to 10 mg / day, for example 500 μg to 5 mg / day, but the precise dosage administered may be determined by the skilled person.

[0134] Compound 1 in crystalline form of polymorph Form 1 is expected to have one or more of the following advantageous attributes:

[0135] ·Exhibits a wide range of thermal stability, as evidenced by a high melting point (>200°C);

[0136] · Easily micronizable while maintaining its polymorphic integrity; and

[0137] • In formulations intended for administration, e.g., other than aqueous suspensions, conversion to Form 2 is prevented.

[0138] Compound 1 in polymorph Form 2 in crystalline form is expected to have one or more of the following advantageous attributes:

[0139] ·Exhibits a wide range of thermal stability, as evidenced by a high melting point (>180°C);

[0140] · Effective bioavailability for topical drugs, particularly those used to treat lung diseases, and showing higher bioavailability than Form 1 when administered by inhalation in an aqueous suspension;

[0141] · Easily micronizable while maintaining its polymorphic integrity; and

[0142] • In aqueous suspensions intended for administration to subjects, conversion to Form 1 is prevented.

[0143] experiment

[0144] abbreviation

[0145] Any undefined abbreviation is intended to convey its generally accepted meaning.

[0146] aq water

[0147] AUC Area under the curve

[0148] BW group average body weight

[0149] conc. concentration

[0150] c-hexane cyclohexane

[0151] D. Exposure time

[0152] DME 1,2-Dimethoxyethane

[0153] DMSO Dimethyl sulfoxide

[0154] DSC Differential Scanning Calorimetry

[0155] Et2O Ether

[0156] EtOH

[0157] Exp

[0158] Evap

[0159] 1 H-NMR Proton Nuclear Magnetic Resonance (Spectroscopy)

[0160] HPLC High Performance Liquid Chromatography

[0161] i-BuOH iso-butanol (2-methyl-1-propanol)

[0162] IPA Isopropyl alcohol (2-propanol)

[0163] K2EDTA Dipotassium Ethylenediaminetetraacetate

[0164] KF Karl Fischer (titration)

[0165] LC-MS / MS Liquid chromatography-mass spectrometry

[0166] LGC Liquid Gas Chromatography

[0167] 2-ME 2-Methoxyethanol

[0168] MeCN Acetonitrile

[0169] MEK Methyl Ethyl Ketone

[0170] MeNO2 Nitromethane

[0171] MIBK Methyl Isobutyl Ketone

[0172] min minutes

[0173] MMAD Mass median aerodynamic diameter

[0174] MTBE Methyl tert-butyl ether

[0175] N / A Not applicable

[0176] NMP N-Methylpyrrolidone

[0177] No

[0178] PLM Polarized Light Microscope

[0179] PrOAc Propyl acetate

[0180] RH Relative humidity

[0181] RSD Relative standard deviation

[0182] RT Room temperature

[0183] RV Reversal

[0184] TBME tert-butyl methyl ether

[0185] temp Temperature

[0186] TGA Thermogravimetric Analysis

[0187] THF Tetrahydrofuran

[0188] vol volume

[0189] SC Single Crystal

[0190] TBME tert-butyl methyl ether

[0191] sec seconds

[0192] UPLC Ultra-Performance Liquid Chromatography

[0193] VT variable temperature

[0194] XRPD X-ray powder diffraction (crystallography)

[0195] Instruments and methods

[0196] The description of the following instruments, methods and steps is relevant to the experiments completed in multiple different laboratories and locations, and therefore it is necessary to use various identical and equivalents with regard to their effects. It will be appreciated by those skilled in the art that many of the parameters described can be changed without hindering the expected results. Therefore, although such instruments, methods and steps described below have been used in some cases, they also constitute exemplary experimental conditions that can be used for implementing the present invention. Therefore, the following instruments, methods and steps should not be interpreted as limiting the present invention in any way.

[0197] X-ray powder diffraction

[0198] X-ray powder diffraction patterns were collected on a PANalytical diffractometer using Cu Kα radiation (45 kV, 40 mA), a θ-θ goniometer, a focusing mirror, a divergence slit (1 / 2”), soller slits (4 mm) at the incident and divergent beams, and a PIXcel detector. The software used for data collection was X’Pert Data Collector, version 2.2f, and data were presented using X’Pert DataViewer, version 1.2d. XRPD patterns were obtained using a PANalytical X’Pert PRO under ambient conditions through a transmission foil stage (polyimide-Kapton, 12.7 μm thick film). The data collection range was 2.994-35° 2θ with a continuous scan rate of 0.202 s -1 .

[0199] Alternatively, X-ray powder diffraction patterns were collected on a Bruker AXS C2 GADDS diffractometer with Cu Ka radiation (40 kV, 40 mA) using an automated XYZ stage, a laser video microscope for automatic sample positioning, and a HiStar 2D area detector. X-ray optics consisted of a single The X-ray beam was composed of a multilayer mirror and a 0.3 mm pinhole collimator. Performance checks were performed weekly using certified standard NIST 1976 corundum (flat plate). The beam divergence was approximately 4 mm. A θ-θ continuous scan mode was used with a sample-to-detector distance of 20 cm, providing an effective 2θ range of 3.2°-29.7°. Typically, the sample was exposed to the X-ray beam for 120 seconds. The software used for data collection was GADDS for XP / 2000 4.1.43, and the data were analyzed and presented using DiffracPlus EVA v15.0.0.0.

[0200] Samples used under ambient conditions were prepared as flat sheets using the unground powder as received. Approximately 1-2 mg of sample was gently pressed onto a glass slide to obtain a flat surface. Samples used under non-ambient conditions were mounted on a silicon wafer with a thermally conductive compound. The sample was then heated to the appropriate temperature at 20°C / min and then held constant for 1 minute before starting data collection.

