N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide in solid form

Abstract

This invention relates to novel forms of N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide (compound 1). In particular, this invention relates to type A and type B crystalline polymorphs and amorphous forms.

Description

Technical Field

[0001] This invention relates to novel forms (including crystalline forms) of compounds having activity as a positive modulator of calcium-activated chloride ion channels (CaCC), TMEM16A. The invention also relates to methods for preparing novel forms and pharmaceutical compositions containing them, and their use in treating diseases and conditions in which TMEM16A functions (particularly respiratory diseases and conditions). Background Technology

[0002] Humans inhale up to 12,000 liters of air daily, which means there's a possibility that airborne pathogens, such as bacteria, viruses, and fungal spores, can enter the airways. To prevent these airborne pathogens, the lungs have evolved innate defense mechanisms to minimize the possibility of airway infection and colonization. One such mechanism is the mucus clearance system, where secreted mucus is propelled upwards into the airways by the coordinated pulsation of cilia and coughing. This continuous lung "cleaning" constantly removes inhaled particles and microorganisms, thereby reducing the risk of infection.

[0003] In recent years, it has become clear that the hydration of mucus gels is crucial for mucus clearance (Boucher 2007; Matsui et al., 1998). In normal, healthy airways, mucus gels typically contain 97% water and 3% w / v solids, under which conditions mucus is cleared via mucociliary action. Hydration of the airway mucosa is regulated by the synergistic activity of multiple ion channels and transport proteins. Anion (Cl) transport is mediated via cystic fibrosis transmembrane conduction regulator (CFTR) and calcium-activated chloride ion transport (CaCC; TMEM16A). - / HCO3 - ) secretion balance and through epithelial Na + Na through channel (ENaC) + Absorption determines the hydration state of the airway mucosa. When ions are transported across the epithelium, water must follow due to osmotic pressure, thus fluid is either secreted or absorbed.

[0004] In respiratory diseases such as chronic bronchitis and cystic fibrosis, the solid percentage of mucus gel increases with decreased hydration and mucus clearance (Boucher, 2007). In cystic fibrosis, loss-of-function mutations in the CFTR impair the airway's ability to secrete fluid, with the solid percentage potentially increasing to 15%, which is believed to lead to small airway obstruction and mucus clearance failure. Strategies to increase airway mucus hydration include stimulating anion secretion, thereby leading to fluid secretion, or inhibiting sodium secretion. +Absorption. To this end, stimulating the activity of TMEM16A channels will increase anion secretion, thereby increasing fluid accumulation in the airway mucosa, hydrating mucus, and enhancing mucus clearance mechanisms.

[0005] TMEM16A (also known as Anoctamin-1 (Ano1)) is a molecular property of calcium-activated chloride channels (Caputo et al. 2008; Yang et al. 2008). TMEM16A channels open in response to elevated intracellular calcium levels, allowing bidirectional flow of chloride, bicarbonate, and other anions across the cell membrane. Functional TMEM16A channels have been proposed to regulate transepithelial ion transport, gastrointestinal motility, nociception, and cell migration / proliferation (Pedemonte & Galietta, 2014).

[0006] The TMEM16A channel is expressed in epithelial cells of various organs, including the lungs, liver, kidneys, pancreas, and salivary glands. In the airway epithelium, TMEM16A is highly expressed in mucus-producing goblet cells, ciliated cells, and submucosal glands. Physiologically, TMEM16A is activated by stimulation that mobilizes intracellular calcium ions, particularly purinergic agonists (ATP, UTP), which are released by the respiratory epithelium in response to periodic shear stress induced by respiration and other mechanical stimuli, such as coughing. Besides increasing anion secretion leading to enhanced airway hydration, TMEM16A activation also plays a crucial role in bicarbonate secretion. Bicarbonate secretion has been reported to be an important regulator of mucus properties and controls airway lumen pH, thereby controlling the activity of natural antimicrobial agents such as defensins (Pezzulo et al., 2012).

[0007] Indirect regulation of TMEM16A via elevated intracellular calcium ions has been explored clinically, for example with denufosol (Kunzelmann & Mall, 2003). Although encouraging preliminary results were observed in small patient populations, this approach has not yielded clinical benefit in larger populations (Accurso et al., 2011; Kellerman et al., 2008). This lack of clinical effect is attributed solely to a transient increase in anion secretion, the short half-life of denufosol on the epithelial surface, and the result of receptor / pathway desensitization, as well as the adverse effects of elevated intracellular calcium ions (e.g., increased mucus release from goblet cells) (Moss, 2013). A compound that acts directly on TMEM16A to enhance channel opening caused by elevated low calcium ion levels holds promise for a sustained enhancement of anion secretion and mucociliary clearance in patients and improved innate defenses. Since TMEM16A activity is independent of CFTR function, TMEM16A positive modulators have the potential to provide clinical benefit to all CF patients and non-CF respiratory diseases characterized by myxemia (including chronic bronchitis and severe asthma).

[0008] TMEM16A modulation is considered a treatment for xerostomia, which is caused by salivary gland dysfunction, dry eye, cholestasis, and gastrointestinal motility disorders resulting from Sjorgen's syndrome and radiation therapy.

[0009] The inventors have developed novel compounds and new forms of these compounds that are positive modulators of TMEM16A, and therefore can be used to treat diseases and conditions in which TMEM16A plays a role, particularly respiratory diseases and conditions. These compounds were first described in our earlier application WO2019 / 145726, the contents of which are incorporated herein by reference in their entirety. In particular, WO2019 / 145726 discloses N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide (compound 1), which has the following structural formula:

[0010] Summary of the Invention

[0011] The inventors have now developed a new form of compound 1.

[0012] In a first aspect of the invention, compound 1 is provided in the form of its type A anhydrous solid crystalline polymorph.

[0013] In a second aspect of the invention, compound 1 is provided in the form of its type B hydrated solid crystalline polymorph, particularly type B(I) hydrated solid crystalline pseudopolymorph or type B(II) hydrated solid crystalline pseudopolymorph.

[0014] In a third aspect of the invention, an anhydrous solid amorphous compound 1 is provided. Attached Figure Description

[0015] Figure 1a The XRPD diffraction pattern of solid crystalline polymorph A of compound 1 is shown, ranging from 5 to 60 2-θ, with a step size of 0.02 s (reflection mode, offset 500 count).

[0016] Figure 1b for Figure 1a A magnified view.

[0017] Figure 2 The DSC thermogram of polymorph A of compound 1 is shown. The thermogram was obtained at a rate of change of +10 °C / min (where the input weight of compound 1A is 12.7 mg) and shows: integral -378.65 mW °C, melting onset temperature 192.94 °C and melting termination temperature 202.19 °C.

[0018] Figure 3a and Figure 3b The diagram shows the dynamic vapor adsorption (DVS) of polymorph A of compound 1. Figure 3a The diagram shows the DVS isotherm, where ■ represents desorption and ◆ represents adsorption; and Figure 3b This is a graph showing the change in mass.

[0019] Figure 4 The XRPD diffraction pattern of solid crystalline polymorph B of compound 1 is shown, ranging from 5 to 60 2-θ, with a step size of 0.02 s (reflection mode, offset 500 count).

[0020] Figure 5 The DSC thermogram of polymorph B of compound 1 is shown. The thermogram was obtained at a rate of change of +10 °C / min and shows an endothermic peak attributable to water loss from the hydrate: integral -96.97 mW °C, onset temperature 90.90 °C, and termination temperature 114.65 °C.

[0021] Figure 6 The XRPD diffraction pattern of the unsolvated polymorph C of compound 1, recrystallized from trifluorotoluene (upper trace) and toluene (lower trace) in Example 3.

[0022] Figure 7 The XRPD diffraction pattern of polymorph D of the ether solvate of compound 1 isolated from THF (upper trace) and tert-butyl methyl ether (lower trace) in Example 3 is shown.

[0023] Figure 8 The image shows the XRPD diffraction pattern of polymorph E of the methyl ethyl ketone semisolvent of compound 1 isolated from methyl ethyl ketone in Example 3.

[0024] Figure 9 The XRPD diffraction pattern of anhydrous polymorph F of compound 1, which was isolated from ethanol in Example 3.

[0025] Figure 10 The XRPD diffraction pattern of solvate form H of compound 1 isolated from cumene in Example 4.

[0026] Figure 11 The image shows the XRPD diffraction pattern of the amorphous product of Example 3 before oven drying.

[0027] Figure 12 The image shows the XRPD diffraction pattern of the amorphous product of Example 3 after oven drying.

[0028] Figure 13 The XRPD diffraction patterns of compound 1 isolated from water (upper trace), DCM / heptane (middle trace), and acetonitrile / water (lower trace) in Example 4 are shown, and it is shown that all these products are identical and consistent with type B hydrates (as shown in the image). Figure 4 (Compared to). The formation of hydrates from DCM / heptane must be attributed to the presence of water or moist heptane.

[0029] Figure 14 The DSC thermogram of the product separated from DCM / heptane in Example 4, with a temperature range of 20℃-300℃ and a heating rate of 10℃ / min, shows an endothermic peak attributable to water loss from the hydrate and is consistent with type B, indicating the ingress of water during suspension equilibrium, possibly from a large amount of heptane; Lobe 1: integral -114.32 mW℃, onset temperature 98.45℃, peak temperature 110.50℃, termination temperature 115.32℃; Lobe 2: integral 9.95 mW℃, onset temperature 115.33℃, peak temperature 116.17℃, termination temperature 117.26℃; Lobe 3: integral -132.83 mW℃, onset temperature 194.49℃, peak temperature 197.17℃, termination temperature 199.06℃.

[0030] Figure 15a Overlapped DSC and TGA thermograms of the ether solvate type D isolated from tetrahydrofuran in Example 4 are shown, and the first weight loss transition at approximately 97.27 °C, corresponding to approximately 14% w / w to 15% w / w, is shown and is attributed to solvent release (via...). 1H NMR (sample contains 12.5% ​​w / w THF). DSC, lobe 1: integral -90.50 J / g, onset temperature 98.44℃, peak 118.9℃; lobe 2: integral -101.8 J / g, onset temperature 194.06℃, peak 196.72℃.

[0031] Figure 15b Overlapping DSC and TGA thermograms of the ether solvate type D isolated from tert-butyl methyl ether in Example 4 are shown, and the first weight loss transition at approximately 95.92 °C, corresponding to approximately 15% w / w to 16% w / w, is shown and attributed to solvent release (via...). 1 H NMR, sample contains 14.8% w / w tBME. DSC, lobe 1: integral -151.4 J / g, onset temperature 98.56℃, peak 106.35℃; lobe 2: integral -130.1 J / g, onset temperature 194.82℃, peak 196.82℃.

[0032] Figure 16 Overlapped DSC and TGA thermograms of the partially solvated form E isolated from methyl ethyl ketone in Example 4 are shown. 1 ¹H NMR: The sample contained 6.9% w / w MEK. A significant weight loss transition occurred upon melting, accompanied by solvent release. DSC: Lobe: integral -102.8 J / g, onset temperature 191.06 °C, peak temperature 194.36 °C.

[0033] Figure 17 The DSC thermogram of the H-type isolated from cumene in Example 5 is shown; the temperature range is 20℃-300℃, heated at 10℃ per minute; Lobe 1: integral -26.14mW℃, starting temperature 94.78℃, ending temperature 111.97℃; Lobe 2: integral 17.72mW℃, starting temperature 112.79℃, ending temperature 117.25℃; Lobe 3: integral -47.09mW℃, starting temperature 183.96℃, ending temperature 190.15℃.

[0034] Figure 18 The XRPD diffraction pattern of the G-type separated from THF in Example 7 is shown.

[0035] Figure 19 The TGA plot (upper trace) of the material obtained from Example 1 shows the onset temperature of the weight loss transition at 195.0 °C; and the DSC thermogram (lower trace) shows lobe 1: integral -3 J / g; onset temperature 112.5 °C; peak 121.1 °C; lobe 2: integral 12 J / g; onset temperature 124.2 °C; peak 130.4 °C; lobe 3: integral -91 J / g; onset temperature 192 °C; peak 194.2 °C.

[0036] Figure 20 The XRPD diffraction pattern of the material obtained from Example 1 is shown for preparation (lower trace) and after storage at 40°C and 75% relative humidity for 7 days (upper trace).

[0037] Figure 21a The DSC chart is of type B(I), with a temperature range of 20℃-350℃ and a heating rate of 10℃ per minute; Lobe 1: Integral -23.23 W℃g -1 Initial temperature 107.16℃, peak temperature 119.83℃, termination temperature 125.41℃; Lobe 2: integral 5.21 W℃g -1 Initial temperature 125.42℃, peak temperature 129.50℃, termination temperature 136.09℃; Lobe 3: integral -26.07W℃g -1 The initial temperature was 196.66℃, the peak temperature was 199.83℃, and the final temperature was 201.37℃.

[0038] Figure 21b The DSC chart is for type B(II), with a temperature range of 20℃-350℃ and a heating rate of 10℃ per minute; Lobe 1: Integral -4.90 W℃g -1 Initial temperature 75.22℃, peak temperature 90.50℃, termination temperature 111.38℃; Lobe 2: integral -8.37W℃g -1 Initial temperature 102.87℃, peak temperature 115.50℃, termination temperature 120.74℃; Lobe 3: integral 1.59 W℃g -1 Initial temperature 121.83℃, peak temperature 131.42℃, termination temperature 135.55℃; Lobe 4: integral -13.35W℃g -1 The initial temperature was 194.06℃, the peak temperature was 195.92℃, and the final temperature was 196.70℃.

[0039] Figure 22a The TGA chromatograms of type B(I) obtained by heating from 20°C to 600°C at a rate of 5°C per minute are shown; Step 1: -4.9539%, -0.1498 mg; Step 2: -95.1942%, -2.8792 mg.

[0040] Figure 22b The TGA chromatograms of type B(II) obtained by heating from 20°C to 600°C at a rate of 5°C per minute are shown; Step 1: -0.8685%, -0.02707 mg; Step 2: -3.8748%, -0.1208 mg; Step 3: -95.1186%, -2.9649 mg.

[0041] Figure 23a The XRPD diagram is of type B(I).

[0042] Figure 23b The XRPD diagram is for type B(II).

[0043] Figure 24 The DSC chart for type B(II) after compression at 10 tons for 20 min (heated at a rate of 5°C per minute from 20°C to 350°C); note the transformation to a single dehydration cleft, indicating that the sample has been transformed into type B(I). Cleft 1: integral -127.53 J / g, initial temperature 87.12°C, peak temperature 103.67°C, termination temperature 114.36°C; Cleft 2: integral 11.64 J / g, initial temperature 118.97°C, peak temperature 128.08°C, termination temperature 134.72°C; Cleft 3: integral -168.37 J / g, initial temperature 193.86°C, peak temperature 195.75°C, termination temperature 196.80°C.

[0044] Figure 25 The image shows the DSC plot of type B(II) after 23 hours of suspension in acetonitrile / water (4 to 1 v / v). The plot was generated over a temperature range of 25°C–280°C with a heating rate of 5°C per minute, indicating that the sample had converted to type B(I). Lobe 1: integral -189.83 J / g, initial temperature 106.25°C, peak temperature 113.92°C, final temperature 116.65°C; Lobe 2: integral -9.99 J / g, initial temperature 117.12°C, peak temperature 118.25°C, final temperature 120.00°C; Lobe 3: integral 11.82 J / g, initial temperature 120.98°C, peak temperature 127.25°C, final temperature 132.87°C; Lobe 4: integral -239.87 J / g, initial temperature 194.78°C, peak temperature 196.25°C, final temperature 197.08°C.

[0045] Figure 26a The image shows the DSC plot of type B(II) after 2 hours of suspension and equilibrium in pure water. The plot was generated within a temperature range of 25℃-350℃, with a heating rate of 5℃ per minute, indicating that the sample had converted to type B(I). Lobe 1: integral -154.53 J / g, initial temperature 101.15℃, peak temperature 113.92℃, termination temperature 120.00℃; Lobe 2: integral 46.14 J / g, initial temperature 120.27℃, peak temperature 129.42℃, termination temperature 132.79℃; Lobe 3: integral -144.408℃, initial temperature 194.51℃, peak temperature 195.92℃, termination temperature 196.80℃.

[0046] Figure 26b The image shows the XRPD plot of type B(II) after 2 hours of suspension and equilibration in pure water, indicating that the sample is still type B; upper trace: input material; lower trace: output material.

