Psilocybin, different polymorphs, intermediates, preparation of formulations and their use

Abstract

To provide a method for treating a disease. [Solution] The present invention relates to the large-scale production of psilocybin for use in pharmaceuticals. More specifically, the present invention relates to a method for obtaining high-purity crystalline psilocybin, particularly in the form of polymorph A. The present invention further relates to a method for producing psilocybin, intermediates in its production, and formulations containing psilocybin. Large-scale means producing batches of psilocybin having a weight greater than 10 g, more preferably greater than 100 g, more preferably greater than 250 g, and up to and above the kilogram level.

Description

[Technical Field]

[0001] This invention relates to the large-scale production of psilocybin for use in pharmaceuticals.

[0002] Large scale means producing batches of psilocybin having weights greater than 10g, more preferably greater than 100g, more preferably greater than 250g, and up to and above the kilogram level.

[0003] The present invention relates to intermediates containing but not limited to psilocybin, different polymorphic forms of psilocybin including isomorphic variants, and, in combination with psychological support that can be provided alone or digitally, specifically, but not limited to, Diagnostic and Statistical Manual, 5 th This also relates to the production of those formulations for use in medicine for the treatment of treatment-resistant depression as defined in Edition. [Background technology]

[0004] Psilocybin was first synthesized by Sandoz in 1958 (see British Patent No. 912714 and U.S. Patent No. 3075992) and was widely available as a research chemical until the mid-1960s.

[0005] The plant-based hallucinogen psilocybin has been used as an adjunct to psychotherapy for the treatment of mood disorders and alcohol-related disorders, and recently, three clinical trials have reported the use of psilocybin for depressive symptoms.

[0006] Griffiths et al. 2016;J Psychopharmacol 30(12):1181-1197;

[0007] Ross et al. 2016; J Psychopharmacol 30(12):1165-1180; and

[0008] Carhart-Harris et al. 2016, Lancet Psychiatry 3(7):619-627.

[0009] The production methods of psilocin are limited and include the following.

[0010] J Nat Prod 2003, 66, 885-887;

[0011] Helv Chim Acta 1959, 42, 2073-2103;

[0012] Experientia 1958, 15, 397-399; and

[0013] Synthesis 1999, 935-938.

[0014] Based on this literature, the applicant considered that the method disclosed in J Nat Prod 2003, 66, 885-887 (hereinafter referred to as JNP) was the most suitable method for developing into a commercial-scale method.

[0015] The method disclosed therein produced an amount in the order of 10 g and consisted of six steps numbered (i)-(vi).

[0016] Similar to the applicant's method, hereinafter, steps ii and iii are discussed as a single step (step 2), and the JNP method is reproduced as Figure 1 in this specification.

[0017] Step 1 (i) involved reacting 4-hydroxyindole ("3") with acetic anhydride (Ac2O) in pyridine and anhydrous dichloromethane (CH2Cl2) at 0 °C. Water was added, the mixture was evaporated, the resulting concentrate was dissolved in ethyl acetate, washed with water and saturated sodium chloride, the organic phase was dried with sodium sulfate, evaporated to obtain 4-acetylindole ("4"), which was collected by filtration and washed with water and ethyl acetate.

[0018] Steps 2(ii and iii), specifically the acylation (ii)-amidation step (iii) of step 2, involved reacting (ii)4-acetylindole ("4") with oxalyl chloride ((COCl)2) in anhydrous diethyl ether, stirring, adding n-hexane, maintaining the temperature at -20°C, and producing the intermediate 3-(2-chloro-2-oxoacetyl)-1H-indole-4-ylacetate ("5") separated by filtration, thereby forming 3-dimethylaminooxalyl-4-acetylindole ("6"). This intermediate was dissolved in anhydrous tetrahydrofuran (THF) and reacted with dimethylamine ((CH3)2NH) in tetrahydrofuran and pyridine. Due to solidification, anhydrous ether was added, the reaction product was separated by filtration, and washed with n-hexane, ethyl acetate and water to obtain 3-dimethylaminooxalyl-4-acetylindole ("6").

[0019] Step 3(iv) involved the formation of psilocine ("1") by reacting 3-dimethylaminooxalyl-4-acetylindole ("6") with lithium aluminum hydride (LiAlH4) in anhydrous THF under an argon atmosphere. After reflux and cooling, anhydrous sodium sulfate was added, followed by a solution of sodium sulfate and then anhydrous sodium sulfate. The reaction mixture was diluted with ethyl acetate and rapidly concentrated under vacuum, and the resulting psilocine crystals were briefly washed with methanol.