[0201] X-ray powder diffraction patterns were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), a θ-2θ goniometer, a V4 divergence and receiving slits, a Ge monochromator, and a Lynxeye detector. Instrument performance was checked using a certified corundum standard (NIST 1976). The software used for data collection was Diffrac Plus XRD Commander v2.6.1, and the data were analyzed and presented using Diffrac Plus EVA v15.0.0.0.

[0202] The samples were measured as flat sheet samples under ambient conditions using powder as received. The sample was gently packed into a cavity cut into a polished zero background (510) silicon wafer. The sample was rotated on its own plane during analysis. Data was collected as follows:

[0203] Angle range: 2°~42°2θ

[0204] Step size: 0.05° 2θ

[0205] Acquisition time: 0.5sec / step

[0206] Diamond Light Source X-ray Crystallography

[0207] Data were collected on a Fluid Film Devices 3-circle diffractometer using a Dectris Pilatus 2M detector. Crystals were mounted in MiTeGen rings using perfluoropolyether oil. The wavelength used was All data were collected at 295 K. The structure was solved by routine automatic direct methods and all unique measured F 2 Values ​​are refined.

[0208] 1 H NMR spectrum

[0209] NMR spectra were collected on a Bruker 400MHz instrument equipped with an autosampler and controlled by a DRX400 console. Standard Bruker loading experiments were used and automated experiments were acquired using ICON-NMR v4.0.7 running with Topspin v1.3. For unconventional spectra, data were acquired by using a separate Topspin. Unless otherwise stated, samples were prepared in DMSO-d6. Offline analysis was performed using ACD Spectrus Processor 2012.

[0210] Differential Scanning Calorimetry

[0211] DSC data were collected on a TA Instrument Q2000 equipped with a 50-position autosampler. Heat capacity calibration was performed using sapphire, and energy and temperature calibration was performed using certified indium. Typically, 0.5 to 3 mg of sample contained in a pinhole aluminum pan was heated at 10 ° C / min from 25 ° C to 300 ° C (for slow scan rates), or at 40 ° C / min from 25 ° C to 300 ° C (for fast scan rates). Dry nitrogen was kept purged on the sample at 50 mL / min. The instrument control software was Advantage and Thermal Advantage v5.5.3 of Q Series v2.8.0.394, and the data were analyzed using Universal Analysis v4.5A.

[0212] Thermogravimetric analysis

[0213] TGA data were collected on a TA instrument Q500 TGA equipped with a 16-bit autosampler. Instrument temperature calibration was performed using certified Alumel and nickel. Typically 5-10 mg of sample was loaded into a pre-tared aluminum DSC pan and heated to 350 °C from ambient temperature at 10 °C / min. Nitrogen was purged on the sample at 60 mL / min. The instrument control software was Advantage and Thermal Advantage v5.5.3 of Q Series v2.5.0.256, and Universal Analysis v4.5A was used to analyze the data.

[0214] Chemical purity determination by HPLC

[0215] Purity analysis was performed on an Agilent HP1100 series system equipped with a diode array detector using ChemStation software vb.04.03. The samples were prepared by dissolving in aqueous MeCN (1:1 mixture) heated to 50°C to obtain a 0.5 mg / mL solution. The samples were tested using the parameters given below (Table 2).

[0216] Table 2: HPLC parameters / method for chemical purity determination.

[0217]

[0218] Gradient elution schedule

[0219]

[0220] Preparation of Form 1 and Form 2 polymorphs

[0221] The "crude input material" referred to herein refers to Form 3 synthesized and isolated according to any of the above methods. A representative reference XRPD pattern of this material is shown in Figure 7 as shown and is consistent with a low crystallinity material.

[0222] Micronized Form 1 was prepared by recrystallization in aqueous acetone (5% H2O v / v)

[0223] A crude input material sample (100 g, Form 3) was suspended in a mixture of 5% water in acetone solution (1.00 L), Form 1 seeds (100 mg) were added, and the mixture was stirred at RT overnight. The suspension was filtered, and the filter cake was washed with a mixture of 5% water in acetone solution (20 mL), and the solid was dried at 40° C. to give polymorphic Form 1 in the form of a white powder (85.5 g, 85.5% recovery, purity 98.7%, HPLC detection), as determined by DSC spectrum ( Fig. 9 The product thus obtained is micronized to provide a powder whose particle size distribution expressed as D value is as follows: D 10 =0.75μm; D 50 =1.59μm and D 90 =3.23 μm. XRPD spectrum of micronized solid ( Fig.10 ) confirmed that its polymorphic integrity was preserved during this process.

[0224] Preparation of Form 2 polymorph by ripening in MIBK

[0225] The crude input material (10 g) was suspended in MIBK at RT. The suspension was aged at 25°C to 50°C (hot-cold cycles, 8 h / cycle) for 24 hours. After 3 hours at 50°C, an aliquot was collected, filtered, air-dried and analyzed by XRPD. After 24 hours, the remaining solid was filtered and air-dried for 2 hours at RT. The resulting solid was called Form 2 polymorph. The XRPD pattern of a representative sample is shown in Figure 2. Figure 2 shown.

[0226] Micronized Form 2 was prepared by recrystallization from THF / TBME

[0227] The crude input material (476.4 g) was dissolved in THF (3.82 L) at 45-55° C. under nitrogen atmosphere. The hot solution was filtered through an in-line filter while maintaining an internal temperature of 45-55° C. Over 1.5 hours, TBME (0.72 L) was added dropwise while maintaining an internal temperature of 45-55° C. Seed crystals of Form 2 polymorph (500 mg) were added, and then clear TBME (5.01 L) was added dropwise at 45-55° C. over 10.5 h. After the addition was complete, the mixture was cooled to 0° C. over 3.5 hours. At this temperature, stirring was continued for an additional 7.5 h, and the resulting suspension was vacuum filtered and the solids washed with a mixture of clear THF (380 mL) and TBME (570 mL). The filter cake was sucked dry and the isolated solid was dried under vacuum at 45°C under nitrogen for 21 h to give an off-white solid (401.4 g, 84.2%), which was confirmed to be the Form 2 polymorph by XRPD and DSC analysis ( Fig.11 and Fig.12 ).