[0047] Figure 26cThe DSC chromatogram for type B(II) after 200 hours of suspension and equilibrium in pure water is shown. The chromatogram was generated within a temperature range of 25℃-280℃, with a heating rate of 5℃ per minute, indicating that the sample has converted to type B(I). Lobe 1: Integral -109.07 J / g; initial temperature 100.76℃, peak temperature 109.92℃, termination temperature 114.05℃; Lobe 2: Integral 5.54 J / g, initial temperature 114.05℃, peak temperature 114.58℃, termination temperature 116.23℃; Lobe 3: Integral 25.08 J / g, initial temperature 116.86℃, peak temperature 126.25℃, termination temperature 131.82℃; Lobe 4: Integral -147.30 J / g, initial temperature 193.90℃, peak temperature 195.92℃, termination temperature 197.01℃.

[0048] Figure 26d The image shows the XRPD plot of type B(II) after 20 hours of suspension and equilibrium in pure water, indicating that the sample still has the crystalline form of B; upper trace: input material; middle trace: sample after 2 hours in pure water; lower trace: output material.

[0049] Figure 27 The image shows the XRPD plot of type B(II) after equilibration in anhydrous acetonitrile for 3 hours, indicating that the sample has been converted to type A. Upper trace: input material; middle trace: output material; lower trace: true type A as a reference.

[0050] Figure 28 The image shows the XRPD plot of type B(I) after equilibration in anhydrous acetonitrile for 5 hours, indicating that the sample has been converted to type A. Upper trace: True type A (reference); Lower trace: Type B(I) after equilibration in anhydrous acetonitrile for 5 hours.

[0051] Figure 29 For citrate buffer XRPD plots (middle trace) of 80 suspended type A samples, which are compared with the true type A (lower trace) and the true type B(I) (upper trace) after 14 days of thermal cycling at a rate of 1℃ / min from -10℃ to +40℃; and show that the samples have been converted to type B.

[0052] Figure 30 For citrate buffer 20 / XRPD plot of suspended type A (middle trace) in 22, which is a comparison of the XRPD of true type A (lower trace) and true type B(I) (upper trace) after 14 days of thermal cycling at a rate of 1℃ / min from -10℃ to +40℃; and shows that the sample has been converted to type B.

[0053] Figure 31The image shows the XRPD plot of the material obtained from Example 14. Upper trace: Type A; Middle trace: Separated product from Example 14; Lower trace: Type B(I).

[0054] Figure 32 The DSC chart for the material obtained from Example 14 was generated over a temperature range of 25°C–280°C at a heating rate of 5°C per minute. Lobe 1: Integral -134 J / g, initial temperature 105.21°C, peak temperature 115.92°C, final temperature 121.05°C; Lobe 2: Integral 13.47 J / g, initial temperature 121.05°C, peak temperature 122.58°C, final temperature 126.45°C; Lobe 3: Integral 23.1 J / g, initial temperature 127.43°C, peak temperature 130.17°C, final temperature 134.67°C; Lobe 4: Integral -125.89 J / g, initial temperature 190.72°C, peak temperature 193.08°C, final temperature 194.16°C.

[0055] Figure 33 Hydrogen bonds between water and the molecular structure of compound 1 in type B(I) crystals are shown – thermal ellipsoids are plotted at a 50% probability level. Detailed Implementation

[0056] In this specification, unless the context requires otherwise due to the language of expression or necessary implication, the word "comprising" or variations such as "including" or "containing" are used in an inclusive sense, that is, specifying the presence of the stated features but not excluding the presence or addition of other features in various embodiments of the invention.

[0057] All references and patent documents cited in this article are incorporated by way of citation as completely as possible.

[0058] When the specification refers to the solvent volume per unit mass of compound 1, this refers to the mg of compound 1 and the μL of solvent, such that 50 mg of compound 1 in 20 volumes of solvent constitutes a 1 mL sample; 30 mg of compound 1 in 60 volumes of solvent constitutes a 1.8 mL sample; and 30 mg of compound 1 in 70 volumes of solvent constitutes a 2.1 mL sample.

[0059] In this specification, references to "medicinal use" refer to the purpose of administration to humans or animals, particularly humans or mammals (e.g., domestic or livestock mammals), for the treatment or prevention of disease or medical condition. The term "medicinal composition" refers to a composition suitable for medicinal use, and "pharmaceutical use" refers to a pharmaceutical preparation suitable for a pharmaceutical composition. Other similar terms should be interpreted accordingly.

[0060] The compound 1 disclosed herein is N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide, which has the structure shown above. This compound is illustrated in our earlier application WO2019 / 145726, and the method illustrated in that document results in the production of compound 1 as a mixture of at least two crystalline forms (see Example 1).

[0061] The inventors have discovered various crystalline and amorphous forms of compound 1, namely forms A, B, C, D, E, F, G, and H, as well as an amorphous form. Polymorphs A and B are particularly useful because they are thermodynamically stable.

[0062] Therefore, in one aspect of the invention, compound 1 is provided in the form of its anhydrous solid crystalline polymorph (type A polymorph), characterized for example, substantially as follows: Figure 1a and Figure 1b The XRPD diffraction pattern shown.

[0063] The peak at position 7.25 (±0.2 degrees, 2-θ value) in the XRPD diffraction pattern of the type A polymorph of compound 1 is particularly useful for distinguishing it from the type B hydrated solid crystalline polymorph of compound 1.

[0064] Suitablely, in the XRPD diffraction pattern of the type A polymorph of compound 1, a main peak at 7.25 (±0.2 degrees, 2-θ value) and at least three (e.g., three, four, five, six, seven, eight or all nine) other peaks selected from those at positions 14.44, 20.42, 21.68, 24.38, 27.21, 29.01, 30.82, 36.46 and 41.49 (±0.2 degrees, 2-θ value) can be observed.

[0065] The peaks at positions 21.68 and 29.01 (±0.2 degrees, 2-θ value) are particularly characteristic of type A crystalline polymorphs, and therefore at least one of these two peaks can usually be observed, and preferably both peaks.

[0066] Appropriately, clusters of peaks at 24.09, 24.22, and 24.38 (±0.2 degrees, 2-θ value) can also be observed in the XRPD diffraction pattern, as these are also characteristic of type A crystalline polymorphs.

[0067] More commonly, in the XRPD diffraction pattern of the type A polymorph of compound 1, a main peak at 7.25 (±0.2 degrees, 2-θ value) and at least three (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen) other peaks selected from those at positions 10.20, 14.44, 17.79, 20.42, 20.69, 21.68, 24.22, 24.38, 26.13, 27.21, 29.01, 30.82, 36.46 and 41.49 (±0.2 degrees, 2-θ value) can be observed.

[0068] Similarly, at least one of the peaks at positions 21.68 and 29.01 (±0.2 degrees, 2-θ value) can usually be observed, and preferably both peaks, as well as a cluster of peaks at 24.09, 24.22 and 24.38 (±0.2 degrees, 2-θ value).

[0069] In some cases, in the XRPD diffraction pattern of the type A polymorph of compound 1, a main peak at 7.25 (±0.2 degrees, 2-θ value) and at least three (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or all nineteen) other peaks selected from those at positions 10.20, 14.44, 16.13, 17.79, 20.42, 20.69, 21.07, 21.68, 24.09, 24.22, 24.38, 26.13, 27.21, 29.01, 29, 30, 30.82, 32.50, 36.46, and 41.49 (±0.2 degrees, 2-θ value) can be observed.

[0070] Similarly, at least one of the peaks at positions 21.68 and 29.01 (±0.2 degrees, 2-θ value) can usually be observed, and preferably both peaks, as well as a cluster of peaks at 24.09, 24.22 and 24.38 (±0.2 degrees, 2-θ value).

[0071] The XRPD diffraction pattern of the type A polymorph has θ values ​​of 7.25, 10.20, 12.64, 14.44, 14.81, 15.27, 16.13, 16.47, 16.90, 17.79, 19.86, 20.42, 20.69, 21.07, 21.68, 24.09, 24.22, 24.38, and 25.46. 26.13, 26.69, 27.21, 27.71, 29.01, 29.30, 30.16, 30.82, 31.55, 32.50, 33.02, 34.14, 34.42, 36.46, 36.96, 38.92, 39.82, 40.26, 41.49, 42.28, 44.76, 47. Peaks at 34, 47.92, 51.61, and 51.84 (±0.2 degrees, 2-θ value) are observed, and suitably, at least three of the remaining peaks (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, or all forty-three) are observed in the resulting XRPD diffraction pattern.

[0072] Similarly, at least one of the peaks at positions 21.68 and 29.01 (±0.2 degrees, 2-θ value) can usually be observed, and preferably both peaks, as well as a cluster of peaks at 24.09, 24.22 and 24.3801 (±0.2 degrees, 2-θ value).

[0073] The 2-θ values ​​and intensities of the XRPD peaks of type A are shown in Table 1 below.

[0074] Table 1 - 2-θ values ​​for type A

[0075]

[0076]

[0077] Type A polymorphs can be micronized, and the inventors have demonstrated that the crystal structure is maintained during micronization. Micronization suitably produces particles with D50 ≤ 5 μm, more preferably ≤ 3 μm, and D90 ≤ 10 μm, more preferably ≤ 5 μm. For example, D50 can be about 1 μm to 5 μm, more preferably about 1 μm to 3 μm;

[0078] D50 represents the median particle size based on volume; such that the particle size of 50% of the total volume is less than or equal to D50. Similarly, D90 is defined as such that the particle size of 90% of the volume is less than or equal to D90.

[0079] Several suitable methods can be used to measure D50 and D90, such as laser diffraction. These methods are well known and appreciated by those skilled in the art.

[0080] During micronization, the intensity of some peaks in the XRPD diffraction pattern may increase or decrease. Specifically, the intensity of the peaks at 14.44 and 29.01 (±0.2 degrees, 2-θ values) may decrease, while the intensity of the peaks at 17.79 and 27.21 (±0.2 degrees, 2-θ values) may increase. This is thought to be caused by orientation effects as well as variations in texture and crystallite size and distribution.

[0081] XRPD analysis can be performed using a Bruker D2 Phaser powder diffractometer equipped with a LynxEye detector.

[0082] Suitablely, the type A polymorph is substantially free of other forms of compound 1, such that, for example, in a sample of compound 1, at least 97 wt%, 97.5 wt%, 98 wt%, 98.5 wt%, 99 wt%, 99.5 wt%, 99.6 wt%, 99.7 wt%, 99.8 wt%, or 99.9 wt% of compound 1 is present as a type A polymorph.

[0083] The inventors have discovered that type A polymorphs can be obtained, for example, by crystallization from any of a variety of solvents, including acetone, butanol, ethanol, ethyl formate, isopropyl acetate, methyl acetate, nitromethane, 2-propanol, propionitrile, and acetonitrile. A variety of crystallization conditions can be used.

[0084] Crystallization can be achieved by heating and cooling, and this method includes the following steps:

[0085] i. Prepare a saturated solution of compound 1 in a solvent at a temperature of approximately 50°C to 70°C;

[0086] ii. Cool the solution to a temperature of approximately 5°C to 20°C;

[0087] iii. Allow the cooled solution to stand until crystals of compound 1 form; and

[0088] iv. Separate the crystallized products;

[0089] The solvent is selected from acetone, butanol, ethanol, ethyl formate, isopropyl acetate, methyl acetate, nitromethane, 2-propanol, propionitrile, and acetonitrile.

[0090] More preferably, the solvent is acetonitrile, ethanol, ethyl acetate, methyl acetate, butanol, 2-propanol, or isopropyl acetate.

[0091] The appropriate amounts of ethanol, butanol, and 2-propanol are 4 to 7 volumes per unit mass of compound 1, more preferably about 4 to 6 volumes.

[0092] Suitable amounts of methyl acetate, ethyl acetate, and isopropyl acetate are 10 to 20 volumes per unit mass of compound 1. For example, the amount of methyl acetate may be about 10 to 12 volumes, particularly about 10.5 to 11.5 volumes, for example, 11 volumes per unit mass of compound 1. The amount of ethyl acetate may be about 19 to 21 volumes, particularly about 19.5 to 20.5 volumes, for example, about 20 volumes per unit mass of compound 1, and the amount of isopropyl acetate may be about 15 to 17 volumes, particularly about 15.6 to 16.5 volumes, for example, about 16 volumes per unit mass of compound 1.

[0093] Most preferably, the crystallization solvent is ethanol, ethyl acetate or methyl acetate, preferably at the concentrations mentioned above.

[0094] Crystallization is not suitable to be performed in anisole, butyl methyl ether, cumene, chlorobenzene, ethyl acetate, methyl ethyl ketone, propionitrile, toluene, trifluorotoluene tetrahydrofuran, dichloromethane, or dichloromethane / heptane, as these will result in other forms of compound 1.

[0095] It is also not suitable to perform crystallization in aqueous solvents, as this would result in the formation of a type B hydrated solid crystalline polymorph of compound 1.

[0096] Suitablely, the compound 1 used to prepare the saturated solution in step (i) is an amorphous compound 1.

[0097] Alternatively, crystallization can be carried out via diffusion from a binary solvent in a method that includes the following steps:

[0098] i. Prepare a saturated solution of compound 1 in a less volatile solvent;

[0099] and

[0100] ii. Transfer the saturated solution to the first container;

[0101] iii. Place the first container into the second container, which is larger than the first container and contains a second solvent with a higher volatility than the first solvent;

[0102] iv. Cover the container and allow it to stand at 15°C to 25°C, preferably 18°C ​​to 23°C, for 1 to 10 days, allowing the second solvent to diffuse into the first container and for compound 1 to crystallize within the first container; and

[0103] v. To separate the crystallized product from the first container;

[0104] The first solvent is ethanol and the second solvent is pentane.

[0105] Alternatively, type A polymorphs can be prepared by a cold crystallization method, which involves heating the amorphous compound 1 to a temperature greater than 97°C and then cooling it.

[0106] Particularly suitable crystallization methods include:

[0107] i. Prepare a mixture of compound 1 in ethyl acetate;

[0108] ii. Heat the mixture to 55°C to 70°C, particularly 55°C to 65°C, and more particularly about 60°C, and stir until a solution of compound 1 in ethyl acetate is obtained;

[0109] iii. Cool the solution to 40°C to 50°C, particularly to about 45°C to 49°C, and even more particularly to about 47°C;

[0110] IV concentrated solution;

[0111] v. Cool at a constant rate to about 15°C to 30°C, especially about 20°C to 25°C, and even more especially about 23°C, for 1 to 4 hours, particularly about 2 to 3 hours, and let stand for 4 to 6 hours, for example 5 hours;

[0112] vi. Add n-heptane at a temperature of 15°C to 30°C, particularly about 20°C to 25°C, and even more particularly about 23°C, over a period of 0.75 hours to 2.5 hours, for example, 1 hour to 2 hours, and then let stand for 20 minutes to 80 minutes, for example, about 30 minutes to 60 minutes;

[0113] vii. The mixture is cooled at a constant rate to -5°C to 10°C, particularly to 0°C to 5°C, and even more particularly to 2.5°C, over a period of 1 to 5 hours, particularly to about 2 to 4 hours, and usually to about 3 hours, and then allowed to stand to obtain a solid crystalline type A polymorph of compound 1.

[0114] viii. Separate and dry the solid crystalline type A polymorph of compound 1.

[0115] In this method, in step (i), the volume of ethyl acetate per unit mass of compound 1 is suitably about 15 to 25 volumes, more suitably about 20 volumes of ethyl acetate per unit mass of compound 1.

[0116] Suitably, in step (ii), stirring is performed for at least 10 minutes. The solution of compound 1 is a turbid solution, and after step (iii), the solution can be clarified by filtration (e.g., through a 1 μm filter). The method suitably includes an additional step of rinsing with ethyl acetate. Suitably, the rinsing volume is about 10% to 20% of the original amount of ethyl acetate used in step (i), for example, about 15% by volume.

[0117] In step (iv), the solution is suitably concentrated to about 40% to 60%, for example about 50%, of the volume of ethyl acetate used in step (i). Concentration may be achieved by vacuum distillation and is suitably performed at a temperature of about 40°C to 50°C, typically about 45°C.

[0118] Polymorph A is the most thermodynamically stable form of compound 1. It has a relatively high melting point, from Figure 2 The DSC thermal spectrum shows that the melting initiation temperature is approximately 193℃.