[0020] Step 4(v) involved reacting psilocine dissolved in anhydrous THF with n-butyllithium (n-BuLi) in n-hexane at -78°C and tetrabenzyl pyrophosphate [(BnO)2PO]2O, heating the reaction to 0°C, and monitoring the production of the intermediate dibenzyl 3-[(2-dimethylamino)ethyl]-1H-indole-4-yl phosphate ("7") to form benzyl[2-(4-oxyindole-3-yl)ethyl]dimethylammonio-4-O-benzylphosphate ("8"). Immediately after checking for the presence of "7", aminopropyl silica gel was added, the mixture was diluted with ethyl acetate, filtered through a Celite pad by suction, the filtrate was concentrated under vacuum, redissolved in CH2Cl2, and the precipitate was collected by filtration.

[0021] Step 5(vi) involved the formation of psilocybin ("2") by the reaction of ("8") with hydrogen (H2) in methanol (MeOH) using a palladium-activated carbon catalyst (Pd / C). Due to product precipitation, water was added, and along with the appearance of psilocybin ("8"), its mono-debenzylated derivative was monitored, and the reaction solution was filtered through a Celite pad. The product was collected by filtration and washed with ethanol to obtain white needle-shaped crystals with a melting point of 190°C to 198°C.

[0022] Unlike many methods, such as those by JNP, which use non-aqueous solvents such as methanol or ethanol, Experientia 1958, 15, 397-399 used a single recrystallization from water to obtain psilocybin from a mushroom extract. The teaching uses boiling water to dissolve the starting material obtained on a small scale by chromatography, and the resulting high-vacuum dried material is described as melting indistinctly between 185 and 195°C and showing a weight loss of 25.4%, suggesting that it is distinctly different in purity and form from what was obtained by the applicant.

[0023] During the development of the synthesis for producing psilocybin, the applicant carried out numerous hydrogenation reactions on a 5g scale, yielding psilocybin in different crystalline forms. The first hydrogenation reaction gave hydrate A (JCCA2157E), showing the XRPD diffractogram in Figure 7d and the DSC and TGA thermograms in Figure 8d. The DSC shows an endothermic event with a starting temperature of approximately 216°C, which was estimated to be melting, and the DSC shows an endothermic event with a weight decrease of TGA at approximately 97°C, suggesting dehydration. Another hydrogenation reaction gave ethanol solvate (JCCA2158D), which shows the XRPD (Figure 7e), DSC (Figure 8e), TGA (Figure 8e) and 1 Analysis by 1H NMR revealed 11% captured ethanol. The DSC thermogram showed endothermic activity with an onset of approximately 154°C, which was thought to be melting occurring simultaneously with approximately 13% weight loss in the TGA. In another experiment conducted during development, the applicant performed crystallization of psilocybin. Precipitation occurred at high temperatures (>90°C) rather than being kept in solution in hot water to allow for a polish filtration step. The formed solid did not redissolve upon further heating or addition of further water. XRPD was performed upon cooling and isolation of the solid (CB646E). The XRPD diffractogram (Figure 7f) suggested a mixed phase of polymorph A' (JCCA2160-F-D4) and polymorph B (JCCA2160-F-TM2-C5). These findings highlight the importance of developing a method that can consistently produce the desired crystalline form; therefore, the applicant undertook experiments to determine which of these forms is capable of producing chemically pure psilocybin in a controlled form suitable for pharmaceutical use.

[0024] For clinical trials, each novel active substance (NAS) should be capable of large-scale production (typically in batches of 100g or more, more typically more than 250g, more preferably more than 500g, up to 1kg or more) depending on the amount of active substance to be administered to human subjects. Furthermore, the novel active substances should be chemically pure, well-established, and stable during storage.

[0025] Furthermore, all manufacturing methods must be easily reproducible and provide batch-to-batch consistency.

[0026] The first objective of the present invention is to provide psilocybin in a consistent polymorphic form for administration to human subjects.

[0027] Another object of the present invention is to provide chemically pure psilocybin in a consistent polymorphic form for administration to human subjects.

[0028] Since pure psilocybin must be produced on a large scale for commercial use, a further objective is to provide chemically pure psilocybin in large batch quantities.