[0228] The product thus obtained is micronized to provide a powder having a particle size distribution expressed as D values ​​as follows: D 10 =0.42μM; D 50 =1.24μM and D 90 =3.57 μM. Those skilled in the art will appreciate that these characteristics apply to products intended for administration by inhalation. The maintenance of polymorphic integrity during micronization was confirmed from the XRPD spectra obtained after grinding ( Fig.13 ), the XRPD spectrum did not change significantly compared to the spectrum obtained before particle size reduction ( Fig.11 ). Therefore, the Form 2 polymorph is amenable and stable to micronization processes to obtain a material having the desired properties for the intended purpose.

[0229] Characteristics of Form 1 and Form 2 Polymorphs

[0230] X-ray powder crystallography

[0231] The polymorphic form 1 ( Figure 1 ) and polymorphic form 2 ( Figure 2 ) showed distinct patterns, characteristic of two alternative solid states. The experimentally determined patterns were found to be consistent with those calculated from data collected at a synchrotron source ( Figure 5 and 6 ), from which the crystal structure was obtained. Comparison of the two spectra ( Figure 3 and 4 ) demonstrates the reliability of XRPD analysis in determining the polymorphic nature of a given sample. The following table (Table 3) lists the 2θ values ​​(in degrees), d-spacings, and relative intensities of peaks characterizing Form 1 and Form 2 polymorphs, as derived from their respective XRPD spectra.

[0232] Table 3: Powder X-ray diffraction peaks of polymorphic forms 1 and 2 of Compound I

[0233]

[0234]

[0235] The structures of Forms 1 and 2 were determined by synchrotron X-ray crystallography

[0236] The structure determination of polymorphs Forms 1 and 2 was performed at Diamond Light Source, Oxfordshire, UK. Form 1 data was collected from a 7x12x140 μm single crystal on beam I19. Crystal data and structural optimization parameters for the Form 1 crystallized polymorph are listed in the table below (Table 4), and images of the solved structure viewed along its a-axis are included herein ( Fig.14 ).

[0237] Table 4: Crystal data and structural optimization parameters of polymorph form 1

[0238]

[0239] The Form 2 data was collected from a 5x5x50 μm single crystal on beam I19. The crystal data and structure optimization parameters for the Form 2 crystalline polymorph are listed in the table below (Table 5), and an image of the solved structure viewed along its b-axis is included herein ( Fig.15 ).

[0240] Table 5: Crystal data and structural optimization parameters of polymorph form 2

[0241]

[0242]

[0243] Thermodynamic stability relationship

[0244] Thermodynamic stability of polymorphic forms 1 and 2 at different temperatures

[0245] An equal amount of form 1 and form 2 mixture (100 mg) was added to a crystallization tube, followed by the addition of the selected solvent. The suspension was heated to the temperature shown (Table 6), maintained for 72 h, during which time samples were taken at 24 h intervals. The solid was separated by filtration at the ripening temperature, then vacuum dried at 45 ° C for 20 h. In both cases (Table 6, item 1 and item 2), the suspension was maintained at 135 ° C, and form I was included in the suspension from the 24 h time point to the end of the experiment. In the remaining cases (Table 6, item 4-6), at 72 hours, the solution was formed, and the solid was recovered by slight cooling, and all solids were determined as form 1. Those ripened at lower temperatures all obtained form 2 within 24 h.

[0246] Table 6: XRPD analysis of Form 1 and Form 2 suspensions matured at different temperatures.

[0247]

[0248] Footnotes: a. Determined by XRPD analysis after 72 hours; b. Dissolution occurred at the stated temperature; slight cooling gave a solid.

[0249] Differential Scanning Calorimetry Analysis

[0250] DSC studies were performed according to the method described above.

[0251] Differential scanning calorimetry was performed on a sample of the crude input material at 98.4% purity and the results showed that there were many thermal events when the sample was heated. A small endotherm at 166°C preceded a second larger endotherm at 180°C, followed almost immediately by an exotherm at -183°C, and a final endotherm at -201°C (see Figure 8 These analyses revealed that a series of thermally induced transformations occurred during which the initial, poorly crystalline material (Form 3) transformed into polymorphic Form 2 (mp 180-185°C) and then melted and transformed into Form 1 (mp 201-202°C).

[0252] In order to obtain the heat of fusion values ​​(ΔH f ), a fast scan DSC (using a heating rate of 40°C / min) was used to eliminate the recrystallization events of Form 1 from the Form 2 melt at a slower scan rate (10°C / min). Replicate analysis of the same sample of polymorph (n=10) showed that the average heat of fusion for Form 1 and Form 2 was 72.28 J / g and 77.15 J / g, respectively (Table 7).

[0253] Table 7: Melting data of Form 1 and Form 2 fast scan DSC patterns

[0254]

[0255] Fig.16 A fast scan DSC pattern of the polymorph Form 1 sample is included and is shown to have a melting point of 202.6°C. Similarly, Fig.17 A fast scan DSC graph of a sample of the polymorph Form 2 is shown and shows Form 2 to have a melting point of 182.6°C.

[0256] The Burger and Ramburger (1979) rule states: "If the higher melting form has the lower heat of fusion, then the two forms are generally enantiotropic, otherwise they are unitropic". DSC analysis of multiple batches confirmed that the heat of fusion of Form 1 was always lower than that of Form 2, from which it can be concluded that the polymorphs Forms 1 and 2 are enantiotropically related. It can be seen that Form 2 is the more thermodynamically stable state (with the lowest Gibbs free energy) from absolute zero to the transition point, which is the temperature at which Forms 1 and 2 have equal thermodynamic stability. Above this transition temperature, before the melting point is reached, the Form 1 polymorph is thermodynamically more preferred.