[0119] As shown in the DVS diagram in Figure 3, the type A polymorph exhibits low water affinity and is non-hygroscopic. Figure 3 shows that at 90% relative humidity (RH), the mass change (representing water absorption) is only 0.02%. Furthermore, when XRPD analysis was repeated after the DVS test, the results were identical to those of the sample before the DVS analysis, indicating that no hydrates formed. This demonstrates that type A is stable to water, and indeed, the inventors have shown that water must enter the crystal lattice to form hydrate type B. This is why hydrate type B forms when compound 1 is recrystallized from an aqueous solvent, as discussed in more detail below.

[0120] Surprisingly, however, although type A is the most thermodynamically stable crystalline form at low water activity and is kinetically stable to water via DVS, it has been found to be thermodynamically unstable under elevated water activity conditions (e.g., when suspended in an aqueous solvent). Under these conditions, it transforms into the type B hydrated crystalline polymorph within a period of several days.

[0121] In the aqueous suspension of the type A crystalline polymorph, type B was not detected at the 24-hour time point. However, after 8 days, virtually all of compound 1 existed as the type B hydrated crystalline form.

[0122] In another aspect of the invention, compound 1 is provided in the form of its type B hydrated solid crystalline polymorph (type B polymorph), characterized, for example, substantially as Figure 4 The XRPD diffraction pattern shown.

[0123] When compound 1 is crystallized from an aqueous solvent or when water enters with sufficient activity under solvent-mediated conditions, type B is a hydrate derived from type A or an amorphous form.

[0124] XRPD patterns of hydrates are often more variable than those of amorphous forms because variations in the amount of water within the structure can lead to changes in interplanar spacing, which in turn causes changes in the reflection angle, particularly to smaller angles. Therefore, the following values ​​have an error of ±0.4 degrees, 2-θ, although an error of ±0.3 degrees, 2-θ, or even ±0.2 degrees, 2-θ would be more appropriate.

[0125] The peak at 11.03 (±0.4 degrees, 2-θ value) in the XRPD diffraction pattern is particularly useful for distinguishing the type B hydrated crystalline polymorph of compound 1 from the type A polymorph of compound 1.

[0126] Suitablely, in the XRPD diffraction pattern of the type B polymorph of compound 1, a main peak at position 11.03 (±0.4 degrees, more preferably ±0.3 degrees or ±0.2 degrees, 2-θ value) and at least three (e.g., three, four, five, six, seven, eight or all nine) peaks selected from those at positions 5.56, 14.04, 17.28, 18.03, 18.86, 22.08, 23.69, 24.12 and 24.93 (±0.4 degrees, more preferably ±0.3 degrees or ±0.2 degrees, 2-θ value) can be observed.

[0127] The peaks at positions 5.56 and 22.08 (±0.4 degrees, more preferably ±0.3 degrees or ±0.2 degrees, 2-θ values) are particularly characteristic of type A crystalline polymorphs, and therefore at least one of these two peaks can usually be observed, and preferably both.

[0128] More commonly, in the XRPD diffraction pattern of the type B polymorph of compound 1, a main peak at position 11.03 (±0.2 degrees, 2-θ value) and at least three (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen) peaks selected from those at positions 5.56, 14.04, 17.28, 18.03, 18.86, 19.34, 22.08, 23.69, 24.12, 24.93, 25.98, 26.53, 27.28 and 28.79 (±0.4 degrees, more preferably ±0.3 degrees or ±0.2 degrees, 2-θ value) can be observed.

[0129] As described above, it is suitable to observe at least one of the peaks at positions 5.56 and 22.08 (±0.4 degrees, more preferably ±0.3 degrees or ±0.2 degrees, 2-θ values), and preferably both peaks.

[0130] More commonly, in the XRPD diffraction pattern of the type B polymorph of compound 1, a main peak at position 11.03 (±0.2 degrees, 2-θ value) and at least three (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or all nineteen) peaks can be observed at positions 5.56, 14.04, 16.58, 17.28, 18.03, 18.86, 19.34, 21.19, 22.08, 23.69, 24.12, 24.93, 25.98, 26.53, 27.28, 28.22, 28.79, 30.69, and 30.90 (±0.4 degrees, more preferably ±0.3 degrees or ±0.2 degrees, 2-θ value)

[0131] As described above, it is suitable to observe at least one of the peaks at positions 5.56 and 22.08 (±0.4 degrees, more preferably ±0.3 degrees or ±0.2 degrees, 2-θ values), and preferably both peaks.

[0132] The XRPD diffraction pattern of the type B polymorph has the following values: 5.56, 11.03, 14.04, 16.58, 17.28, 18.03, 18.86, 19.34, 20.51, 21.19, 22.08, 23.69, 24.12, 24.93, 25.98, 26.53, 27.28, 28.22, 28.79, 30.69, 30.90, 32.07, 32.40, 35.69, 36.54, 37.95, 38.11, 38.77, 3 Peaks at 8.85 and 40.09 (±0.2 degrees, 2-θ value) are observed, and appropriately, at least three (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, or all thirty) peaks are observed in the resulting XRPD diffraction pattern.

[0133] As described above, at least one peak, or preferably two peaks, can be observed, including the main peak at position 11.03 and the peaks at positions 5.56 and 22.08 (±0.2 degrees, 2-θ value).

[0134] The XRPD spectra of types B(I) and B(II) were rerun, and the 2θ values ​​and peak intensities are shown in Table 2 below. Some variations exist in the values ​​given above, but as discussed, this is within the range of experimental variation typically found in the XRPD spectra of hydrates.

[0135] Table 2 - 2-θ values ​​for type B

[0136]

[0137]

[0138] Obviously, considering experimental variation, the 2θ values ​​of 5.4903, 10.9682, 17.0775, 17.8749, 18.6669, 19.1626, 21.9937, 23.5468, 23.9220, 24.7841, 25.7789, 26.3371, 27.0946, and 28.5839 are shown in Table 2. The peaks in the values ​​correspond to the peaks mentioned above at 5.56, 11.03, 14.04, 17.28, 18.03, 18.86, 19.34, 22.08, 23.69, 24.12, 24.93, 25.98, 26.53, 27.28, and 28.79 (±0.4 degrees, more appropriately ±0.3 degrees or ±0.2 degrees, 2-θ values).

[0139] The other peaks in Table 2 similarly correspond to the peaks mentioned above.

[0140] Type B polymorphs can be micronized, and the inventors have demonstrated that the crystal structure is maintained during micronization. Micronization suitably produces particles with D50 ≤ 5 μm, more preferably ≤ 3 μm, and D90 ≤ 10 μm, more preferably ≤ 5 μm. For example, D50 can be about 1 μm to 5 μm, more preferably about 1 μm to 3 μm;

[0141] Suitablely, the type B polymorph is substantially free of other forms of compound 1, such that, for example, in a sample of compound 1, at least 97 wt%, 97.5 wt%, 98 wt%, 98.5 wt%, 99 wt%, 99.5 wt%, 99.6 wt%, 99.7 wt%, 99.8 wt%, or 99.9 wt% of compound 1 exists as a type B polymorph.

[0142] The inventors have discovered that type B polymorphs can be obtained, for example, by crystallizing compound 1 from an aqueous solvent, typically water or water mixed with acetonitrile.

[0143] Crystallization can be achieved by heating and cooling, and this method includes the following steps:

[0144] i. Prepare a saturated solution of compound 1 in a solvent at a temperature of approximately 60°C to 80°C;

[0145] ii. Cool the solution to a temperature of approximately 5°C to 30°C;

[0146] iii. Allow the cooled solution to stand until crystals of compound 1 form; and

[0147] iv. Separate the crystallized products;

[0148] The solvent is an aqueous solvent, such as water or water mixed with one or more other solvents, such as acetonitrile, cumene, dichloromethane, nitromethane, trifluorotoluene, or a mixture of dichloromethane and heptane.

[0149] Preferably, the solvent is not pure water.

[0150] Any other form of compound 1 can be used as a starting material for crystallization. For example, compound 1 used in step (i) to prepare a saturated solution can be an amorphous compound 1 or compound 1 in the form of its type A crystalline polymorph.

[0151] Particularly suitable solvents are mixtures of acetonitrile and water, for example, acetonitrile / water ratios of 5:1 to 1:5 v / v, suitably 5:1 to 3:1, and typically about 4:1 acetonitrile / water.

[0152] Crystallization is not suitable in anisole, butyl methyl ether, cumene, chlorobenzene, ethyl acetate, methyl ethyl ketone, propionitrile, toluene, trifluorotoluene tetrahydrofuran, dichloromethane, or dichloromethane / heptane, as these will result in other forms of compound 1. Crystallization is also not suitable in acetone, butanol, ethanol, ethyl formate, isopropyl acetate, methyl acetate, nitromethane, 2-propanol, propionitrile, or acetonitrile, as this will result in polymorph A of compound 1.

[0153] Surprisingly, the inventors have discovered that the type B polymorph of compound 1 can take two pseudopolymorphs. These forms are isomorphic and cannot be distinguished by XRPD. However, their dehydration characteristics differ when measured by thermogravimetric analysis and differential scanning calorimetry. The first pseudopolymorph undergoes unimodal dehydration and is designated type B(I), while the second pseudopolymorph undergoes bimodal dehydration and is designated type B(II). The DSC plot of type B(I) shows a single dehydration lobe, while the DSC plot of type B(II) shows two dehydration lobes, wherein the onset temperature of the first dehydration lobe is lower than that of the dehydration lobe of type B(I) (see [link to DSC]). Figure 21a and Figure 21b The sum of the enthalpies of the two lobes of type B(II) is approximately equal to the enthalpy of a single lobe of type B(I). Similarly, Figure 22aThe TGA plot for type B(I) shows a single weight change (-4.95% mg) attributable to water loss, while Figure 22b The TGA plot for type B(II) shows a smaller weight change (-0.87%) followed by a larger weight loss (-3.87%).

[0154] The dehydration behavior of these two forms is thought to be caused by the different positions and bonds of the associated water molecules. To determine their stability, both forms were subjected to 10 tons of compaction, after which the endothermic dehydration of the compacted B(II) form was found to exhibit a broad singlet event, similar to the singlet event exhibited by the B(I) form. Therefore, it is hypothesized that the two forms are related through isomorphic pseudopolymorphism, differing only slightly in the orientation and position of their constituent water molecules.

[0155] Type B(I) appears after type B(II) compaction, and type B(I) remains unchanged after the same treatment. Furthermore, in the DSC diagram ( Figure 21a and Figure 21b The dehydration initiation temperature of valve I in type B(II) is lower than that of dehydration valve (I) in type B(I), indicating that type B(II) is a less stable hydrate form (metastable hydrate form), and tends to become a more stable hydrate form, type B(I), when a higher compressive force is applied.

[0156] To further investigate the stability grades of types B(I) and B(II), equal portions of types B(I) and B(II) were competitively matured in a 4:1 MeCN / water (w / w) slurry for 3 days. DSC confirmed the resulting solid to be type B(I), indicating that the type B unimodal pseudopolymorph is the more stable of the two forms.

[0157] As mentioned above, both types A and B are thermodynamically stable, depending on their storage conditions. Type A is thermodynamically stable when stored in solid form, even under humid conditions. Therefore, type A, as a solid, is suitable as a micronized solid, particularly for transport and storage.

[0158] However, in an aqueous suspension, form A transforms into form B, more specifically into form B(I). It appears that water molecules adsorb onto the surface of the form A crystals prior to this transformation, and the rate-determining step is a water-related bimolecular process, rather than proceeding via the conversion of form A into anhydrous form B, which is subsequently hydrated. If a pharmaceutical composition containing an aqueous suspension of form A is desired, it is most suitable to prepare the suspension just before administration to the patient so that compound 1, as a hydrated form B, does not recrystallize prior to administration.

[0159] Type B(I) has been shown to be thermodynamically stable in aqueous suspensions over extended periods. This makes it particularly suitable for pharmaceutical formulations that are aqueous suspensions.

[0160] Single-crystal data for a type B(I) pseudopolymorph were obtained. This crystal is orthorhombic, space group Pna21, with the following lattice parameters:

[0161]

[0162] In another aspect of the invention, compound 1 is provided in its solid amorphous form.

[0163] Suitablely, the amorphous form of compound 1 is substantially free of other forms of compound 1, such that, for example, in a sample of compound 1, at least 97 wt%, 97.5 wt%, 98 wt%, 98.5 wt%, 99 wt%, 99.5 wt%, 99.6 wt%, 99.7 wt%, 99.8 wt%, or 99.9 wt% of compound 1 may exist as an amorphous form.

[0164] Solid amorphous compound 1 can be prepared, for example, by dissolving compound 1 in a solvent (e.g., ethyl acetate) and removing the solvent under reduced pressure at a temperature of about 30°C to 45°C.

[0165] Suitable, compound 1 used as a starting material will have type A or type E (described below).

[0166] Other polymorphic forms of compound 1 include:

[0167] • Type C, a non-solventized form isolated from trifluorotoluene or toluene, which has the following properties: Figure 6 The XRPD diffraction pattern shown;

[0168] Type D, an ether solvate, which has the following properties: Figure 7 The XRPD diffraction pattern shown;

[0169] Type E, a methyl ethyl ketone semisolvent, which has the following properties: Figure 8 The XRPD diffraction pattern shown;

[0170] • Type F, which is an anhydrous form derived from ethanol treatment; and has the following properties: Figure 9 The XRPD diffraction pattern shown;

[0171] • Type G is a solvate obtained by suspending and equilibrating Type A in THF at 40°C, and has the following properties: Figure 18 The XRPD diffraction pattern shown.

[0172] • The H-type is the solvate form obtained by recrystallization from cumene, and has the following properties: Figure 10 The XRPD diffraction pattern shown

[0173] Compound 1 is a modulator of TMEM16A. Therefore, in another aspect of the invention, it is provided in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form for the treatment or prevention of diseases and conditions affected by the regulation of TMEM16A.

[0174] Compound 1 is also provided in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, in the manufacture of medicaments for the treatment or prevention of diseases and conditions regulated by TMEM16A.

[0175] Methods for treating or preventing diseases and conditions regulated by TMEM16A are also provided, comprising administering to a patient in need of such treatment an effective amount of the compound 1 in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0176] Diseases and conditions regulated by TMEM16A include respiratory diseases and conditions, xerostomia (dry mouth), hyperactive bowel, cholestasis, and eye diseases.

[0177] Also provided:

[0178] • Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, for the treatment or prevention of respiratory diseases and conditions.

[0179] • Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, for the treatment or prevention of dry mouth (xerostomia).

[0180] • Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, for the treatment or prevention of intestinal hyperactivity.

[0181] • Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, for the treatment or prevention of cholestasis.

[0182] • Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, for the treatment or prevention of eye diseases.

[0183] The present invention also provides:

[0184] It also provides the use of Compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, in the manufacture of medicaments for the treatment or prevention of respiratory diseases and conditions.

[0185] • Use of Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, in the manufacture of a medicament for the treatment or prevention of dry mouth (xerostomia).

[0186] • Use of Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, in the manufacture of a medicament for the treatment or prevention of intestinal hyperactivity.

[0187] • Use of Compound 1 in the manufacture of a medicament for the treatment or prevention of cholestasis, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0188] • Use of Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, in the manufacture of a medicament for the treatment or prevention of eye diseases.

[0189] Further details were provided:

[0190] • A method for treating or preventing respiratory diseases and conditions, comprising administering to a patient in need of such treatment an effective amount of the compound 1 in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0191] • A method for treating or preventing dry mouth (xerostomia), comprising administering to a patient in need of such treatment an effective amount of compound 1 in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0192] • A method for treating or preventing intestinal hyperactivity, comprising administering to a patient requiring such treatment an effective amount of compound 1 in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0193] • A method for treating or preventing cholestasis, comprising administering to a patient requiring such treatment an effective amount of the compound 1 in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0194] • A method for treating or preventing an eye condition, comprising administering to a patient in need of such treatment an effective amount of compound 1 in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0195] Respiratory diseases and conditions that can be treated or prevented by Compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, include cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis including noncystic fibrotic bronchiectasis, asthma, and primary ciliary dyskinesia.

[0196] Dry mouth (xerostomia), which can be treated or prevented by compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, can be caused by Sjorgens syndrome, radiation therapy, and drugs that cause dry mouth.