[0029] A further object of the present invention is to provide a method for crystallizing psilocybin in a desired polymorphic form. A further object of the present invention is to provide an expandable method for producing psilocybin from psilocine or 4-hydroxyindole.

[0030] In developing an appropriate methodology, the applicant encountered numerous problems and difficulties that had to be overcome. Overcoming the problems identified in each step and using the invention alone or in combination is another independent objective. A further object of the present invention is to formulate psilocybin of the present invention in a form suitable for administration to human subjects, either alone or in combination with digital health products or digital solutions, particularly in the treatment of central nervous system disorders (CNS), and more specifically, but not limited to, in the treatment of depression, particularly drug-resistant depression, and to use such formulation in pharmaceuticals. [Prior art documents] [Patent Documents]

[0031] [Patent Document 1] British Patent No. 912714 [Patent Document 2] U.S. Patent No. 3075992 [Non-patent literature]

[0032] [Non-Patent Document 1] Griffiths et al. 2016;J Psychopharmacol 30(12):1181-1197 [Non-Patent Document 2] Ross et al. 2016; J Psychopharmacol 30(12):1165-1180 [Non-Patent Document 3] Carhart-Harris et al. 2016, Lancet Psychiatry3(7):619-627 [Non-Patent Document 4] J Nat Prod 2003, 66, 885-887 [Non-Patent Document 5] Helv Chim Acta 1959,42,2073-2103 [Non-Patent Document 6] Experientia 1958, 15, 397-399 [Non-Patent Document 7] Synthesis 1999, 935-938 [Overview of the project] [Means for solving the problem]

[0033] According to a first aspect of the present invention, crystalline psilocybin is provided in the form of polymorph A or polymorph A' characterized by one or more of the following: a. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ; b. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ (further characterized by at least one additional peak at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ); c. The XRPD diffractogram substantially illustrated in Figure 7a or Figure 7b; or d. Endothermic events in a DSC thermogram with a starting temperature of 205-220°C, substantially illustrated in Figure 8a or Figure 8b. Polymorph A

[0034] According to a preferred embodiment of the present invention, crystalline psilocybin is provided in the form of polymorph A characterized by one or more of the following: a. Peaks in the XRPD diffractogram at 11.5, 12.0, 14.5, and 17.5°2θ±0.1°2θ; b. Peaks in the XRPD diffractogram at 11.5, 12.0, 14.5 and 17.5°2θ±0.1°2θ (further characterized by at least one additional peak at 19.7, 20.4, 22.2, 24.3 or 25.7°2θ±0.1°2θ); c. The XRPD diffractogram substantially illustrated in Figure 7a; or d. Endothermic events in a DSC thermogram with an onset temperature of 205–220°C, substantially illustrated in Figure 8a.

[0035] The peak at 17.5°2θ±0.1°2θ has a relative intensity of at least 5%, preferably at least 6%, more preferably at least 7%, 8%, 9%, and at least 10% compared to the peak at 14.5°2θ±0.1°2θ.

[0036] In one embodiment, psilocybin polymorph A exhibits an XRPD diffractogram characterized by the diffractogram summarized in Table 1. In one embodiment, the crystalline psilocybin polymorph A described herein includes (±0.1°²θ) at least three peaks in Table 1. In one embodiment, the crystalline psilocybin polymorph A described herein includes (±0.1°²θ) at least four peaks in Table 1. In one embodiment, the crystalline psilocybin polymorph A described herein includes (±0.1°²θ) at least five peaks in Table 1. In one embodiment, the crystalline psilocybin polymorph A described herein includes (±0.1°²θ) at least six peaks in Table 1. In one embodiment, the crystalline psilocybin polymorph A described herein includes (±0.1°²θ) at least eight peaks in Table 1. In one embodiment, crystalline psilocybin polymorph A described herein includes at least 10 peaks in Table 1 at (±0.1°2θ). In one embodiment, crystalline psilocybin polymorph A described herein includes at least 15 peaks in Table 1 at (±0.1°2θ). The peak at approximately 17.5°2θ±0.1°2θ distinguishes psilocybin polymorph A from polymorph A' in which this peak is absent or substantially absent (i.e., having a relative intensity of less than 2%, more preferably less than 1%, compared to the peak at 14.5°2θ±0.1°2θ).