[0257] Thermogravimetric analysis (TGA)

[0258] TGA analysis was performed according to the method described above and was used to determine the amount of residual solvent associated with the polymorphs of Compound 1. In addition, it was also used to measure the amount of solvent released by the solvated forms of Compound 1 upon heating. Typical residual solvent levels found in samples of polymorphs of Compound 1 are as follows.

[0259] The Form 1 polymorph prepared from toluene lost 0.3% w / w weight when heated at 25-120°C, consistent with the loss of 0.1 mol of water. Degradation was evident at 320°C (data not shown).

[0260] The Form 2 sample from EtOH showed a weight loss of 0.2% w / w in the range of 25-150°C, which was attributed to the loss of 0.03 mol of EtOH. Compound I began to degrade at 320°C. The Form 2 polymorph material isolated from MIBK showed no weight loss until the sample began to degrade at 320°C ( Fig.18 When the sample was heated from 25°C to 160°C, the anisole solvate experienced a loss of 13.4% w / w (data not shown).

[0261] Pulping and cross-inoculation experiments

[0262] A mixture of Form 1 and Form 2 (50% w / w) was prepared and a sample of the mixture (20 mg) was then suspended in a solvent system (50 vol, 1.0 mL) and shaken at different temperatures for 96 h. The solid was filtered, dried in air and analyzed by XRPD. The results obtained (Table 8) show that in all the systems studied, except one (entries 10-12), the polymorphic mixture had transformed to Form 2 when kept at room temperature and 50°C. The transformation was found to be particularly easy in propyl acetate, as Form 2 (entry 1) had been isolated from the suspension when kept at 5°C. It is noteworthy that the transformation was found to occur not only in the solvent and TBME (entries 4-6), but also in the aromatic solvent toluene (entries 7-9), which previously delivered Form 1 from the crude input material (Form 3). The material recovered from the aqueous isopropanol solution was a mixture of the two forms (entries 10-12).

[0263] Table 8: Cross-inoculation experiments of Form 1 and Form 2 at 5°C, 25°C and 50°C.

[0264]

[0265] Footnote: All samples remained in suspension during the ripening process.

[0266] This observation suggests that either the solubility of Compound 1 in this solvent system is too low to allow complete conversion, or that the conditions are inherently unfavorable for the conversion of Form 1 to Form 2. To determine the effect of water in determining the yield of the conditioning experiments described, the behavior of the Form 2 polymorph was examined in nominally dry organic media, and also in a single-phase aqueous mixture of a water-miscible solvent.

[0267] When the Form 2 solid was suspended in heptane, TBME, toluene as the sole solvent or in cyclohexane at 25°C or 50°C for 24 h, there was no apparent change in the crystal structure. The same results were obtained when the conditioning medium was pure acetone (Table 9, entries 1 and 6) or water alone (Table 9, entries 5 and 10). However, over the course of 24 h in an acetone-water mixture at 50°C, there was clear evidence that Form 2 had transformed to Form 1 in both cases (Table 9, entries 7 and 9). This result was unexpected because this polymorph of Compound I is metastable compared to Form 2 in this temperature range.

[0268] Table 9: Fate of Form 2 slurried in acetone, water and acetone / water mixtures.

[0269]

[0270] To investigate the universality of this unforeseen transformation, similar experiments were performed in THF and THF / water mixtures. Consistent with the data disclosed above, it was found that polymorph Form 2 of Compound I alone in THF at 25°C and 50°C for at least 4h had no effect on the polymorph composition of the mixture. However, the addition of water showed a significant effect on the conversion of Form 2 polymorph to Form 1, with the conversion observed to occur at both lower and higher temperatures (Table 10). At 25°C, those mixtures containing 10% water and 20% water returned to Form 1 after 4h and 24h, respectively (Table 10, entries 2 and 3). Remarkably, at 50°C, all aqueous THF mixtures resulted in the formation of Form 1 crystalline state (Table 10, entries 6-8).

[0271] Table 10: Fate of Form 2 slurried in THF and THF / water mixtures.

[0272]

[0273] Footnote: nd means not determined (data not collected)

[0274] It is clear that the presence of water induces the polymorphic transitions reported above. However, except at low levels, water is an effective antisolvent in the system and significantly reduces the solubility of the drug in aqueous solvent mixtures as its proportion increases. The result of this solubility profile is that the conversion process from Form 2 to Form 1 in water alone or in water-rich solvent mixtures either does not occur at all or is inhibited by marginal solubility, especially at ambient temperature. For acetone, and especially in THF, increasing temperature increases the solubility of the input Form 2 and accelerates its conversion to the Form 1 state.

[0275] A similar, revealing pattern of interconversion was derived from an experiment in which Form 2 was slurried in various mixtures of dioxane and water at ambient and elevated temperatures (Table 11). In the presence of 10% water, Form 1 polymorph was evident in the slurry mixture within 1 hour at 25°C. However, when the water content was increased to 40%, the transformation was delayed to such an extent that only the sample collected at 24 h showed the presence of Form 1. The ternary system behaved similarly at 50°C. However, at a higher temperature of 65°C, the conversion rate increased, so that Form 1 was produced after 3 h. Consistent with the data obtained from the above experiments, increasing the proportion of water to 80% in this system was sufficient to stop the interconversion of the polymorphs at 25°C, 50°C, and even 65°C.

[0276] Table 11: Fate of Form 2 slurried in 1,4-dioxane / water mixtures.