[0197] Compound 1 is typically administered as part of a pharmaceutical composition, and therefore the present invention further provides a pharmaceutical composition comprising, in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form, compound 1 and a pharmaceutical excipient.

[0198] The pharmaceutical composition can be formulated for oral, rectal, intranasal, topical (including topical application to the lungs, skin, transdermal, ophthalmic, sublingual, and sublingual) administration, vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration, and can be prepared by any method well known in the pharmaceutical field. Compositions for oral or topical application to the lungs are particularly suitable.

[0199] The composition can be prepared by combining the active agent as defined above with the excipient. Generally, the preparation method involves uniformly and tightly binding the active agent with a liquid carrier or a fine-particle solid carrier, or both, and then, if necessary, shaping the product. The invention extends to a method for preparing a pharmaceutical composition, comprising coordinating or combining compound 1, in the form of its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form, with a pharmaceutical carrier or medium.

[0200] The formulations for oral administration in this invention may be represented as: discrete units, such as capsules, sachets or tablets, each containing a predetermined amount of active agent; powders or granules; solutions or suspensions of the active agent in aqueous or non-aqueous liquids; or oil-in-water emulsions or water-in-oil emulsions; or pills, etc.

[0201] For compositions intended for oral administration (e.g., tablets and capsules), the term "acceptable carrier" includes media such as common excipients, such as binders like syrups, gum arabic, gelatin, sorbitol, tragacanth gum, polyvinylpyrrolidone (polyvinyl ether), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose, and starch; fillers and carriers such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginate; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, glyceryl stearate, stearic acid, silicone oil, talc, oils, and silica gel. Flavoring agents such as peppermint, wintergreen oil, cherry flavoring, etc., may also be used. Coloring agents may be added to make the dosage form easily identifiable. Tablets may also be coated using methods well known in the art.

[0202] Tablets can be made by compression or molding, optionally with one or more excipients. Compressed tablets can be prepared by compressing an active agent in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant. Molded tablets can be made by molding a mixture of powdered compounds wetted with an inert liquid diluent in a suitable machine. The tablet may optionally be coated or scored, and optionally formulated to allow for sustained-release or controlled-release of the active agent.

[0203] Other formulations suitable for oral administration include lozenges containing the active agent in a flavoring base (typically sucrose and gum arabic or astragalus gum); lozenges containing the active agent in an inert base (such as gelatin and glycerin, or sucrose and gum arabic); and mouthwashes containing the active agent in a suitable liquid carrier.

[0204] Particularly suitable compositions for oral administration comprise an aqueous suspension of Compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form. When Compound 1 is present as a type A polymorph, it is best to suspend it in an aqueous solvent just before administration to the patient to avoid recrystallization into the type B hydrated polymorph. On the other hand, when Compound 1 is present as its type B polymorph (especially the type B(I) pseudopolymorph), the aqueous suspension can be stored for a longer period of time because the type B(I) pseudopolymorph is thermodynamically stable in the aqueous suspension. The aqueous suspension may also include other additives discussed below.

[0205] For topical application to the skin, compound 1, in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, can be formulated as creams, ointments, gels, solutions, or suspensions. Cream or ointment formulations that can be used for pharmaceuticals are conventional formulations well known in the art, for example, as described in standard textbooks of pharmaceutics (e.g., the British Pharmacopoeia).

[0206] Topical application to the lungs can be achieved using aerosol formulations. Aerosol formulations typically contain an active ingredient suspended or dissolved in a suitable aerosol spray, such as chlorofluorocarbons (CFCs) or hydrofluorocarbons (HFCs). Suitable CFC sprays include trichlorofluoromethane (Spray 11), dichlorotetrafluoromethane (Spray 114), and dichlorodifluoromethane (Spray 12). Suitable HFC sprays include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227). The spray typically constitutes 40% to 99.5% by weight of the total inhaled composition, for example, 40% to 90% by weight. The formulation may contain excipients, including co-solvents (e.g., ethanol) and surfactants (e.g., lecithin, sorbitan trioleate, etc.). Other possible excipients include polyethylene glycol, polyvinylpyrrolidone, glycerin, etc. The aerosol formulation is packaged in a can and the appropriate dose is delivered via a metering valve (e.g., provided by Bespak, Valois, or 3M, or alternatively by Aptar, Coster, or Vari).

[0207] Topical application to the lungs can also be achieved using non-pressurized formulations, such as aqueous solutions or suspensions. These can be administered via nebulizers (e.g., handheld and portable or home / hospital (i.e., non-portable) nebulizers). Formulations may contain excipients such as water, buffers, tension modifiers, pH adjusters, surfactants, and co-solvents. Suspensions and aerosol formulations (whether pressurized or non-pressurized) will typically contain compounds of the invention in fine particulate form, such as D having a particle size of 0.5 μm–10 μm (e.g., about 1 μm–5 μm). 50D can be used. 10 D 50 and D 90 The value is used to represent the particle size distribution. The particle size distribution is represented by D. 50 The median is defined as the particle size, measured in micrometers, that divides the distribution in two. Measurements derived from laser diffraction more accurately describe the volume distribution; therefore, the D obtained using this procedure... 50 The value is more meaningfully called Dv 50 The Dv value (median of the volume distribution). As used in this article, the Dv value refers to the particle size distribution measured using laser diffraction. Similarly, Dv in the context of laser diffraction... 10 and D 90 The values ​​are interpreted as Dv respectively. 10 and Dv 90 The value refers to particle size, with 10% of the distribution being below D. 10 The value and 90% of the distribution are lower than D. 90 value.

[0208] When compound 1 is present as a type A polymorph, it is best to suspend it in an aqueous solvent just before administration to the patient to avoid recrystallization into a type B hydrated polymorph. On the other hand, when compound 1 is present as its type B polymorph (particularly type B(I) pseudopolymorph), the aqueous suspension can be stored for a longer period of time because the type B(I) pseudopolymorph is thermodynamically stable in the aqueous suspension. The aqueous suspension may also include other additives discussed below.

[0209] Topical application to the lungs can also be achieved using dry powder formulations. The dry powder formulation will contain the disclosed compound in fine particulate form, typically having a mass-average particle size (MMAD) of 1 μm-10 μm or a density of 0.5 μm-10 μm (e.g., about 1 μm-5 μm). 50 Powders of the compounds of the present invention in fine-grained form can be prepared by micronization or similar size reduction methods. Micronization can be performed using an air jet mill (such as those manufactured by Hosokawa Alpine). The resulting particle size distribution can be measured using laser diffraction (e.g., with a Malvern Mastersizer 2000S instrument). The formulation typically contains locally acceptable diluents, such as lactose, glucose, or mannitol (preferably lactose), and generally has a large particle size, such as a mass average particle size (MMAD) of 50 μm or greater (e.g., 100 μm or greater) or a D0 of 40 μm–150 μm. 50As used herein, the term "lactose" refers to components containing lactose, including α-lactose monohydrate, β-lactose monohydrate, α-anhydrous lactose, β-anhydrous lactose, and amorphous lactose. Lactose components can be processed by micronization, sieving, grinding, compression, agglomeration, or spray drying. It also encompasses commercially available forms of lactose in various forms, such as... (Inhalation-grade lactose; DFE Pharma) 70 (for screening lactose in dry powder inhalers; Meggle) (DFEPharma) and (Sieve-inhaled lactose; DFE Pharma) product. In one embodiment, the lactose component is selected from the group consisting of α-lactose monohydrate, α-anhydrous lactose, and amorphous lactose. Preferably, the lactose is α-lactose monohydrate.

[0210] Dry powder formulations may also contain other excipients. Therefore, in one embodiment, a dry powder formulation according to this disclosure comprises magnesium stearate or calcium stearate. Such formulations can have excellent chemical and / or physical stability, particularly when such formulations also contain lactose.

[0211] Dry powder inhaler (DPI) devices are typically used to deliver dry powder formulations. Exemplary dry powder delivery systems include... and 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.

[0212] In one embodiment, Compound 1, in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, is provided as a micronized dry powder formulation (e.g., containing a suitable grade of lactose).

[0213] Therefore, as one aspect of the present invention, a pharmaceutical composition is provided, which is in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, in combination with particulate lactose to form compound 1 in particulate form, said composition optionally comprising magnesium stearate.

[0214] In one embodiment, Compound 1, in its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form, is provided as a micronized dry powder formulation (containing suitable grades of lactose and magnesium stearate) and filled into a device (e.g., DISKUS). Suitably, such a device is a multi-dosing device, for example, filling the formulation into a blister pack for use in a multi-unit dosing device (e.g., DISKUS).

[0215] In another embodiment, the compound 1, in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, is provided as a micronized dry powder formulation (e.g., containing a suitable level of lactose), and filled into a hard-shell capsule for use in a single-dose device (e.g., AEROLISER).

[0216] In another embodiment, the compound 1, in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, is provided as a micronized dry powder formulation (e.g., containing suitable levels of lactose and magnesium stearate), and filled into hard-shell capsules for use in a single-dose device (e.g., AEROLISER).

[0217] In another embodiment, compound 1, in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, is provided as a fine powder for an inhalation formulation, wherein the powder is in D 50 Fine particles of 0.5 μm–10 μm (e.g., about 1 μm–5 μm) are produced by size reduction methods other than air jet milling (e.g., spray drying, spray freezing, microfluidization, high-pressure homogenization, supercritical fluid crystallization, ultrasonic crystallization, or combinations thereof, or other suitable particle forming methods known in the art for producing fine particles with an aerodynamic particle size of 0.5 μm–10 μm). The resulting particle size distribution can be measured using laser diffraction (e.g., with a Malvern Mastersizer 2000S instrument). The particles may contain a single compound or a compound in combination with other suitable excipients that may facilitate processing. The resulting fine particles may be formulated into a final formulation for delivery to humans, or optionally further formulated with other suitable excipients to facilitate delivery in an acceptable dosage form.

[0218] Compound 1, in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, may also be administered rectally, for example, as a suppository or enema, comprising aqueous or oily solutions, as well as suspensions, emulsions, and foams. Such compositions are prepared according to standard procedures well known to those skilled in the art. For example, suppositories can be prepared by mixing the active ingredient with a conventional suppository base (such as cocoa butter or other glycerides). In such cases, the drug is mixed with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thus melting in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycol.

[0219] Typically, for compositions intended for topical application to the eye in the form of eye drops or ointments, the total amount of compound 1, in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in its amorphous form, will be from about 0.0001% to less than 4.0% (w / w).

[0220] Preferably, for topical ocular application, compositions comprising Compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, will be formulated as solutions, suspensions, emulsions, and other dosage forms. Aqueous solutions are generally preferred due to their ease of formulation and the ability of the patient to easily apply such compositions by instilling one or two drops of solution into the affected eye. However, compositions may also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions. Suspensions are preferred for compounds that are poorly soluble in water.

[0221] An alternative for ocular application is intravitreal injection of a solution or suspension of Compound 1 in its type A or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form. Alternatively, Compound 1 can be introduced via ocular implants or inserts in its type A or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0222] Compositions containing Compound 1 in its A-type or B-type polymorph (especially its B(I) type pseudopolymorph), as defined above, or in an amorphous form, may further contain various other components, including but not limited to tonics, buffers, surfactants, stabilizing polymers, preservatives, cosolvents, and thickeners. Suitable pharmaceutical compositions containing Compound 1 in its A-type or B-type polymorph (especially its B(I) type pseudopolymorph), as defined above, or in an amorphous form, may be formulated with tonics and buffers. Pharmaceutical compositions containing Compound 1 in its A-type or B-type polymorph (especially its B(I) type pseudopolymorph), as defined above, or in an amorphous form, may further optionally contain surfactants and / or modifiers and / or stabilizing polymers.

[0223] Various tonic agents can be used to adjust the tension of the composition, preferably the tension of the natural tear film in the ophthalmic composition. For example, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, monosaccharides (such as dextrose, fructose, galactose) and / or simple polyols (such as sugar alcohols mannitol, sorbitol, xylitol, lactitol, isomaltitol, maltitol) and hydrogenated starch hydrolysate can be added to the composition to approximate physiological tension. The amount of such tonic agents will vary depending on the specific pharmaceutical agent to be added. However, generally, the composition will have a sufficient amount of tonic agent to give the final composition an ophthalmologically acceptable osmotic pressure (typically about 150 mOsm-450 mOsm, preferably 250 mOsm-350 mOsm, most preferably about 290 mOsm). Generally, the tonic agents of the present invention will be present in the range of 2% w / w to 4% w / w. Preferred tonic agents of the present invention include monosaccharides or sugar alcohols, such as D-mannitol.

[0224] A suitable buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate, or boric acid) can be added to the composition to prevent pH drift under storage conditions. The specific concentration will vary depending on the reagent used. However, preferably, a buffer solution will be selected to maintain the target pH in the range of pH 5 to 8, and more preferably in the range of pH 5 to 7.

[0225] Surfactants may optionally be used to deliver higher concentrations of the compound 1 in its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form. The surfactant acts to dissolve the compound and stabilize colloidal dispersions such as micelle solutions, microemulsions, emulsions, and suspensions. Examples of surfactants that may be used optionally include polysorbates, poloxamer, polyethylene glycol 40 stearate, polyoxyethylene castor oil, teroxazolidinium, Triton, and sorbitan laurate. Preferred surfactants used in this invention have a hydrophilic / lipophilic / balanced “HLB” in the range of 12.4 to 13.2 and are acceptable for ophthalmic use, such as Triton X114 and teroxazolidinium.

[0226] Additional agents that can be added to ophthalmic compositions of Compound 1 in its A-type or B-type polymorph (especially its B(I)-type pseudopolymorph), as defined above, or in an amorphous form, serve as moderating agents to stabilize the polymer. The stabilizing polymer should be an ionic / charged instance, preferably for topical ophthalmic use, and more specifically a polymer with a negatively charged surface, exhibiting a (-)10mV-50mV zeta potential for physical stability and capable of forming a dispersion in water (i.e., water-soluble). Preferred stabilizing polymers of the present invention are polyelectrolytes, or more than one polyelectrolyte derived from the cross-linked polyacrylate family, such as carbomer and Pemulen(R), particularly carbomer 974p (polyacrylic acid), at 0.1% w / w-0.5% w / w.

[0227] Other compounds may also be added to ophthalmic compositions of Compound 1 in its type A or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, to increase the viscosity of the carrier. Examples of thickeners include, but are not limited to: polysaccharides (such as hyaluronic acid and its salts), chondroitin sulfate and its salts, dextran, various polymers of the cellulose family; vinyl polymers; and acrylic polymers.

[0228] Topical ophthalmic products are typically packaged in multiple doses. Therefore, preservatives are required to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, chlorobutanol, benzyl dimethyl dodecyl ammonium bromide, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, polyquaternium-1, or other agents known to those skilled in the art. Such preservatives are typically used at levels from 0.001% w / v to 1.0% w / v. Unit dose compositions of Compound 1, in its type A polymorph or type B polymorph (particularly its type B(I) pseudopolymorph), as defined above, or in an amorphous form, will be sterile but generally not preservative-free. Therefore, such compositions will typically not contain preservatives.

[0229] Parenteral preparations are usually sterile.

[0230] A licensed physician or other skilled worker will be able to determine the appropriate dosage of the compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, and therefore the amount of the compound of the present invention should be included in any particular pharmaceutical preparation (whether in unit dosage form or otherwise).

[0231] Compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, may be used in combination with one or more other active agents for the treatment or prevention of respiratory diseases and conditions.

[0232] Another active agent of this type may be included in the above-described pharmaceutical composition, but alternatively, it may be administered alone, concurrently with, or at an earlier or later time, compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form.

[0233] Therefore, in another aspect of the invention, a product is provided comprising compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, and additional pharmaceutical agents for the treatment or prevention of respiratory conditions, which are used as a combination formulation for simultaneous, sequential, or separate use in the treatment of diseases or conditions affected by TMEM16A and, in particular, respiratory diseases or conditions (e.g., one of the aforementioned diseases and conditions).

[0234] It is also provided that the compound 1, in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, is combined with other pharmaceutical agents that can be used to treat or prevent respiratory conditions, as a combination preparation for simultaneous, sequential, or separate use in the treatment of diseases or conditions regulated by TMEM16A, and especially respiratory diseases or conditions (such as one of the aforementioned diseases and conditions).