[0037] [Table 1]

[0038] In one embodiment, crystalline psilocybin polymorph A is characterized by XRPD diffractogram peaks at 11.5, 12.0, 14.5 and 17.5°2θ±0.1°2θ. In another embodiment, crystalline psilocybin polymorph A is characterized by at least one additional peak appearing at 19.7, 20.4, 22.2, 24.3 or 25.7°2θ±0.1°2θ. It is further characterized by the peaks. In another embodiment, crystalline psilocybin polymorph A is further characterized by at least two additional peaks appearing at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ. In another embodiment, crystalline psilocybin polymorph A is further characterized by at least three additional peaks appearing at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ. In a further embodiment, crystalline psilocybin polymorph A exhibits an XRPD diffractogram substantially identical to the XRPD diffractogram shown in Figure 7a.

[0039] In one embodiment, crystalline psilocybin polymorph A is characterized by XRPD diffractogram peaks at 14.5° and 17.5°2θ ± 0.1°2θ, wherein the peak at 17.5°2θ has an intensity of at least 5%, more preferably at least 6%, at least 7%, to at least 8%, at least 9%, and at least 10% of the intensity of the peak at 14.5°2θ.

[0040] In one embodiment, crystalline psilocybin polymorph A is either absent or substantially absent from the XRPD diffractogram peak at 10.1. Substantially absent means that any XRPD diffractogram peak at 10.1 is less than 2% of the peak intensity at 14.5°2θ, for example less than 1%, or is not detectable in the XRPD diffractogram.

[0041] In one embodiment, crystalline psilocybin polymorph A is characterized by an endothermic event in a DSC thermogram having an onset temperature of 205-220°C, e.g., 210-220°C, e.g., 210-218°C, or e.g., 210-216°C. In another embodiment, crystalline psilocybin polymorph A is further characterized by an endothermic event in a DSC thermogram having an onset temperature of 145-165°C, e.g., 145-160°C, or e.g., 145-155°C. In yet another embodiment, crystalline psilocybin polymorph A is characterized by an endothermic event in a DSC thermogram having an onset temperature of 205-220°C, e.g., 210-220°C, e.g., 210-218°C, or e.g., 210-216°C, and an endothermic event having an onset temperature of 145-165°C, e.g., 145-160°C, or e.g., 145-155°C. In yet another embodiment, crystalline psilocybin polymorph A exhibits a DSC thermogram substantially identical to the DSC thermogram in Figure 8a.

[0042] In another embodiment, crystalline psilocybin polymorph A is characterized by having a water content of less than 0.5% w / w, e.g., less than 0.4% w / w, e.g., less than 0.3% w / w, e.g., less than 0.2% w / w, or e.g., less than 0.1% w / w. Those skilled in the art will know methods for measuring the water content of a compound, e.g., Karl Fischer titration. In one embodiment, crystalline psilocybin polymorph A is characterized by having a loss of less than 0.5% w / w, e.g., less than 0.4% w / w, e.g., less than 0.3% w / w, e.g., less than 0.2% w / w, e.g., less than 0.1% w / w, in a TGA thermogram at an ambient temperature of ~200°C, such as about 25°C. In one embodiment, crystalline psilocybin polymorph A loses less than 2% by weight, e.g., less than 1% by weight, e.g., less than 0.5% by weight, in a loss on drying test. The loss on drying test is performed at 70°C.

[0043] In one embodiment, crystalline psilocybin polymorph A is an extremely pure crystalline form of polymorph A, for example, psilocybin contains at least 90% by weight, e.g., 95% by weight, e.g., 99% by weight, e.g., 99.5% by weight of polymorph A.

[0044] In one embodiment, crystalline psilocybin polymorph A is a white to grayish-white solid.

[0045] In another embodiment, crystalline psilocybin polymorph A is chemically pure, for example, silocybin Silicin has a chemical purity of over 97%, for example, over 98%, or for example, over 99%, as determined by HPLC. In one embodiment, crystalline psilocybin polymorph A is 31 It is free from any single impurity greater than 1%, more preferably less than 0.5%, including phosphoric acid as measured by 3P NMR and psilocine as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A has a chemical purity greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A is free from any single impurity greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A does not contain psilocine at a level greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A is 31 When measured by 1P NMR, it does not contain phosphoric acid at a level greater than 1% by weight, more preferably less than 0.5% by weight. In one embodiment, crystalline psilocybin polymorph A has a chemical assay of at least 95% by weight, for example, at least 96% by weight, or for example, at least 98% by weight. Polymorph A'

[0046] According to another embodiment of the present invention, crystalline psilocybin polymorph A' is provided, characterized by one or more of the following: a. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ (where a peak is absent or substantially absent at 17.5°2θ±0.1°2θ); b. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ (where the peak is absent or substantially absent at 17.5°2θ±0.1°2θ, and further characterized by at least one additional peak at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ); c. The XRPD diffractogram substantially illustrated in Figure 7b; or d. Endothermic events in a DSC thermogram with an onset temperature of 205–220°C, substantially illustrated in Figure 8b.