[0277]

[0278]

[0279] Trace Form 2 Remaining

[0280] The maturation of Form 2 solids in MEK at 25°C and 50°C revealed the interaction of the nature of the organic solvent, the presence of adventitious water, and the slurry temperature (and by inference the contact time), where the water content in MEK was gradually increased from 1% (v / v) to 8% (v / v) (Table 12). At lower temperatures, the integrity of Form 2 was observed to be maintained for at least 24 h until the water content exceeded 6%. This was despite the solubility of Compound 1 increasing from 19 mg / ml to 30 mg / ml (data not shown). Once the proportion of water reached 8%, the slurry mixture returned to the Form 1 polymorph even though the solubility of the drug was reduced to 23 mg / mL.

[0281] Increasing the temperature to 50°C accelerates the conversion process, so for mixtures with a water content of more than 2%, polymorphic interconversion can be seen at the 24 hour time point. It is clear from the data that the environmental conditions to which Compound I is exposed not only determine which of the two polymorphs (Form 1 or Form 2) is produced, but also determine the rate at which the interconversion occurs.

[0282] Table 12: Fate of Form 2 slurried in MEK / water mixtures.

[0283]

[0284]

[0285] The effect of the initial polymorphic composition on the fate of the samples was investigated by equilibrating either a single polymorph or an equal mixture of the two polymorphs in anhydrous MEK and THF, as well as the same solvents spiked with 5% water.

[0286] Table 13: Equilibria of Form 1 and Form 2 alone and as a mixture in MEK and THF alone and in aqueous MEK and THF.

[0287]

[0288] a. The results are the same at both equilibrium temperatures unless otherwise stated. b. Some evidence for Form 2 at 10°C.

[0289] The results obtained (Table 13) show that in anhydrous media, both polymorphs retain their structural integrity if present alone (Table 13, entries 1, 2, 4 and 5). Some weak signs of conversion were found in THF at 10°C (Table 13, entry 4), which may be due to the presence of trace amounts of water in the highly hygroscopic solvent. However, at both temperatures, the mixture of the two polymorphs converted to Form 2 in MEK or THF (Table 13, entries 3 and 6), indicating that Form 2 is preferred under anhydrous or near anhydrous conditions.

[0290] When 5% water was mixed into both solvents, the results observed indicated that under these conditions, the dominance of the two forms was opposite, taking into account kinetic factors. In aqueous MEK (5% water), the single polymorph again remained unchanged during the experiment (Table 13, entries 7 and 8). In contrast, in the medium, the binary polymorph mixture converted to Form 1 (Table 13, entry 9). In aqueous THF (5% water), the dominance of Form 1 as the most preferred polymorph was particularly evident. In this environment, the Form 1 suspension remained unchanged (Table 13, entry 10), consistent with the results observed for aqueous MEK under the same conditions.

[0291] However, in THF spiked with 5% water, suspensions containing the binary mixture, as well as suspensions consisting of the Form 2 polymorph alone, transformed into suspensions containing only Form 1 (Table 13, entries 11 and 12). These data are particularly surprising because the presence of water, a minor component in the mixture, overwhelms the stability ranking in favor of Form 2 when it is absent. This result is unexpected because we have disclosed elsewhere herein evidence that the Form 2 polymorph is the more thermodynamically stable enantiotrope of Compound 1 within the temperature range of this experiment (5-10°C) or below temperatures of at least 70°C.

[0292] Comparison of the pharmacokinetics of Form 1 and Form 2 administered by inhalation to rats

[0293] A study was conducted to compare the time course of unchanged drug concentrations in plasma following a single inhaled dose of Form 1 or Form 2 in rats.

[0294] Male RccHan TM:WIST rats are considered to be a suitable species and strain because they are approved by regulatory agencies. The inhalation route of administration is selected to simulate clinical application conditions. A total of 14 animals (6 in each group, 2 spare animals) were used in this study, the animals were 10 to 12 weeks old, and the weight was 306g to 341g when the drug treatment began. Treatment included a single 2h inhalation exposure, and the micronized form 1 (98.94% purity) aerosol formulation was applied to the first group of animals or the micronized form 2 (98.10% purity) was applied to the second group of animals. The pharmacokinetic parameters of the two polymorphic forms were determined to support the pharmacology and toxicology studies of animals and clinical studies of humans.

[0295] The suspension is produced by first preparing a concentrated solution of the surfactant (wetting solution) and a saline solution. This is then used to prepare the vehicle. The saline solution is prepared as follows. Sodium chloride (50 g) is weighed into a 5000 mL volumetric flask, made up to volume with water, stirred with a magnetic stirrer for 10 min, and then filtered through a 0.22 μm membrane filter. The wetting solution is prepared as follows. Lipoid S100 (10 g) is weighed into a 2000 mL Duran bottle, 1000 g of propylene glycol is added, and the mixture is stirred at 8000 rpm for 1 min on a Silverson mixer equipped with a 1" high shear screen, and then filtered through a 0.22 μm membrane filter. The vehicle is prepared as follows. The wetting solution (500 mL) is transferred to a 5000 mL volumetric flask, made up to volume with saline solution, stirred with a magnetic stirrer for 10 min, and then filtered through a 0.22 μm membrane filter.

[0296] A 1000 mL batch of a 4 mg / mL Compound I suspension was prepared as follows. Compound I (4.00 g) was weighed into a small beaker, 100 mL of wetting solution was added, and the mixture was stirred at 8000 rpm for 5 min using a Silverson mixer equipped with a 1" high shear screen. The mixture was then transferred to a 5000 mL Duran bottle with saline solution and stirred at 8000 rpm for 5 min using a Silverson mixer equipped with a 1" high shear screen and then stored at 2-8°C. Higher or lower strength suspensions (0.2 mg / mL to 20 mg / mL) were prepared as needed by adjusting the input weight of the API for higher mg / mL suspensions or further diluting with the solvent for lower mg / mL suspensions. All formulations were magnetically stirred for at least 30 min and visually evaluated before administration.