[0235] Suitable additional active agents that may be included in a pharmaceutical composition or in a combination formulation of compound 1 in the form of its type A polymorph or type B polymorph (especially its type B(I) pseudopolymorph), as defined above, or in an amorphous form, include:

[0236] β2-adrenergic receptor agonists, such as metaproterenol, isoproterenol, isoprenaline, salbutamol, salbutamol, formoterol, salmeterol, indacaterol, terbutaline, isoproterenol, bitoterol mesylate, pibuterol, olodaterol, vilanterol, and abedaterol;

[0237] Antihistamines, such as histamine H1 receptor antagonists like loratadine, cetirizine, desloratadine, levocetirizine, fexofenadine, astemizole, azelastine and chlorpheniramine or H4 receptor antagonists;

[0238] Alpha-chain enzyme;

[0239] Corticosteroids, such as prednisone, prednisolone, flunisolone, triamcinolone, beclomethasone dipropionate, budesonide, fluticasone propionate, mometasone furoate, and fluticasone furoate;

[0240] Leukotriene antagonists, such as montelukast and zafirlukast;

[0241] Anticholinergic compounds, especially muscarinic antagonists, such as ipratropium bromide, tiotropium bromide, glycopyrronium bromide, adecyl bromide and fudecyl bromide;

[0242] CFTR repair agents (such as CFTR enhancers, correctors, or amplifiers), such as Ivacaftor, QBW251, Bamacaftor (VX659), Elexacaftor (VX445), VX561 / CPT-656, VX152, VX440, GLP2737, GLP2222, GLP2451, PTI438, PTI801, PTI808, FDL-169, and FDL-176, and CFTR correctors, such as Lumacaftor and Tezacaftor, or combinations thereof (e.g., combinations of Ivacaftor, Tezacaftor, and Elexacaftor);

[0243] ENaC modulators, especially ENaC inhibitors;

[0244] antibiotic;

[0245] Antiviral drugs, such as ribavirin, and neuraminidase inhibitors such as zanamivir;

[0246] Antifungal agents, such as PUR1900;

[0247] Airway moisturizers (penetrating agents), such as hypertonic saline and mannitol. as well as

[0248] Mucus-dissolving agents, such as N-acetylcysteine.

[0249] When the other active agent is an ENaC modulator, it can be an ENaC inhibitor, such as amiloride, VX-371, AZD5634, QBW276, SPX-101, BI443651, BI265162, and ETD001. Other suitable ENaC blockers disclosed in our applications WO 2017 / 221008, WO 2018 / 096325, WO2019 / 077340, and WO 2019 / 220147, and any of the exemplary compounds of these applications, can be used in combination with compounds of general formula (I). Particularly suitable compounds for use in combination with compounds of general formula (I) include compounds having a cation selected from:

[0250] 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamoyl)ethyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazole-3-onium;

[0251] 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamoyl)methyl]-6-{[2-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazole-3-onium;

[0252] 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamoyl)methyl]-5-[4-({bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}methyl)piperidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazole-3-onium;

[0253] 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamoyl)methyl]-6-[(3R)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidin-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazole-3-onium;

[0254] 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamoyl)methyl]-6-[(3S)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidin-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazole-3-onium;

[0255] 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamoyl)methyl]-1,3-diethyl-6-{[(1r,4r)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazole-3-onium;

[0256] 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamoyl)methyl]-1,3-diethyl-6-{[(1s,4s)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazole-3-onium;

[0257] And suitable anions, such as halide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate, succinate, mandelic acid, methanesulfonate or p-toluenesulfonate.

[0258] The invention will now be described in more detail with reference to examples.

[0259] Instruments and general conditions

[0260] For Example 1 and Example 2:

[0261] The starting materials and intermediates, as well as compound 1, can be separated and purified using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography. Unless otherwise stated, all starting materials were obtained from commercial suppliers and were ready for use without further purification. Salts can be prepared from the compounds using known salt-forming procedures.

[0262] MS

[0263] Mass spectrometry was performed on an LC-MS system using electro-jet ionization. These were performed using either a Waters Acquity uPLC system with Waters PDA and ELS detectors or a Shimadzu LCMS-2010EV system. [M+H]+ refers to the molecular weight of a single isotope.

[0264] NMR

[0265] NMR spectra were recorded on a Bruker Avance III HD at 500 MHz or 250 MHz using a solvent as an internal deuterium lock. Unless otherwise specified, the spectra were recorded at room temperature, and the solvent peaks were used for reference.

[0266] HPLC

[0267] The analytical HPLC conditions are as follows:

[0268] Method A

[0269] Column: Phenomenex Kinetix-XB C18 2.1x100mm, 1.7μm

[0270] Column temperature 40℃

[0271] Eluent: A: H2O, 0.1% formic acid; B: acetonitrile, 0.1% formic acid

[0272] Flow rate: 0.6 mL / min

[0273] Gradient: 0-5.3 minutes 5%-100% B, 5.3 minutes-5.8 minutes 100% B, 5.8 minutes-5.82 minutes 100%-5% B, 5.82 minutes-7.00 minutes 5% B

[0274] Method E

[0275] Pillar: Kinetex Core-Shell C18 2.1x50mm 5μm

[0276] Column temperature 40℃

[0277] Eluent: A: H2O + 0.1% formic acid, B: acetonitrile + 0.1% formic acid

[0278] Flow rate: 1.2 mL / min

[0279] Gradient: 0-1.20 minutes 5%-100% B, 1.20-1.30 minutes 100% B, 1.30-1.31 minutes 100%-5% B

[0280] Method F

[0281] Column: Phenomenex Gemini-NX C18 2x50mm 3μm

[0282] Column temperature 40℃

[0283] Eluent: A: 2mM ammonium bicarbonate, buffered to pH 10; B: acetonitrile

[0284] Flow rate: 1 mL / min

[0285] Gradient: 0-1.80 minutes 1%-100% B, 1.80 minutes-2.10 minutes 100% B, 2.10 minutes-2.30 minutes 100%-1% B

[0286] For Examples 3 to 12

[0287] DSC

[0288] The Mettler Toledo DSC 821 instrument is used for thermal analysis operated using STARe™ software. The analysis is performed under nitrogen atmosphere in a 40 μL open aluminum dish, with sample volumes ranging from 1 mg to 10 mg. Typical analytical rates are 20 to 250 °C at 10 °C / min.

[0289] DVS

[0290] The hygroscopic properties of the feed API were analyzed using a DVS (DVS Intrinsic, Surface Measurement System). Approximately 50 mg of API was weighed into an aluminum pan and loaded into the instrument at 25°C. The sample was equilibrated in a dry atmosphere (0% relative humidity) for 1 hour, then the humidity was increased from 0% to 30% in 5% increments, and from 30% to 90% in 10% increments. Desorption cycles were also applied from 90% to 30% (10% increments) and from 30% to 0% (5% increments). The rate of change over time was set as the equilibration parameter (1 hour per step). Kinetics and isotherms were calculated.

[0291] FT-IR

[0292] FT-IR spectra were acquired using a PerkinElmer Spectrum One FT-IR spectrometer. Samples were analyzed directly in the frequency range of 4000 to 600 cm⁻¹ using a universal ATR attachment. Spectra were processed using Spectrum CFD, vs. 4.0 PerkinElmer Instruments LLC.

[0293] LC-MS

[0294] Conventional liquid chromatography-mass spectrometry (LC-MS) data were collected using Agilent 1260 Infinity II and Agilentseries 1260 Infinity II binary pumps that interact with the 1260 Infinity II DAD HS.

[0295] This instrument uses a single quadrupole InfinityLab MSD. The instrument is calibrated to 2000 Da.

[0296] 1H NMR

[0297] ¹H NMR spectra were acquired using a Bruker 400MHz spectrometer and TopSpin was used. TM (Bruker) was used to process the data. Samples were prepared in DMSO-D6 at typical concentrations of 10 mg / mL to 20 mg / mL and up to 50 mg / mL for 1H NMR w / w determination, with the corresponding non-deuterated solvent residue calibrated to 2.50 ppm.

[0298] 1H NMR w / w determination

[0299] API (w / w) determinations by 1H NMR spectroscopy were performed by the project chemists.

[0300] Internal standard 2,3,5,6-tetrachloronitrobenzene (TCNB) (approximately 20 mg, FW260.89) and API (approximately 20 mg) were dissolved in DMSO-D6 (2.0 ml), and 1H NMR spectra were obtained using the extended relaxation method to measure the assay according to the following equation:

[0301] %w / w=[m(std.) / Mw(std.)*∫(sample) ÷∫(std.)*Mw(sample) / m(sample)]

[0302] Optical (polarized light) microscope

[0303] The instrument used for digital capture was an Olympus BX41 microscope with a digital camera attachment. Magnification was 100x and 400x. Samples were observed under both plane-polarized and cross-polarized light.

[0304] thermal microscope

[0305] The instrument used for digital capture was an Olympus BX41 microscope equipped with a digital camera and a Linkam hot stage accessory. Magnification was 100x and 400x. Samples were observed under plane-polarized and cross-polarized light.

[0306] TG Analysis

[0307] Thermogravimetric analysis (TGA) was performed using a combined differential technique combining TGA and DSC signals (SDT, Q600, TA instruments). Approximately 5 mg of sample was placed in a ceramic dish. The sample was heated from room temperature to 600 °C at a rate of 10 °C / min under a nitrogen atmosphere. The TGA and DSC signals were analyzed using TA Universal analysis software.

[0308] XRPD Analysis

[0309] X-ray powder diffraction (XRPD) analysis was performed using a Bruker D2 Phaser powder diffractometer equipped with a LynxEye detector. Specimens were prepared to a minimum, but if necessary, they were lightly ground with a pestle and mortar before collection. The specimens were placed in the center of a silicon sample holder within a 5 mm (approximately 5 mg to 10 mg) bag.

[0310] Samples were fixed during data collection and scanned in steps of 0.02° / 2θ (2θ) between 4° and 40° and 5° and 60°2-θ. Data were acquired using either a 3-minute or 20-minute acquisition method. BrukerDiffrac.Suite was used to process the data.

[0311] For Examples 13 to 17

[0312] DSC

[0313] Mettler Toledo DSC 3 instrument for use with STARe TM Thermal analysis was performed using software. The analysis was conducted under nitrogen atmosphere in a 40 μL open aluminum dish, with sample volumes ranging from 1 mg to 10 mg. Typical analytical rates were 20 to 250 °C at 10 °C / min.

[0314] DVS

[0315] Perform this operation as described above for Examples 3 through 12.

[0316] FT-IR

[0317] FT-IR spectra were acquired using a PerkinElmer Frontier FT-IR spectrometer. Samples were directly analyzed in the mid-to-long frequency range of 4000 to 30 cm⁻¹ using a universal ATR accessory. Spectrum IR was used. TM The instrument uses software (PerkinElmer Instruments LLC) to process the spectra. A standard KBr window is used for mid-IR applications; polyethylene and polyethylene / diamond windows are used for far-IR operations. Other features of the instrument include a liquid flow cell with a ZnSe window for rapid reaction monitoring. This is consistent with Spectrum. TM When used in conjunction with TimeBase software (PerkinElmer), time-resolved measurements can be performed.

[0318] LC-MS

[0319] Perform this operation as described above for Examples 3 through 12.

[0320] 1H NMR

[0321] Perform this operation as described above for Examples 3 through 12.

[0322] 1H NMR w / w determination

[0323] Perform this operation as described above for Examples 3 through 12.

[0324] Optical (polarized light) microscope

[0325] Perform this operation as described above for Examples 3 through 12.

[0326] thermal microscope

[0327] Perform this operation as described above for Examples 3 through 12.

[0328] TG Analysis

[0329] A Mettler Toledo TGA 2 instrument was used to measure weight loss as a function of temperature from 25°C to 500°C. The scan rate was typically 5°C or 10°C per minute. STARe was used. TM The software was used to perform the experiments and analyses. The analyses were conducted under nitrogen atmosphere in a 100 μL open aluminum dish, with sample volumes ranging from 1 mg to 10 mg.

[0330] XRPD Analysis

[0331] X-ray powder diffraction (XRPD) analysis was performed using a Bruker D2 Phaser powder diffractometer equipped with a LynxEye detector. Specimens were prepared to a minimum, but if necessary, they were lightly ground with a pestle and mortar before collection. The specimens were placed in the center of a silicon sample holder within a 5 mm (approximately 5 mg to 10 mg) bag.

[0332] The sample was stationary during data collection and scanned in steps of 0.02° / 2θ (2θ) within the range of 4° to 40° / 2θ. Data were acquired using either a 3-minute or 20-minute acquisition method. Bruker Diffrac Suite was used to process the data.

[0333] abbreviation

[0334] br wide peak

[0335] d double peak

[0336] dd double peak

[0337] DCM dichloromethane

[0338] DIPEA (diisopropylethylamine)

[0339] DMF N,N-dimethylformamide

[0340] DSC Differential Scanning Calorimetry

[0341] EtOAc (ethyl acetate)

[0342] h hours

[0343] HPLC (High Performance Liquid Chromatography)

[0344] IR infrared spectroscopy (FT-IR is Fourier transform infrared spectroscopy)

[0345] m multiplet

[0346] MeCN Acetonitrile

[0347] mg

[0348] min minutes

[0349] mL

[0350] mol

[0351] MS mass spectrometry

[0352] m / z mass-to-charge ratio

[0353] N / A Not Applicable

[0354] NMR (Nuclear Magnetic Resonance)

[0355] Rt retention time

[0356] s Single peak

[0357] Saturated

[0358] t triple peak

[0359] TBTU N,N,N',N'-Tetramethyl-O-(benzotriazol-1-yl)uranium tetrafluoroborate

[0360] TEA Triethylamine

[0361] TGA thermogravimetric analysis

[0362] Example 1 -N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide (compound) Preparation of substance 1) - Method WO2019 / 145726

[0363] Step 1: 4-Amino-N-tert-butyl-pyridine-2-carboxamide

[0364]

[0365] 2-Methylpropane-2-amine (1.69 mL, 69.5 mmol) was added to a mixture of 4-aminopyridine-2-carboxylic acid (8.0 g, 57.92 mmol), TBTU (22.32 g, 69.5 mmol), and TEA (24.22 mL, 173.76 mmol) in DMF (100 mL). The resulting mixture was stirred at room temperature for 22 hours, and then concentrated under vacuum. The starting material was purified by silica gel chromatography, eluted with a solution of 3.5 M methanol-ammonia in DCM, and the product fractions were combined and concentrated under vacuum to yield the title compound, which is a pale yellow solid.

[0366] 1H NMR (500MHz, Methanol-d4) δ7.99 (d, J = 5.6 Hz, 1H), 7.23 (d, J = 2.2 Hz, 1H), 6.62 (dd, J = 5.6, 2.4 Hz, 1H), 1.45 (s, 9H).

[0367] LC-MS (Method F): Rt 1.47 mins; MS m / z 194.3 = [M + H] + (100% @ 215 nm)

[0368] Step 2: N-tert-butyl-4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]pyridine-2-carboxamide

[0369]

[0370] A solution of 2-(5-chloro-2-methoxy-phenyl)acetic acid (2.26 g, 11.27 mmol) in thionyl chloride (8.13 mL, 92.21 mmol) was heated at 70 °C for 30 min. After cooling to room temperature, excess thionyl chloride was removed under vacuum and azeotropically with toluene. The resulting residue was dissolved in DCM (5 mL) and a solution of 4-amino-N-tert-butyl-pyridine-2-carboxamide (step 1) (2.0 g, 10.25 mmol) and DIPEA (2.15 mL, 12.29 mmol) in DCM (25 mL) was added. The mixture was stirred at room temperature for 1 h, then diluted with water (50 mL) and extracted with DCM. The combined organic extracts were washed with brine (50 mL), dried over Na₂SO₄, and concentrated under vacuum. The crude residue was purified by silica gel chromatography, eluting with a solution of 0-50% EtOAc in heptane to give the title compound as a pale orange powder.

[0371] 1H NMR(500MHz,Chloroform-d)δ8.39(d,J=5.6Hz,1H),8.20(dd,J=5.6,2.2Hz,1H),8.10(br s,1H),7.98(br s,1H),7.56(d,J=2.1Hz,1H),7.29-7.26(m,2H),6.89(d,J=9.5Hz,1H),3.94(s,3H),3.70(s,2H),1.47(s,9H).