[0047] The substantially absence of a peak at 17.5°2θ±0.1°2θ means that, if this peak were present, it would have a relative intensity of less than 5%, more preferably less than 4%, less than 3%, 2%, 1%, or less than 3%, compared to the peak at 14.5°2θ±0.1°2θ.

[0048] In one embodiment, psilocybin polymorph A' exhibits an XRPD diffractogram characterized by the diffractogram summarized in Table 2. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least three peaks in Table 2 at (±0.1°2θ), but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least four peaks in Table 2 at (±0.1°2θ), but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least five peaks in Table 2 at (±0.1°2θ), but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least six peaks in Table 2 at (±0.1°2θ), but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least eight peaks in Table 2 at (±0.1°2θ), but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline Psilocybin polymorph A' contains at least 10 peaks (±0.1°2θ) in Table 2, but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least 15 peaks (±0.1°2θ) in Table 2, but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least 20 peaks (±0.1°2θ) in Table 2, but the peak is absent or substantially absent at 17.5°2θ±0.1°2θ. In one embodiment, the crystalline psilocybin polymorph A' described herein contains at least 25 peaks in Table 2 at (±0.1°2θ), but the peak at 17.5°2θ±0.1°2θ is absent or substantially absent.

[0049] [Table 2]

[0050] In one embodiment, crystalline psilocybin polymorph A' is characterized by XRPD diffractogram peaks at 11.5, 12.0, and 14.5°2θ±0.1°2θ, with substantially no peak at 17.5°2θ±0.1°2θ. In another embodiment, crystalline psilocybin polymorph A' is further characterized by at least one additional peak appearing at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ. In yet another embodiment, crystalline psilocybin polymorph A' is further characterized by at least two additional peaks appearing at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ. In another embodiment, crystalline psilocybin polymorph A' is further characterized and distinguished from polymorph A by the presence of a peak appearing at 10.1°2θ ± 0.1°2θ. In yet another embodiment, crystalline psilocybin polymorph A' exhibits an XRPD diffractogram substantially identical to the XRPD diffractogram shown in Figure 7b.

[0051] In one embodiment, crystalline psilocybin polymorph A' is characterized by XRPD diffractogram peaks at 14.5° and 17.5°2θ ± 0.1°2θ, where the intensity of the peak at 17.5°2θ is less than 5% of the intensity of the peak at 14.5°2θ, e.g., less than 4%, e.g., less than 3%, e.g., less than 2%, e.g., less than 1%, or e.g., about 1%.

[0052] In one embodiment, crystalline psilocybin polymorph A' is characterized by XRPD diffractogram peaks at 10.1° and 14.5°2θ ± 0.1°2θ, where the intensity of the peak at 10.1°2θ is at least 1%, e.g., at least 2%, e.g., at least 3%, or e.g., about 4%, of the intensity of the peak at 14.5°2θ.

[0053] In one embodiment, crystalline psilocybin polymorph A' is characterized by an endothermic event in a DSC thermogram having an onset temperature of 205-220°C, e.g., 210-220°C, e.g., 210-218°C, or e.g., 210-216°C. In another embodiment, crystalline psilocybin polymorph A' is further characterized by an endothermic event in a DSC thermogram having an onset temperature of 145-165°C, e.g., 145-160°C, or e.g., 145-155°C. In yet another embodiment, crystalline psilocybin polymorph A' is characterized by an endothermic event in a DSC thermogram having an onset temperature of 205-220°C, e.g., 210-220°C, e.g., 210-218°C, or e.g., 210-216°C, and an endothermic event having an onset temperature of 145-165°C, e.g., 145-160°C, or e.g., 145-155°C. In yet another embodiment, crystalline psilocybin polymorph A' exhibits a DSC thermogram substantially identical to the DSC thermogram in Figure 8b.