[0297] Before administration, rats were adapted to the inhalation administration procedure for three consecutive days. Animals were treated with the test substance for 120 min by aerosol inhalation administration with only oral and nasal exposure at a target aerosol concentration of 25.2 μg / L. The estimated inhalation dose was calculated using the following formula with an exposure time of 2 hours and an assumed body weight of 300 g:

[0298]

[0299] Where: C is the aerosol concentration (μg / L); RMV is the respiratory volume per minute (L / min); D is the exposure time (120 min), and BW is the group mean body weight (kg). The summary data of the average aerosol concentration, estimated inhaled dose and particle size distribution (PSD) obtained are shown below (Table 14).

[0300] Table 14: Indoor aerosol concentrations and estimated inhaled doses of Compound I obtained during aerosol inhalation administration.

[0301]

[0302] Footnotes: MMAD. Mass median aerodynamic diameter; σg. Geometric standard deviation

[0303] As shown in Table 14, a target aerosol concentration of 25.2 μg / L was selected to deliver a nominal dose of 2.2 mg / kg. An actual aerosol concentration of 28.8 μg / L was obtained for Form 1, providing an estimated inhaled dose of approximately 2.48 mg / kg to Group 1 animals. The Form 2 aerosol concentration was 32.6 μg / L, thus delivering an estimated inhaled dose of approximately 2.82 mg / kg to Group 2 animals.

[0304] The aerosol concentrations obtained were close to the target values, and the estimated inhaled doses were 113% and 128% of the target, respectively, for Group 1 and Group 2. The particle size distribution confirmed that the generated aerosol was inhalable to rats.

[0305] Venous blood samples (0.3 mL) were collected from the tail vein of the animals and after administration (2 h after the start of administration), then 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 28 h, 32 h, 36 h and 48 h after treatment. The samples were treated with K2EDTA anticoagulant, spun at 2000 g for 10 minutes at 4 ° C, and then stored frozen (-20 ° C ± 10) and waited for analysis. Plasma samples were then analyzed for compound I using LGC using a validated LC-MS / MS method.

[0306] Extraction and analytical methods

[0307] The rat plasma sample was vortexed and a 25 μL aliquot was taken and treated with 12.5 μL of internal standard working solution (containing 20 ng / mL compound 2) and 25 μL of 10 mM aqueous ammonium formate solution. The mixture was vortexed at 1400 rpm for 5 min, after which 300 μL of MTBE was added and the sample was tumbled for 10 min. After centrifugation at 3500 g for 5 min, a 150 μL portion of the organic layer was taken and evaporated to dryness at 50 ° C. under a nitrogen atmosphere for approximately 15 min. The residue was reconstituted in a mixture of 100 μL of acetonitrile and water (50:50) containing 0.1% formic acid and vortexed at 1400 rpm for 5 min. The samples prepared in this manner were then analyzed using validated LC-MS / MS to determine the original plasma concentration of compound I.

[0308] The LC system consisted of an Acquity Binary SolventManager equipped with an Acquity UPLC C8 (50 x 2.1 mm) analytical column maintained at a nominal temperature of 40° C. Samples were analyzed by gradient elution (Table 15) with a run time of 2.3 minutes using acetonitrile containing 0.1% formic acid (mobile phase a) and water containing 0.1% formic acid (mobile phase B) at a flow rate of 0.8 mL / min.

[0309] Table 15: HPLC eluent gradient profiles.

[0310]

[0311] Compound 1 was used as a reference standard and the tetradeuterium derivative Compound 2 was used as an internal standard. Compound 2 is: 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl-2,3,5,6-d4)benzamide.

[0312]

[0313] All instrument control, data collection, peak area integration, and storage were performed using MassLynx (version 4.1). The mass spectrometer response (peak area ratio of analyte to internal standard) for each calibration standard was calculated using Watson LIMS (version 7.2) and plotted relative to the labeled (prepared) concentration. Weighted (1 / x 2 ) The calibration curve equation was calculated by least squares linear regression analysis. The concentration of compound I in the sample was back-calculated to 3 significant figures from the calibration curve. Microsoft Excel 2010 was used for further calculations. All percentages were displayed to one decimal place.

[0314] Pharmacokinetic analysis was performed using Phoenix WinNonlin. The maximum mean plasma concentration (C max ) and its occurrence time (T max The area under the mean plasma concentration-time curve until the time of the last quantifiable sample (AUC t There were no adverse reactions after drug administration, and serial blood samples were collected within 48 hours after the start of inhalation exposure.

[0315] The mean pharmacokinetic parameters of the two polymorphs following their inhalation administration to rats are summarized in Table 16 and described in more detail below.

[0316] Table 16: Mean pharmacokinetic parameters of Form 1 and Form 2 of Compound I following inhalation administration to rats.

[0317]

[0318] Footnote: a. Two AUCs t The values ​​are all 0-48h; b. AUC of Form 2 normalized to the dose of Form 1 (2.48 mg / kg) t .

[0319] Plasma concentration

[0320] Following inhalation administration of Form 1 and Form 2, drug plasma concentrations were quantifiable up to 48 h after the start of inhalation exposure in both groups and are shown below (Tables 17 and 18, respectively). The resulting mean plasma concentration-time curves are presented graphically ( Fig.19 and 20 for Form 1 and Form 2 respectively).

[0321] Table 17: Plasma drug concentrations following inhalation administration of Form 1 to male rats at a nominal dose level of 2.2 mg / kg.

[0322]

[0323] Footnotes: a. From inhalation exposure; IAD: immediately after administration; NS: no sample available.

[0324] Table 18: Plasma drug concentrations following inhalation administration of Form 2 to male rats at a nominal dose level of 2.2 mg / kg.

[0325]

[0326]

[0327] Footnotes: a. From inhalation exposure; IAD: immediately after administration; NS: no sample available.