[0372] LC-MS (Method E): Rt 1.21 mins; MS m / z 376.1 / 378.1 = [M + H] + (92% @ 215 nm)

[0373] Step 3:N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide was added dropwise at 0 °C to a solution of N-tert-butyl-4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]pyridine-2-carboxamide (step 2) (2.7 g, 6.82 mmol) in DCM (10 mL) with 1 MBBr3 in DCM (27.3 mL, 27.3 mmol). Once the addition was complete, the mixture was warmed to room temperature and stirred for 1 hour. The reaction was quenched by the slow addition of water (10 mL) and the DCM was removed under vacuum. The resulting residue was dissolved in EtOAc and washed with saturated NaHCO3 solution (50 mL) and brine (50 mL). The organic fraction was separated, dried over Na2SO4, and concentrated under vacuum. The crude residue was purified by silica gel chromatography, eluting with a solution of 0-70% EtOAc in heptane to give the product as an orange powder. This was further purified by reversed-phase chromatography, eluting with a solution of 0-100% MeCN in water (containing 0.1% formic acid) to give the product as a colorless powder. The product was recrystallized from MeCN to give the title compound. A second batch of product was separated by adding water dropwise to the MeCN filtrate, followed by heating and cooling the mixture.

[0374] 1 H NMR(500MHz,DMSO-d6)δ10.69(br s,1H),9.82(br s,1H),8.44(d,J=5.5Hz,1H),8.17(d,J=1.9Hz,1H),8.03(s,1H),7.82(dd,J=5.5,2.2Hz,1H),7. 22(d,J=2.7Hz,1H),7.12(dd,J=8.6,2.7Hz,1H),6.80(d,J=8.6Hz,1H),3.67(s,2H),1.40(s,9H).

[0375] LC-MS (Method A): Rt 3.28 mins; MS m / z 362.1 / 364.1 = [M+H]+(99%@215nm).

[0376] XRPD analysis (see...) Figure 20 The diagram shows that compound 1 prepared by this method is crystalline, exhibiting multiple unique peaks as well as several peaks consistent with type A, indicating the presence of a mixture of at least two different polymorphs (including type A). (DSC thermogram) Figure 19 This indicates that the material contains at least one polymorph that is not type A, as specifically demonstrated by exothermic and endothermic events between 112.5°C and 130.4°C. Thermogravimetric analysis also shows that the product is not a hydrate or other solvate.

[0377] Example 2 - Preparation of crystalline polymorph A

[0378] A batch of compound 1 (14.6 g) was prepared according to the method described in Example 1. The material was recrystallized by suspending it in MeCN (200 mL) and heating it under reflux until all solids dissolved. It was then slowly cooled to room temperature over several hours or overnight. The resulting crystalline solid was filtered and dried in a vacuum oven to give N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide (compound 1) (12.9 g, 353 mmol, yield 33%), which was a colorless crystalline solid (polymorph A). The XRPD diffraction pattern, DSC thermogram, and DVS pattern of this material are shown below. Figure 1a , Figure 1b , Figure 2 , Figure 3a and Figure 3b As shown.

[0379] Example 3 - Preparation of amorphous compound 1

[0380] Compound 1A (1.50 g, 1.0 wt) was dissolved in ethyl acetate (30 ml, 20 v / v). The solution was filtered through a PTFE membrane and rapidly evaporated under reduced pressure at 40 °C, and then subjected to XRPD and... 1 Analysis was performed by 1H NMR. The crude product was dried under reduced pressure at 40 °C overnight. XRPD was then used for analysis. Figure 11 and Figure 12 The product was analyzed to confirm that the amorphous product did not recrystallize after oven drying, and the product was analyzed by 1H NMR to confirm that the solvent content was reduced.

[0381] Example 4 - Anhydrous suspension equilibrium of amorphous phase at 20°C

[0382] Amorphous compound 1 (approximately 50 mg, 1.0 wt) and a suitable solvent (1000 μl, 20 volumes) were placed in separate containers and stirred at 20 °C for 7 days. The product was then cooled, separated by filtration, washed with the recovered aging solvent, dried under reduced pressure at 40 °C, and evidence of alternative crystalline forms was analyzed by XRPD. The results are shown in Table 3.

[0383] Table 3 - Phase Equilibrium in Anhydrous Solvents at 20°C

[0384]

[0385]

[0386] *Pure water is included in this screening.

[0387] pass 1¹H NMR confirmed that all products were chemically related to compound 1. No significant chemical degradation was observed; therefore, the observed different diffraction patterns were not attributable to the presence of different chemical entities.

[0388] Type A was the most abundant form to be separated. Several other diffraction patterns were observed, some of which were anhydrous and others of solvated form.

[0389] • In this screening, type B (hydrate) is generated when water is present. Figure 5 The B-type DSC shown has an endothermic peak at 90.90 °C to 114.65 °C attributable to water loss from the hydrate. An exception is the product separated from DCM / heptane, in which the B-type still forms despite the absence of water as a co-solvent. The formation of the hydrate can be attributed to the introduction of water or the presence of moist heptane. 1 No solvent was detected by HNMR, and XRPD (see [link to XRPD]) Figure 13 ) and isomorphic type B hydrates (see Figure 4 Consistent. DSC analysis ( Figure 14 ) and BI type (see Figure 21a Consistent.

[0390] • C-type was isolated from trifluorotoluene and toluene, and the XRPD diffraction pattern is as follows: Figure 6 As shown. Both products obtained were non-solventized, and DSC analysis revealed melting events and crystallization into type A, as well as subsequent melting events.

[0391] • Type D corresponds to the isomeric ether solvate, and the XRPD spectrum of this isomeric ether solvate is as follows: Figure 7 As shown. Both are slightly below integer stoichiometry, i.e., 0.8 * THF. Figure 15a ) and 0.8*tBME( Figure 15b ).

[0392] • The E-type separated from MEK was partially solvated (0.4*MEK). Figure 16 The corresponding TG analysis shows no significant weight loss transition prior to the melting event; therefore, the bound MEK was released only after significant reorganization of the matrix crystals (i.e., MEK solvent vapor was released upon melting, accompanied by a significant weight loss associated with compound degradation). The XRPD diffraction pattern is shown below. Figure 8 As shown.

[0393] • The F-type was separated from ethanol, and its XRPD diffraction pattern is as follows: Figure 9 As shown. Type E is an anhydrous form, and DSC analysis shows a similar melting event to Type A, although the diffraction patterns are different; the competitive suspension equilibrium of an equimolar mixture of Types A and F yields only Type A, indicating that Type A is the more stable of the two forms.

[0394] Example 5 - Anhydrous suspension equilibrium of amorphous phase at 40°C

[0395] A separate fraction of amorphous compound 1 (approximately 50 mg, 1.0 wt) and a suitable solvent (1000 μl, 20 volumes) were placed in a separate container and stirred at 40 °C for 7 days. The product was then cooled, separated by filtration, washed with the recovered aging solvent, dried under reduced pressure at 40 °C, and evidence of alternative crystalline forms was analyzed by XRPD. The results are shown in Table 4.

[0396] Table 4 - Phase Equilibrium in Anhydrous Solvents at 40°C

[0397]

[0398]

[0399] *Pure water is included in this screening.

[0400] The same graph as that obtained from the suspension equilibrium test of the amorphous form at 20°C (Example 3) was obtained.

[0401] Type A is the main form.

[0402] • When water is present, it produces type B or a mixture of types A and B.

[0403] • C-type compounds have been observed and formed from toluene and trifluorotoluene.

[0404] • D-type (isomorphic solvates) were observed in the following ether solvents: THF and tBME.

[0405] • A new disordered form was observed and named H-type (see reference). Figure 10 The XRPD shown in the figure) and Figure 17 (DSC shown in the figure). The H-form is partially solvated and contains 9.0% w / w cumene, equivalent to about 0.4* solvate. By DSC, the exothermic event is evident at an initial temperature of 94 °C, which is significantly lower than the boiling point of cumene (152 °C). The endothermic event at 94 °C is the estimated transition temperature from the H-form solvate to the anhydrous form, followed by an exothermic event attributed to crystallization.

[0406] Example 6 - Equilibrium of an amorphous aqueous suspension at 20°C

[0407] A separate container was prepared by mixing amorphous compound 1 (approximately 50 mg, 1.0 wt) with a suitable solvent (950 μl, 19 v) and pure water (50 μl) and stirring at 20 °C for 7 to 10 days. The product was then cooled, separated by filtration, washed with the recovered aging solvent, dried under reduced pressure at 40 °C, and analyzed by XRPD for evidence of alternative crystalline forms. The results are shown in Table 5.

[0408] Table 5 - Phase Equilibrium in Aqueous Solvents at 20°C

[0409]

[0410]

[0411] No new forms were observed. Type B was the most prevalent form observed.

[0412] Example 7 - Further Suspension Equilibrium

[0413] Further suspension equilibrium experiments will be conducted below.

[0414] A. Anhydrous suspension equilibrium of type A at 20℃

[0415] Compound type 1A (approximately 50 mg, 1.0 g / L) and a suitable solvent (1000 μL, 20 volumes) were placed in a separate container and stirred at 20 °C for 7 days. The product was then cooled, separated by filtration, washed with the recovered aging solvent, dried under reduced pressure at 40 °C, and analyzed by XRPD for evidence of alternative crystalline forms. Note that water and acetonitrile / water were also included in this study.

[0416] Unless water is present, the product is consistent with type A, which promotes the formation of hydrate type B.

[0417] • D-type is generated in the presence of tBME, and the samples recovered from other ether THF and 2-MeTHF are insufficient for XRPD analysis.

[0418] Therefore, type A exhibits flexibility for long-term solvent-mediated treatment under anhydrous conditions at 20°C.

[0419] B. Anhydrous suspension equilibrium of type A at 40°C

[0420] The individual fraction of compound 1A (approximately 50 mg, 1.0 g / L) and a suitable solvent (1000 μL, 20 volumes) were placed in a separate container and stirred at 40 °C for 7 days. The product was then cooled, separated by filtration, washed with the recovered aging solvent, dried under reduced pressure at 40 °C, and analyzed by XRPD for evidence of alternative crystalline forms. Note that water was also included in this screening.

[0421] • In addition to tBME (type D) and THF (type G; see also Figure 18 Apart from the products separated from THF, all products correspond to type A. Typically, the product encountered in the presence of THF is type D.

[0422] • The experiment was performed in pure water, and unexpectedly, type A was generated, with no obvious type B.

[0423] C. Equilibrium of type A aqueous suspension at 20°C

[0424] The individual fraction of compound 1A (approximately 50 mg, 1.0 wt) was placed in a separate container with a suitable solvent (950 μl, 19 v) and pure water (50 μl, 1.0 v) and stirred at 20 °C for 7 to 10 days. The product was then cooled, separated by filtration, washed with the recovered aging solvent, dried under reduced pressure at 40 °C, and evidence of alternative crystalline forms was analyzed by XRPD.

[0425] As expected, the presence of water facilitates the conversion from type A to type B, although the rate of interconversion is relatively slow under these conditions.

[0426] • In the presence of pure water alone, partial conversion to type B was observed. This finding contradicts the results observed at 40°C (where type A was just formed) and suggests that a transition temperature may exist under solvent-mediated conditions.

[0427] Unusually, form C is also generated from acetone, anisole, ethanol, and 2-propanol. Form C is an anhydrous form and sometimes occurs in the presence of aromatic solvents (toluene and trifluorotoluene). In the presence of form A, the competitive suspension equilibrium of this form is then used to determine which form is most stable under anhydrous conditions.

[0428] Example 8-A: Aqueous surfactant equilibrium at and below the critical micelle concentration (CMC)

[0429] Type A samples were micronized using an AS100 spiral air jet mill (Alpine) with a ZD9 spiral metering system at a grinding pressure of 5.0 bar (18°C) and an injection pressure of 6.0 bar (18°C). The feed rate was 350 g / hour.

[0430] Using Beckman Coulter LS TM A 13 320 particle size analyzer is used to measure the D50 and D90 values ​​of input and output materials.

[0431] Input: D50 35.74μm, D90 77.74μm.

[0432] Output: D50 1.14μm, D90 2.44μm.

[0433] Type A and micronized Type A were added to pure water containing the surfactants shown in Table 6, and the mixture was stirred at 20°C.

[0434] Table 6 - Surfactant media used in surfactant equilibrium experiments

[0435]

[0436] Secondary sampling samples (300 μl to 500 μl) were collected at appropriate time points. The mixture was centrifuged, the clear supernatant was discarded, and the particles were dried (20 °C, 24 h) and analyzed by XRPD. The conversion rate was monitored by measuring the approximate rate of change of the selected reflectance peak area from each form to calculate the consumption of type A (exhibiting strong reflectance at approximately 7.1°2-θ, reference 1a) and type B (exhibiting strong reflectance at approximately 11°2-θ, reference 1a). Figure 4 The formation of type A is determined by XRPD. When type A is no longer detected, the time required for type A to completely transform into type B is inferred. The results are shown in Tables 8 to 11.

[0437] Table 7 - Results of surfactant equilibrium for non-micronized type A - Time points sampled at 20°C

[0438]

[0439] Table 8 - Results of surfactant equilibrium for non-micronized type A - Time points sampled at 40°C

[0440]

[0441] Table 9 - Results of surfactant equilibrium for micronized type A - Time points sampled at 20°C

[0442]

[0443]

[0444] *The preference-seeking effect may exacerbate the ratio.

[0445] Table 10 - Results of surfactant equilibrium for micronized type A - Time points sampled at 40°C

[0446]

[0447] definition:

[0448] Micelles are aggregates of surfactant molecules dispersed in a liquid colloid. Micelles only form when the concentration of the surfactant exceeds the critical micelle concentration (CMC).

[0449] Surfactant 1: 80 (polysorbate 80; polyoxyethylene sorbitan monooleate) Critical micelle concentration (CMC) 13-15 mg / L [Actual usage concentration = 1 mg / ml, 1000 mg / L > CMC]

[0450] Surfactant 2: 20 (polysorbate 20; polyethylene glycol sorbitan laurate) 20 has a critical micelle concentration (CMC) of approximately 61 mg / L [actual usage concentration = 0.5 mg / ml, 500 mg / L > CMC]

[0451] Surfactant 3: 20 (sorbitan laurate) does not contain CMC in water and is too hydrophobic, but it still inhibits the form conversion.

[0452] Surfactant 4: Critical micelle concentration (CMC) 13-15 mg / L [Actual usage concentration = 0.01 mg / ml, 10 mg / L] <CMC]

[0453] Surfactants are soluble in water, while The surfactant is oil-soluble and cannot form micelles in water. It will be biphase, and therefore will exhibit the coexistence of an aqueous phase and a surfactant phase.

[0454] Surfactant 5: Critical micelle concentration (CMC) of 80 mg / L: 13-15 mg / L [Actual usage concentration = 0.1 mg / ml, 100 mg / L > CMC]

[0455] Surfactant 6: Critical micelle concentration (CMC) 13-15 mg / L [Actual usage concentration = 0.01 mg / ml, 10 mg / L] <CMC]。

[0456] As shown in Tables 8 and 9:

[0457] • The reflection corresponding to type B was only detectable in the control experiment, which was performed at 20°C for 24 hours in pure water, and increased at 40°C for +96 hours.

[0458] • In experiments containing three surfactants, no conversion from type A to type B occurred within a 24-hour period at 20°C and within a +96-hour period at 40°C. Therefore, it is suitable for use in type A aqueous formulations. 80,5ml (10 volumes, @1mg / ml) does not appear to accelerate the conversion from type A to type B, but rather seems to have the opposite effect.

[0459] · Use an aqueous 80 solution (0.01 mg / ml, <CMC) (surfactant 4) to perform additional experiments. Under these conditions, no transformation from Form A to Form B occurred after 24 h at 20 °C or after +96 h at 40 °C

[0460] Therefore, the presence of the surfactant ( 80) seems to inhibit the formal conversion rate from Form A to Form B, even when present at sub-CMC concentrations; thus, under storage conditions of 20 °C to 40 °C, the storage of Form A in the prefabricated composition should be stable for 120 h.

[0461] As shown in Tables 10 and 11:

[0462] · After 72 h at 20 °C (>CMC, <CMC, pure water), the reflection corresponding to Form B was detectable in all experiments and increased after 146 h at 20 °C

[0463] · No transformation from Form A to Form B occurred within a 24-h period at 20 °C in the experiments containing the surfactant; however, under the conditions studied, the micronized Form A (particle size 2 μm) began to transform into Form B after 48 h and was almost completely transformed into Form B after 146 h

[0464] · Therefore, the micronized Form A is prone to morphological changes, and after 146 h at 20 °C, the morphological changes will be completed with or without the use of the surfactant.