[0328] The mean plasma concentration of Form 1 (Group 1) increased to a maximum 6 h after the start of exposure and then steadily decreased until the last sampling time 48 h after the start of exposure. max was 4.95 ng / mL, and the average AUC t The value was 122 ng.h / mL. The mean plasma concentration of form 2 increased to a maximum 6 hours after the start of exposure and then steadily decreased until 48 hours after the start of exposure. The mean C of form 2 max was 8.59 ng / mL, and the average AUC t The value is 230ng.h / mL. The two polymorphs have similar T max values, but the individual mean plasma concentrations at all 11 sampling times were greater in the rat group administered Form 2 compared to the Form 1 data. Therefore, for Form 2, the dose-normalized C max and AUC t The values ​​are 1.5 and 1.7 times higher respectively.

[0329] Relative bioavailability

[0330] The relative bioavailability (RF) of the two polymorphs after inhalation administration was calculated using the following formula:

[0331] RF=AUC t (Form 2) / AUC t (Form 1) x Achieved Dose (Form 1) / Achieved Dose (Form 2)

[0332] Dose-normalized AUC of Form 2 polymorph relative to Form 1 following inhalation administration to rats t The values ​​(Table 16) indicated a relative bioavailability of 166%.

[0333] This study demonstrated that both crystalline forms were systemically available via the inhaled route and that exposure lasted up to 48 h after the start of administration. However, it is noteworthy that Form 2 ( Fig. 20 ) is always better than form 1 ( Fig.19 ) has a higher systemic drug concentration. Dose normalization of Form 2 polymorph C max Value and AUC t The values ​​are approximately 1.5 and 1.7 times higher than those of Form 1, respectively.

[0334] Given its superior bioavailability, the Form 2 polymorph is expected to be useful in the treatment of fungal diseases where rapid dissolution and amplified drug concentrations at the site of therapeutic action are desired. In aqueous suspensions, Form 2 appears to be prevented from converting to Form 1, although Form 2 is believed to be metastable in this environment, as described above. Therefore, the Form 2 polymorph is particularly suitable for administration by inhalation as an aqueous suspension.

[0335] It is also contemplated that Form 1 may be used to treat fungal diseases, as exemplified by the systemic bioavailability of the polymorphic form. In addition, it is contemplated that the Form 1 polymorph may be used in formulations other than aqueous suspensions, such as creams and vaginal suppositories. Such administration may be useful for preventing fungal diseases in individuals at risk of contracting fungal diseases, such as those affecting the skin and other areas where topical administration may be applied.

[0336] Data Summary

[0337] The present application discloses two novel polymorphic forms of Compound 1: Form 1 and Form 2, both of which have good thermal stability, as evidenced by their high melting points (see Table 7). In addition, both polymorphs have acceptable bioavailability when administered by inhalation as an aqueous suspension, although the bioavailability of Form 2 is significantly higher than that of Form 1 (see relative bioavailability data above). Both crystalline forms are suitable for micronization to achieve a particle size distribution consistent with inhalation administration (see Table 7). Fig.10 and Fig.13 ). The Form 1 polymorph is expected to be particularly suitable for use in formulations other than aqueous suspensions. The Form 2 polymorph is particularly suitable for use in aqueous suspension formulations, such as those intended for administration by inhalation.

[0338] Throughout the specification and appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprising" and "containing", will be understood to imply the inclusion of stated integers, steps, groups of integers or groups of steps but not the exclusion of any other integers, steps, groups of integers or groups of steps.

[0339] All patents, patent applications, and references mentioned in the present specification are incorporated herein by reference in their entirety.

[0340] The present invention includes the above-mentioned preferred and more preferred groups, as well as all combinations of suitable and more suitable groups or embodiments of the above-mentioned groups.

[0341] References

[0342] Burger A. and Ramburger R. On the polymorphism of pharmaceuticals and other molecular crystals. I Theory of Thermodynamic Rules. Mikrochim. Acta, 1979, 72, 259-271。

[0343] Sunose M., Colley T.C., Ito K., Rapeport G. and Strong P. WO 2016 / 087878, June 9, 2016; PCT / GB2015 / 053731A1, December 4, 2015。

[0344] Colley T.C., Ito K., Rapeport G., Strong P., Murray P.J., Onions S.T. and Sunose M., WO 2016 / 087880 A1, June 9, 2016; PCT / GB2015 / 053733, December 4, 2015。

[0345] Colley T., Sehra G., Daly L., Kimura G., Nakaoki T., Nishimoto Y., Kizawa Y., Strong P., Rapeport G. and Ito K. Antifungal synergy of a topical triazole, PC945, with a systemic triazole against respiratory Aspergillus fumigatus infection. Nature Scientific Reports, 2019, 9:9482。

[0346] Colley T., Alanio A., Kelly, S.L., Sehra G., Kizawa Y., Warrilow A.G.S., Parker J.E., Kelly D.E., Kimura G., Anderson-Dring L., Nakaoki T., Sunose M., Onions S., Crepin D., Lagasse F., Critall M., Shannon J., Cooke M., Bretagne S., King-Underwood J., Murray J., Ito K., Strong P., Rapeport G. In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole on Aspergillus fumigatus Infection. Antimicrob. Agents Chemother., 2017, 61: e02280-16。

[0347] Shivaprakash M., Rudramurthy S.M., Colley T., Abdolrasouli A., Ashman J., Dhaliwal M., Kaur H., Armstrong-James D., Strong P., Rapeport G., Schelenz S., Ito K. and Chakrabarti A. In vitro antifungal activity of a novel topical triazole PC945 against emerging yeast Candida auris. J. Antimicrob. Chemother., 2019, 74(10); 2943-2949。

[0348] Kimura G.,Nakaoki T.,Colley T.,Rapeport G.,Strong P.,Ito K.,KizawaY.In vivo Biomarker Analysis of the Effects of Intranasally Dosed PC945,aNovel Antifungal Triazole,on Aspergillus fumigatus Infection inimmunocompromised mice.Antimicrob.Agents Chemother.,2017,61:e00124-17。

Claims

1. Compound of formula (I): It is a crystalline form of 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide, wherein the crystalline form is polymorph Form 1, and wherein the crystalline form has an X-ray powder diffraction pattern comprising peaks at (±0.2) 7.0, 7.4, 7.9, 10.17, 11.60, 16.28, 16.66, 17.85, 18.2, 19.7, 20.8, 23.32, 24.7 ° 2-θ as measured using Cu Ka radiation.