[0465] Example 9 - Competitive Suspension Equilibrium

[0466] Suspend Form A of Compound 1 and the appropriate other forms of Compound 1 in acetonitrile (20 volumes). Stir the white suspension at 40 °C for 7 days. When completed, separate the product, dry it, and analyze it by XRPD. The results are shown in Table 11.

[0467] Table 11 - Results of Competitive Suspension Equilibria

[0468]

[0469]

[0470] The results presented in Table 11 show that:

[0471] · When stirred at 40 °C under anhydrous conditions, Form A and the appropriate forms are transformed into Form A.

[0472] · Under anhydrous conditions, a mixture of Form A and Form B (hydrate) is transformed into Form A.

[0473] • All forms of the complex were stirred at 40°C and converted to type A.

[0474] • When stirred under anhydrous conditions at 20°C and 40°C, both the A and F forms of the complex are transformed into forms consistent with the true A form.

[0475] Equimolar amounts of the F and A forms were competitively suspended in anhydrous acetonitrile to determine the fate of the two initiators. The separated products were consistent with the true A form, and no obvious trace of the F form was observed in the diffraction pattern, confirming that the F form transformed into the A form under these conditions.

[0476] Therefore, under production conditions, the form of compound 1 other than type A should be metastable relative to type A and should inevitably be converted into single-phase A.

[0477] Example 10 - Crystallization of Type A via Diffusion

[0478] A solution of compound 1A (approximately 50 mg, 1.0 wt) was prepared in a suitable, low-volatility solvent and clarified through a 2 μm PTFE membrane. The saturated solutions were then transferred to individual containers, each placed inside a larger container. A volatile precipitating solvent was added to the larger container to form a jacket over the smaller container, which was then sealed. The container assembly was allowed to stand undisturbed for several days at 18°C ​​to 23°C. During this period, the volatile solvent diffused into the smaller container until the solvent jacket was exhausted and the solvent composition of the solution was sufficiently saturated to promote the crystallization of ET003861. The separated product was collected by filtration and dried under reduced pressure at 40°C for approximately 20 h.

[0479] The following combinations:

[0480] Ethanol (less volatile) and dichloromethane (more volatile);

[0481] Ethanol (less volatile) and tert-butyl methyl ether (more volatile);

[0482] It did not lead to crystal formation.

[0483] Using a combination of ethanol (less volatile) and pentane (more volatile), needle-like morphology grew from a single point on the vial wall after eight days. The solid was separated by centrifugation, the mother liquor was discarded, and the product was dried overnight at 40°C. The sample was then processed... 1 Analysis was performed using 1H NMR spectroscopy and XRPD, and the compound was identified as a crystalline polymorph of compound 1A.

[0484] Example 11 - Crystallization via heating and cooling

[0485] Nineteen aliquots of amorphous compound 1 (approximately 70 mg, 1.0 wt) were placed into individual scintillation vials. Aliquots of the relevant solvent (solvent A in Table 12) were added to each vial at a temperature of approximately 70 °C until complete dissolution occurred. The solutions were cooled and allowed to stand undisturbed below ambient temperature to promote crystallization. The products were separated by filtration, washed with the recovered aging solvent, dried under reduced pressure at 40 °C, and analyzed for evidence of crystallization by plate XRPD. The results are shown in Table 10.

[0486] Table 12 - Crystallization via Heating and Cooling

[0487]

[0488]

[0489] No new crystalline forms were identified.

[0490] Type A is the primary form derived from this screening, and the screening provides alternative crystallization conditions for generating type A.

[0491] Therefore, future developments in alternative solvent conditions or heating / cooling crystallization studies should include approximately 5 volumes of ethanol and approximately 11 volumes of methyl acetate. Currently, ethyl acetate is the preferred crystallization solvent (see Example 12).

[0492] Example 12 - Crystallizing type A from ethyl acetate

[0493] The method is as follows:

[0494] 1. Add compound 1 (1.00 by weight, 1.0 equivalent) to the container (total volume at this step is 1.0).

[0495] 2. Add ethyl acetate (20 volumes, 18 kg) to the container. (Total volume at this step)

[0496] 3. Heat the mixture to 55°C to 65°C, preferably 60°C.

[0497] 4. Stir the mixture at 55°C to 65°C, preferably 60°C, for at least 10 minutes to obtain a turbid solution.

[0498] 5. Cool the mixture to 45°C to 50°C, preferably 47°C, within 30 minutes to 1 hour. Once the temperature is reached, immediately proceed to step 6.

[0499] 6. Clarify the mixture by passing it through a 1μm filter at 45°C to 50°C.

[0500] 7. Perform a line flush with ethyl acetate (2 volumes, 1.8 g by weight) (the total volume at this step is 23.0 g, but the volume of ethyl acetate can be increased if necessary, as this ethyl acetate will be distilled off later).

[0501] 8. Distill under vacuum at 40°C to 50°C, preferably 45°C, to approximately 10 volumes. Precipitation can be observed when this volume is reached. (The total volume after this step is approximately 10.0).

[0502] 9. Cool to 35°C to 40°C, preferably 37°C, over 30 to 60 minutes. Aging for up to 2 hours, and proceed to step 10 once crystallization is observed.

[0503] 10. Aging the slurry at 35℃ to 40℃ for 1 to 2 hours.

[0504] 11. (Optional) Sample and filter the reaction mixture for analysis by XRPD to ensure that the resulting crystalline form is type A.

[0505] 12. Cool the contents of the container to 20°C to 25°C at a constant rate over 2 to 3 hours, with a target temperature of 23°C.

[0506] 13. Aging the mixture at 20°C to 25°C for 4 to 6 hours, with a target temperature of 23°C and a target time of 5 hours.

[0507] 14. At 20°C to 25°C, add clear n-heptane (5 volumes, 3.4 g) over 1 to 2 hours, with a target temperature of 23°C and a target time of 1.5 hours.

[0508] 15. Age the mixture at 20°C to 25°C for 30 to 60 minutes, with a target temperature of 23°C.

[0509] 16. Cool the contents of the container to 0 to 5°C at a constant rate over 1 to 2 hours, with a target temperature of 2.5°C and a target time of 1.5 hours.

[0510] 17. Age the mixture at 0°C to 5°C for 2 to 4 hours, with a target temperature of 2.5°C and a target time of 3 hours.

[0511] 18. Use a 20μm cloth to filter the reaction mixture. If material passes through the filter, the liquid can be recycled. Filter cake volume: 2 volumes

[0512] 19. Wash the filter cake with a mixture of clear ethyl acetate (1.3 volumes, 1.17 wt) and clear n-heptane (0.7 volumes, 0.48 wt) at 0 to 5 °C.

[0513] 20. Dry the filter cake at 20°C to 25°C. If necessary, the temperature can be increased to 40°C. 13 and 14 can be easily dried on the filter without heating.

[0514] 21. Determine the solvent content, and if the ethyl acetate content is ≤0.3% w / w and the n-heptane content is ≤0.3% w / w, proceed to step 22. If either solvent content is higher than the specified value, continue drying and resample after at least 4 hours.

[0515] 22. Discharge the product

[0516] Further characterization of type B in example 13

[0517] Two separate batches of the type B hydrated polymorph of compound 1 were analyzed by XRPD, DSC, TGA, FT-IR, and SEM. By DSC and TGA, the batches exhibited different dehydration activities (see Figures 21 and 22). The first batch showed a single-peak dehydration event and was named type B(I), while the second batch was characterized by bimodal dehydration and was named type B(II). XRPD spectra were... Figure 23a and Figure 23b As shown in the figure, the two forms are isomorphic and cannot be distinguished by XRPD. Table 2 above shows the angle, d-value, and intensity.

[0518] Assuming the inconsistency between the dehydration behaviors of the two batches is attributable to the different locations and combinations of their water occupancy, both are examined after 10 tons of compaction to determine whether their dehydration characteristics are maintained or altered after the application of isobaric treatment.

[0519] The retention of dehydration features means that there is less space available in the remaining part of the lattice after compaction, and the change in the basic morphology of the dehydration features by DSC may indicate that due to the application of high compressive force and the breakage of adjacent microcrystals, these smaller crystals may be able to occupy the space in the remaining part of the lattice.

[0520] i. Compaction of types B(I) and B(II)

[0521] A fine-grained type B(II) specimen is uniformly applied to the smooth surface of the lower sample anvil located inside the compression unit. A second smooth anvil is placed on top of the first, and the sample is pressed into place using finger pressure. The sealed sample chamber is then evacuated under internal vacuum pressure for at least 5 minutes. An axial load of approximately 10 metric tons is applied to the upper anvil, and the specimen is held under unilateral compression for at least 15 minutes. The unit is then disassembled, the product is retrieved from the sample holder, and analysis is performed to determine if any phase transition has occurred.

[0522] No significant changes were observed after compression using FT-IR; the image was only magnified by 3000 to 3600 cm⁻¹.-1 Signals between.

[0523] Following compression, the DSC curves exhibited a single, widespread dehydration event, rather than two overlapping bimodal events. Furthermore, an increase in dehydration temperature was observed after compression (the initial temperature increased from 75.2℃ to 87.1℃, as shown in the figure). Figure 21b and Figure 24 As shown. A higher dehydration temperature (initial temperature of 107.2℃) was also identified in the DSC curve of type B(I) - see [link to DSC curve]. Figure 21a Furthermore, XRPD resulted in a more disordered material with reduced crystallinity after compression. The crystallinity decreased from 87.2% to 80.3% by calculating the approximate Gaussian peak area using the following formula:

[0524]

[0525] The decrease in intensity and the broadening of peak reflection can be attributed to the reduction in crystallite size. A possible explanation for these observations is the alteration of water occupancy; water is less stable before compression. After compression, type B(II) did not show significant differences by XRPD or FT-IR, but could be distinguished by DSC. Therefore, it is hypothesized that the two forms are related through isomorphic pseudopolymorphism, differing only slightly in the orientation and position of their constituent water molecules. Due to the increased dehydration temperature after compression, it can be hypothesized that type B(II) is the less stable hydrate form, and that it tends towards the more stable hydrate form B(I) upon compression. Type B(I) remained unchanged after the same treatment.

[0526] Furthermore, the dehydration initiation temperature of valve I in type B(II) is lower than that of dehydration valve (I) in type B(I), indicating that type B(II) from the production batch is a less stable hydrate form and tends to become the more stable hydrate form, type B(I), when a higher compressive force is applied.

[0527] ii. Type B(I) and B(II) thermal cycles

[0528] Samples of compounds 1A, B(I), and B(II) were placed into individual Crystal 16 blank glass vials, and pure water (1 ml, 10.0 volume) was added to each vial. Each vial was subjected to thermal cycling from -10°C to +10°C over 75 hours at a rate of ±0.5°C / min.

[0529] After thermal cycling at a rate of ±0.5℃ / min at constant amplitude from -10℃ to +10℃ to -10℃, the micronized type A remained unchanged by XRPD or DSC, indicating that type A is stable within this temperature range.

[0530] The single-peak B(I) type batch remained unchanged by XRPD and DSC, which also indicates that the B-type single peak is stable within this temperature range.

[0531] The bimodal B(II) type did not show any A-type detected by XRPD after 75 hours of thermal cycling. This indicates that the low level of A-type present in this batch (approximately 1.1% w / w) did not increase upon thermal cycling at constant amplitudes from -10°C to +10°C to -10°C and may have reverted to the B type (at a lower measured level). Furthermore, a change was observed by DSC, indicating that the bimodal events had almost entirely transformed into the unimodal events observed in the B(I) type. This suggests that the unimodal B(I) type is the more stable form of the two B-type states under cold conditions, and therefore, the unimodal B type is thermodynamically favorable.

[0532] iii. Suspension equilibrium of type B(II) in acetonitrile / water

[0533] To further investigate the stability grades of types B(I) and B(II), equal portions of type B(I) and type B(II) were competitively matured for 3 days in a 4:1 MeCN / water (w / w) mixture. DSC confirmed the resulting solids to be type B(I), indicating that the type B unimodal form is the more stable of the two.

[0534] One portion of type B(II) (1.06 g) was loaded into 4 to 1 v / v acetonitrile / water (15 volumes, 15 ml). The mixture was stirred with a nitrogen stream at room temperature. The mixture was sampled twice at t = 4 h and t = 23 h. The second sample was centrifuged (13400 rpm for 15 min), the supernatant was removed, and the sample was dried overnight in an oven at 40 °C under reduced pressure.

[0535] Stirring of type B(II) in 4 to 1 v / v acetonitrile / water resulted in the production of type B(I) at time points t = 4 h and t = 23 h. This was confirmed by DSC (see [link]). Figure 25 XRPD and other methods were used to confirm this, indicating that the B-type singlet is the more stable of the two forms. The DSC spectrum did contain an anomalous event with an onset temperature of 117.12 °C. This was attributed to the shock release of superheated water over a narrow temperature range and is thought to be related to sample inhomogeneity or thermal contact in the DSC crucible, rather than to polymorphism.

[0536] As part of the B-type study, various samples derived from the polymorph screening were re-examined. The samples were initially isolated from different pristine solvents and conditions, and their morphology was analyzed by XRPD and DSC to determine the presence of type B(II). Only a single-peak B-type dehydration event was observed by DSC.

[0537] iv. Suspension equilibrium of type B(II) in pure water

[0538] This experiment was conducted to investigate whether type B(II) transforms into type B(I) after stirring in pure water for 20 hours.

[0539] One portion of type B(II) (1.0 g) was added to pure water (15 volume, 15 ml). It was stirred with a nitrogen stream at room temperature for 20 hours. The mixture was sampled twice at t = 2 h and t = 20 h. The sampled mixture was centrifuged (13400 rpm for 15 min) and the supernatant was removed. The remaining solid was dried overnight in a vacuum oven at 40 °C (at t = 3 days). The dried white solid (A0903-184-C1, 0.84 g, yield 83%, uncorrected) was obtained. The second-sampled sample and the final white solid were analyzed by XRPD and DSC. The results of the second-sampled sample were obtained in... Figure 26a , Figure 26b , Figure 26c and Figure 26d The results are shown in the figure. The sample results for day 3 are not shown because these are the same as the secondary samples taken at hour 20.

[0540] After suspending type B(II) in pure water for 2 hours, it was subjected to XRPD (… Figure 26b Type A was not detected and only by DSC ( Figure 26a A single-peak event was observed.

[0541] The same result was observed after 20 hours and three days, although by DSC (for DSC after 20 hours, see...). Figure 26c Two exothermic events were observed. This situation will be investigated.

[0542] In summary, the B-type hydrated polymorph of compound 1 can take on two pseudopolymorphs, B(I) and B(II). The experimental results above indicate that the unimodal B(I) type is the more thermodynamically stable of the two forms.

[0543] Example 14 - Conversion of compound 1A (anhydrous) to form B (monohydrate)

[0544] Compound 1A was recrystallized from acetonitrile:water in a 4:1 ratio to give monohydrate type B. A singlet type B was formed (225.0 g). 校正 (Yield 85.7%). No bimodal B-type was generated during the procedure. If necessary, the product can be micronized using, for example, an air jet mill.

[0545] If the product contains type A or type B(II), pure type B1 can be obtained by stirring the product in pure water (about 20 volumes) overnight at about 20°C.

[0546] Compound 1 (250.06 g) was loaded into a flask. A 4:1 acetonitrile:water mixture (10V, 2.5L) was filtered through a glass fiber filter and then loaded into the flask. The white slurry was heated under reflux (internal 77°C) for 1 hour, during which dissolution occurred at an internal temperature of approximately 55°C. The slurry was then refluxed again for 15 minutes to ensure complete dissolution. The solution was then gradually cooled to 15°C–25°C while stirring (111 rpm) overnight. After stirring overnight, a slow-precipitating, fine white precipitate was observed. DSC and XRPD analysis confirmed the formation of a singlet type B. The white slurry was cooled to 0°C–5°C and aged for two hours. The slurry was filtered under vacuum and dried on a filter under nitrogen for 72 hours to obtain the product (225.0 g). 校正 (Yield 85.7%). DSC and XRPD before and after drying showed the product remained a singlet B-type. Water content by KF analysis was 4.89% w / w (4.74% w / w expected for monohydrate B-type). 1H NMR analysis showed 0.02% w / w residual acetonitrile.

[0547] Samples from crystallized products were micronized using an AS100 spiral air jet mill (Alpine) at a grinding pressure of 2.5 bar (18°C) and an injection pressure of 3.5 bar (18°C). 90 <5μm. A 225g input yielded 207g of material, representing a recovery rate of 92%.

[0548] Using Beckman Coulter LS TM A 13 320 particle size analyzer is used to measure the D50 and D90 values ​​of the output material.

[0549] Output: D50 1.65μm, D90 3.82μm.

[0550] Chemical purity was analyzed by HPLC before and after micronization. No loss of purity was observed. The main difference between micronization and micronization was a decrease in water content from 4.8% w / w to 4.1% w / w.

[0551] Compound 1B is a channel hydrate; the micronization process reduces the crystal size, thus weakening its water-retention capacity. The resulting solid is isomorphic to the B type, which is achieved through XRPD (…). Figure 31 ) and DSC ( Figure 32 XRPD analysis was used to confirm this. Type A was not detected in the XRPD analysis, and no amorphous features were found.

[0552] Example 15 - Conversion of type A to type B(I) in aqueous suspension

[0553] It has been observed that micronized form A transforms into form B upon standing at low temperatures in an aqueous suspension. Since the observed phase transition occurs below ambient temperature, this suggests the process may be exothermic, preceded by water molecules adsorbing onto the surface of form A. Furthermore, the enthalpy of form B crystallizing into form A is at least partially exothermic, and this was confirmed by DSC analysis of form B (see [link to DSC analysis]). Figure 21a ).

[0554] It has been proposed that the crystallization of form A to form B should be induced by applying cold (thermodynamics), and that once initiated, turnover should be promoted by applying heat (kinetics), at least in the short term; the relative solubility product of the two polymorphs may also have an effect. Furthermore, the inventors hypothesize that the rate-determining step from form A to form B under aqueous conditions may be bimolecular, relating to water (form A + water → form B), rather than via form A → form B (anhydrous form) + water → form B. Therefore, in the presence of D₂O, turnover to form B should exhibit a lower hydration rate kinetic due to primary kinetic isotope effects (Thomas and Jennings; Chem. Mater. 1999, 11, 1907-1914).

[0555] program

[0556] Four separate micronized type A samples (4 x 100 mg, particle size 0.6 μm < 80% < 2.5 μm, supported by SEM) were placed into four separate Crystal 16 blank glass vials. Pure water (1 ml, 10.0 V) was added to three of the four vials, and heavy water (1 ml, D2O, NMR solvent, high isotopic purity) was added to the fourth vial.

[0557] One of the vials was subjected to thermal cycling in water from 20°C to 40°C, and the remaining three vials were subjected to thermal cycling between -10°C and +10°C over 40 hours; one of them was inoculated with type B at 10% w / w (see Table 13).

[0558] Table 13 - Thermal cycling conditions for type A in aqueous suspensions

[0559]

[0560]

[0561] T1: Constant amplitude circulation from 20℃ to 40℃ to 20℃, etc., at a rate of ±0.5℃ / minute.

[0562] T2: Constant amplitude circulation from -10℃ to +10℃ to -10℃, etc., at a rate of ±0.5℃ / minute.

[0563] In Experiment A1, after 40 hours of thermal cycling T1, type B was not detected under aqueous conditions.

[0564] In Experiment B1, type B was detected after 40 hours under aqueous conditions and thermal cycling T2, which supports the hypothesis that water adsorption onto the surface of type A is exothermic, i.e., thermodynamically favorable cooling and subsequent crystallization into type B are also assumed to be exothermic.

[0565] In experiment C1, no type B was detected after 40 hours under aqueous conditions (D2O) and thermal cycling T2. This supports the hypothesis that water adsorption onto the surface of type A may be the rate-determining step of hydration, limited by diffusion control and influenced by the different hydrogen bond strengths of heavier isotopes. This implies that the formation of type B hydrates is bimolecular, thus involving two substances (i.e., type A + water) rather than a monomolecular process (i.e., type A recombines into anhydrous type B, and the absorbent immediately absorbs water molecules to generate type B). Furthermore, the rate of hydration is doubled by doubling the concentration of type A or increasing the effective accessible surface area of ​​type A.

[0566] In Experiment D1, where the culture medium was inoculated with type B, as expected, type A converted to type B faster than in Experiment B1.

[0567] The following conclusions can be drawn from this experiment:

[0568] Lower temperatures favor the conversion of type A to type B;

[0569] • The hydration process is bimolecular; therefore, a higher concentration of form A should favor the conversion of form A to form B.

[0570] As mentioned above, a larger A-type surface area should facilitate the conversion to B-type;

[0571] Since the hydrated form is usually less soluble in an aqueous medium than its anhydrous form, the intrinsic solubility product may have a forward effect on equilibrium, i.e., promote the crystallization of solute A from solution as solute B.

[0572] Example 16-B: Suspension Equilibrium in Anhydrous Acetonitrile

[0573] One sample of type B(II) (1.01 g) was loaded into anhydrous acetonitrile (15 volumes, 15 ml). It was stirred with a nitrogen stream at room temperature. The IPC of the mixture was sampled twice at t = 3 h and t = 23 h. The sampled mixture was centrifuged (13400 rpm for 15 min), the supernatant was removed, and the mixture was dried under reduced pressure overnight at 40 °C. The remaining solid was dried under reduced pressure overnight at 40 °C (t = 2 days). The dried white solid (0.54 g, yield 54%, uncorrected) was analyzed by XRPD and DSC. The XRPD plot of the sample sampled at t = 3 h is shown below. Figure 27 As shown, under anhydrous conditions, type B(II) rapidly transforms into type A. The DSC plot is consistent with this finding. XRPD and DSC plots after 23 hours and 3 days are consistent with... Figure 27 The 3-hour chart shown is essentially the same.

[0574] A similar procedure was performed on a portion (1.06 g) of type B(I). In this case, the mixture was sampled a second time at t = 5 h, and separation was performed at t = 5 days. The uncorrected yield was 66%.

[0575] Similar to type B(II), type B(I) also rapidly transforms into type A under these conditions. This can be seen from... Figure 28 As can be seen, this figure is the XRPD plot of the secondary sample obtained at t=5 hours. The DSC plot is consistent with this result, and the XRPD and DSC plots after 5 days are similar.

[0576] This experiment confirms that, under anhydrous conditions, form A is the thermodynamically stable form of compound 1.

[0577] Further thermal cycling of Example 17-B(I) type

[0578] Type B(I) was prepared in the relevant formulation under aseptic conditions. The samples were thermally cycled for approximately 202 complete cycles. Offline analysis of the suspension was performed using polarized light microscopy, and endpoint sampling was performed on the separated solid particles after centrifugation. The thermal cycling covered a wide temperature range (-10°C to +40°C). Micronized Type A was used as a control in parallel studies.

[0579] The experiments conducted are shown in Table 14. In all cases, the suspension volume was 2 ml and thermal cycling was performed at a rate of 1 °C / min over a temperature range of -10 °C to +40 °C for 336 hours (14 days) and 2020 cycles.

[0580] In the table below, surfactants 80. 20 and 20 As described in Example 8.

[0581] Table 14- Thermal cycling of type B(I) in buffered aqueous suspension (type A control)

[0582]

[0583]

[0584] At daily intervals, a drop was taken from each thermally circulated suspension, placed on a microscope slide, and photographed. After 14 days of continuous thermal cycling, the suspensions were filtered, and the separable solids were dried and analyzed for evidence of changes in morphology and chemical purity.

[0585] Type B(I) is stable under constant thermal cycling treatment within 14 days, as shown in the XRPD, DSC, and TGA plots, and its relationship with... Figure 23a , Figure 21a and Figure 22a The same conditions were observed in the A-type suspension, and no changes were detected by polarized light microscopy. At day 8, the A-type suspension began to show morphological changes (observed by polarized light microscopy) and completely transformed by day 14. Analysis of the separated material confirmed that the A-type had transformed into the B(I)-type. After 14 days of thermal cycling, the material from experiment E (citrate buffer) showed... Type A (in 80) and F (citrate buffer) 20 / The XRPD spectrum of the material (type A in 20) is as follows: Figure 29 and Figure 30 As shown, it was confirmed that only type B existed. Similarly, DSC and TGA plots confirmed that the material was essentially completely transformed into type B(I). No decrease in chemical purity occurred as a result of the change in form.

[0586] Characterization of single crystals of type B(I) in Example 18

[0587] A small sample of compound 1B(I) was suspended in perfluoroether oil; a sample with dimensions of 0.110 × 0.040 × 0.022 mm was selected. 3 Colorless flaky crystals and mounted on MiTeGen with perfluoroether oil TM On the clamp, and then equipped with VariMax TM Confocal mirror, AFC11 goniometer, and HyPix 6000 TM Detector Rigaku AFC11 007-HF TM Alignment was performed on the diffractometer. The crystal remained stable at T = 100(2) K during data collection. Using ShelXT... TM (Sheldrick, 2015) Structure solver, using the Intrinsic Phasing solution method and by using Olex2 TM (Dolomanov et al., 2009) used a graphical interface to solve this structure. ShelXT was used. TM The 2018 / 3 version of (Sheldrick, 2015) improves the model using least squares minimization.

[0588] The crystal data is as follows:

[0589] C 18 H 22 ClN3O4, M r =379.83, orthorhombic crystal system, Pna21 (number 33), α=β=γ=90°, T=100(2)K, Z=4, Z'=1, μ(CuK α ) = 2.099mm -1 30,062 reflections were measured, and 3,324 unique (R) values ​​were obtained. int =0.0389) was used for all calculations. The final wR2 was 0.0635 (all data), and R1 was 0.0238 (I>2(I)).

[0590] X-ray diffraction further confirmed that in the B(I) type hydrate, one water molecule was hydrogen-bonded to three molecules of compound 1. This is as follows: Figure 33 As shown.

Claims

1. A pharmaceutical composition comprising a polymorph of N-tert-butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxamide (compound 1) and a pharmaceutical excipient, said compound 1 having the following structural formula: The polymorphic form is: (a) Type A anhydrous solid crystalline polymorph, exhibiting the following XRPD diffraction pattern: having a characteristic main peak at 7.25±0.2 (in 2-θ degrees) and at least three characteristic peaks at 14.44±0.2, 20.42±0.2, 21.68±0.2, 24.38±0.2, 27.21±0.2, 29.01±0.2, 30.82±0.2, 36.46±0.2 and 41.49±0.2; (b) Type B hydrated solid crystalline polymorph, exhibiting the following XRPD diffraction pattern: a characteristic main peak at 11.03 ± 0.2 degrees (in 2-θ degrees) and at least three characteristic peaks at 5.56 ± 0.2, 14.04 ± 0.2, 17.28 ± 0.2, 18.03 ± 0.2, 18.86 ± 0.2, 22.08 ± 0.2, 23.69 ± 0.2, 24.12 ± 0.2, and 24.93 ± 0.2; or (c) Amorphous form.

2. The pharmaceutical composition according to claim 1, wherein the XRPD diffraction pattern of the type A anhydrous solid crystalline polymorph comprises a characteristic main peak at 7.25 ± 0.2 degrees (in 2-θ degrees) and at least three characteristic peaks at 10.20 ± 0.2, 14.44 ± 0.2, 17.79 ± 0.2, 20.42 ± 0.2, 20.69 ± 0.2, 21.68 ± 0.2, 24.22 ± 0.2, 24.38 ± 0.2, 26.13 ± 0.2, 27.21 ± 0.2, 29.01 ± 0.2, 30.82 ± 0.2, 36.46 ± 0.2, and 41.49 ± 0.

2.

3. The pharmaceutical composition according to claim 1, wherein the XRPD diffraction pattern of the type A anhydrous solid crystalline polymorph comprises a characteristic main peak at 7.25 ± 0.2 degrees (in 2-θ degrees) and at least three characteristic peaks at 10.20 ± 0.2, 14.44 ± 0.2, 16.13 ± 0.2, 17.79 ± 0.2, 20.42 ± 0.2, 20.69 ± 0.2, 21.07 ± 0.2, 21.68 ± 0.2, 24.09 ± 0.2, 24.22 ± 0.2, 24.38 ± 0.2, 26.13 ± 0.2, 27.21 ± 0.2, 29.01 ± 0.2, 29.30 ± 0.2, 30.82 ± 0.2, 32.50 ± 0.2, 36.46 ± 0.2, and 41.49 ± 0.

2.

4. The pharmaceutical composition according to claim 2 or 3, wherein the XRPD diffraction pattern further comprises clusters of characteristic peaks at 21.68±0.2 and 29.01±0.2, and characteristic peaks at 24.09±0.2, 24.22±0.2 and 24.3801±0.2, expressed in 2-θ degrees.

5. The pharmaceutical composition according to claim 2 or 3, wherein the anhydrous solid crystalline polymorph of type A is substantially free of other forms of compound 1, such that at least 97% by weight of compound 1 is present as the anhydrous solid crystalline polymorph of type A.

6. The pharmaceutical composition according to claim 2 or 3, wherein the form is micronized.

7. A method for preparing an anhydrous solid crystalline polymorph of type A as defined in any one of claims 2 to 6, the method comprising crystallizing compound 1 from a solvent selected from ethyl acetate, acetone, butanol, ethanol, ethyl formate, isopropyl acetate, methyl acetate, nitromethane, 2-propanol, propionitrile, and acetonitrile.

8. The method according to claim 7, wherein the method comprises the following steps: i. Prepare a saturated solution of compound 1 in a solvent at a temperature of 50°C to 70°C; ii. Cool the solution to a temperature of 5°C to 20°C; iii. Allow the cooled solution to stand until crystals of compound 1 form; and iv. Separate the crystallized products; The solvent is selected from ethyl acetate, acetone, butanol, ethanol, ethyl formate, isopropyl acetate, methyl acetate, nitromethane, 2-propanol, propionitrile, and acetonitrile.

9. The method according to claim 7 or claim 8, wherein the solvent is selected from acetonitrile, ethanol, ethyl acetate, methyl acetate, butanol, 2-propanol and isopropyl acetate.

10. The pharmaceutical composition of claim 1, wherein the XRPD diffraction pattern of the type B hydrated solid crystalline polymorph comprises a characteristic main peak at 11.03 ± 0.2 degrees (in 2-θ degrees) and at least three characteristic peaks at 5.56 ± 0.2, 14.04 ± 0.2, 17.28 ± 0.2, 18.03 ± 0.2, 18.86 ± 0.2, 19.34 ± 0.2, 22.08 ± 0.2, 23.69 ± 0.2, 24.12 ± 0.2, 24.93 ± 0.2, 25.98 ± 0.2, 26.53 ± 0.2, 27.28 ± 0.2, and 28.79 ± 0.

2.

11. The pharmaceutical composition of claim 10, wherein the XRPD diffraction pattern further comprises characteristic peaks at 5.36±0.2 and 22.08±0.2, expressed in 2-θ degrees.

12. The pharmaceutical composition according to claim 10 or 11, wherein the type B hydrated solid crystalline polymorph is substantially free of other forms of compound 1, such that at least 97% by weight of compound 1 is present as the type B hydrated solid crystalline polymorph.

13. The pharmaceutical composition according to claim 10 or 11, wherein the form is micronized.

14. The pharmaceutical composition according to claim 10 or 11, wherein the type B hydrated solid crystalline polymorph is a type B(I) pseudopolymorph and is characterized in that it undergoes unimodal dehydration.

15. A method for preparing a type B hydrated solid crystalline polymorph as defined in any one of claims 10 to 14, the method comprising crystallizing compound 1 from an aqueous solvent comprising water or water mixed with acetonitrile.

16. The method according to claim 15, wherein the method comprises the following steps: i. Prepare a saturated solution of compound 1 in a solvent at a temperature of 60°C to 80°C; ii. Cool the solution to a temperature of 5°C to 20°C; iii. Allow the cooled solution to stand until crystals of compound 1 form; and iv. Separate the crystallized products; The solvent is an aqueous solvent that contains water or water mixed with acetonitrile in a ratio of 5:1 to 1:5 v / v.

17. The pharmaceutical composition according to claim 1, wherein compound 1 is in a solid, anhydrous, amorphous form.

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