2. A compound according to claim 1 which is in a form having an X-ray powder diffraction pattern substantially as shown in Figure 1 (Form 1).

3. The compound according to claim 1 or 2, wherein the crystalline form has and The unit cell size is , and the α, β and γ angles are 90°.

4. Compounds of formula (I): It is a crystalline form of 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide, wherein the crystalline form is polymorph Form 2, and wherein the crystalline form has an X-ray powder diffraction pattern comprising peaks at (±0.2) 10.8, 11.54, 15.88, 16.61, 17.0, 18.72, 19.08, 19.52, 20.3, 21.28, 21.66, 22.7, 23.9, 24.3, 26.56 and 26.88 ° 2-θ as measured using Cu Ka radiation.

5. A compound according to claim 4 which is in a form having an X-ray powder diffraction pattern substantially as shown in Figure 2 (Form 2).

6. The compound according to claim 4 or 5, wherein the crystalline form has and The unit cell dimensions are , as well as an α angle of 90°, a β angle of 101.54° and a γ angle of 90°.

7. Use of a compound according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of fungal diseases or for the prevention or treatment of diseases associated with fungal diseases, wherein the compound according to any one of claims 1 to 6 is administered in combination with a second or further active ingredient.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and optionally one or more pharmaceutically acceptable diluents.

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and optionally one or more pharmaceutically acceptable carriers.

10. A pharmaceutical composition according to claim 8 or 9, wherein the compound of claims 1 to 6 is in the form of particles suspended in an aqueous medium.

11. The pharmaceutical composition of claim 10, wherein the aqueous medium comprises at least 80% water.

12. The pharmaceutical composition of claim 11, wherein the aqueous medium comprises at least 95% water.

13. The pharmaceutical composition of claim 10, wherein the aqueous medium comprises a charged tonicity adjusting agent.

14. The pharmaceutical composition according to claim 13, wherein the charged tonicity adjusting agent is sodium chloride.

15. The pharmaceutical composition of claim 10, wherein the aqueous medium comprises a co-solvent.

16. The pharmaceutical composition of claim 15, wherein the co-solvent is propylene glycol.

17. The pharmaceutical composition of claim 10, wherein the aqueous medium comprises a surfactant.

18. The pharmaceutical composition according to claim 17, wherein the surfactant is Lipoid S100.

19. A pharmaceutical composition according to claim 8 or 9, comprising a second or further active ingredient.

20. The use of a compound according to claim 7 or a pharmaceutical composition according to claim 19, wherein the second or further active ingredient is an antifungal agent or a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor.

21. The use according to claim 20, wherein the antifungal agent is amphotericin B or an echinocandin.

22. The use of a compound according to claim 7 or a pharmaceutical composition according to claim 19, wherein the second or further active ingredient is selected from voriconazole, posaconazole, caspofungin, lovastatin, pravastatin and fluvastatin.

23. The use of a compound according to claim 7 or the pharmaceutical composition according to claim 19, wherein the second or other active ingredient is selected from candida albicans, filipin, hamycin, natamycin, nystatin, cytomycin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, propiconazole, ravuconazole, terconazole, abafungin, amorolfine, butenafine, naftifine, terbinafine, anidulafungin, micafungin, benzoic acid, ciclopirox, flucytosine, griseofulvin, tolnaftate and undecylenic acid.

24. Use of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 8 to 23 in the preparation of a medicament for treating fungal diseases or for preventing or treating diseases associated with fungal diseases.

25. The use according to claim 7 or 24, wherein the fungal disease is caused by Aspergillus spp.

26. Use according to claim 25, wherein the Aspergillus species is Aspergillus fumigatus or Aspergillus pullulans.

27. Use according to claim 26, wherein the Aspergillus species is Aspergillus fumigatus.

28. Use according to claim 27, wherein the Aspergillus species is azole-resistant Aspergillus fumigatus.

29. The use according to claim 7 or 24, wherein the fungal disease is caused by Candida spp.; Rhizopus spp.; Cryptococcus spp.; Chaetomium spp.; Penicillium spp. or Trichophyton spp.

30. The method of claim 7 or 24, wherein the fungal disease is caused by Candida albicans, Candida glabrata, Rhizopus oryzae, Cryptococcus neoformans, Chaetomium globosum, Penicillium chrysogenum or Trichophyton rubrum.

31. A method of preparing a crystalline form of Compound 1, wherein the crystalline form is polymorph Form 2 according to claim 4, the method comprising the step of crystallizing Compound 1 as polymorph Form 2 from a solution of Compound 1 in a nominally dry organic solvent, wherein the solvent is: (i) is selected from the group consisting of MIBK, THF, PrOAc, EtOH, DME, MeOH, MeCN, MeNO2, n-heptane, Et2O, IPA, MEK, acetone, TBME and i-BuOH; (ii) a mixture comprising two or more solvents selected from the group consisting of MIBK, THF, PrOAc, EtOH, DME, MeOH, MeCN, MeNO2, n-heptane, Et2O, IPA, MEK, acetone, TBME and i-BuOH; (iii) a mixture of THF and toluene; or (iv) A mixture of THF and TBME.

32. The process of claim 31 , wherein the solvent is a mixture of THF and TBME.

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