[0054] In another embodiment, crystalline psilocybin polymorph A' is characterized by having a water content of less than 0.5% w / w, e.g., less than 0.4% w / w, e.g., less than 0.3% w / w, e.g., less than 0.2% w / w, or e.g., less than 0.1% w / w. Those skilled in the art will know methods for measuring the water content of a compound, e.g., Karl Fischer titration. In one embodiment, crystalline psilocybin polymorph A' is characterized by having a loss of less than 0.5% w / w, e.g., less than 0.4% w / w, e.g., less than 0.3% w / w, e.g., less than 0.2% w / w, e.g., less than 0.1% w / w, in a TGA thermogram at an ambient temperature of ~200°C, such as 25°C. In one embodiment, crystalline psilocybin polymorph A' loses less than 2% by weight, e.g., less than 1% by weight, e.g., less than 0.5% by weight, in a loss on drying test. The loss on drying test is performed at 70°C.

[0055] In one embodiment, crystalline psilocybin polymorph A' is an extremely pure crystalline form of polymorph A', for example, psilocybin containing at least 90% by weight, e.g., 95% by weight, e.g., 99% by weight, e.g., 99.5% by weight of polymorph A'.

[0056] In one embodiment, crystalline psilocybin polymorph A' is a white to grayish-white solid.

[0057] In another embodiment, crystalline psilocybin polymorph A' is chemically pure, for example, psilocybin has a chemical purity of greater than 97%, more preferably greater than 98%, and most preferably greater than 99%, as determined by HPLC. In one embodiment, crystalline psilocybin polymorph A' is, 31 It is free from any single impurity greater than 1%, more preferably less than 0.5%, including phosphoric acid as measured by 3P NMR and psilocine as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A' has a chemical purity greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A' is free from any single impurity greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A' does not contain psilocine at a level greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A' is, 31 When measured by 1P NMR, it does not contain phosphoric acid at a level greater than 1% by weight, more preferably less than 0.5% by weight. In one embodiment, crystalline psilocybin polymorph A' has a chemical assay of at least 95% by weight, for example, at least 96% by weight, or for example, at least 98% by weight.

[0058] XRPD diffractograms and XRPD peak positions are obtained using Cu Kα radiation.

[0059] DSC and TGA thermograms are obtained using a heating rate of 20°C / min.

[0060] In one embodiment, a high-purity crystalline psilocybin, polymorph A or polymorph A' (12A or 12A'), or a mixture thereof, is provided, showing an XRPD diffractogram substantially illustrated in Figure 7a or Figure 7b and a DSC thermograph substantially illustrated in Figure 8a or Figure 8b.

[0061] Preferably, crystalline psilocybin polymorph A(12A) exhibits the XRPD diffractogram shown in Figure 7a and the DSC thermograph shown in Figure 8a.

[0062] Preferably, the crystalline psilocybin polymorph A'(12A') shows an XRPD diffractogram substantially illustrated in Figure 7b and a DSC thermograph substantially illustrated in Figure 8b.

[0063] Preferably, the high-purity crystalline psilocybin polymorph A(12A) is characterized by the XRPD diffractogram substantially illustrated in Figure 7a and the DSC thermograph substantially illustrated in Figure 8a.

[0064] Preferably, the high-purity crystalline psilocybin polymorph A'(12A') is characterized by the XRPD diffractogram shown in Figure 7b and the DSC thermograph shown in Figure 8b.

[0065] Polymorph A (including its isomorphic variant polymorph A') (Figures 7a and 7b) differ from polymorph B (Figure 7c), hydrate A (Figure 7d), and ethanol solvate (Figure 7e: solvate A), and the relationships between some of these different forms are illustrated in Figure 9.

[0066] Crystalline psilocybin polymorph A or polymorph A' is a white to grayish-white solid and / or has a chemical purity greater than 97%, more preferably greater than 98%, and most preferably greater than 99%, as determined by HPLC, and 31It is free from any single impurity, including phosphoric acid as measured by 3P NMR and psilocine as measured by HPLC, at a level greater than 1%, more preferably less than 0.5%. In one embodiment, high-purity crystalline psilocybin, polymorph A, or polymorph A' is provided. In one embodiment, crystalline psilocybin polymorph A or polymorph A' has a chemical purity greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A or polymorph A' is free from any single impurity, greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A or polymorph A' does not contain psilocine at a level greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph A or polymorph A' is, 31 When measured by 1P NMR, it does not contain phosphoric acid at a level greater than 1% by weight, more preferably less than 0.5% by weight. In one embodiment, crystalline psilocybin polymorph A or polymorph A' has a chemical assay of at least 95% by weight, for example, at least 96% by weight, or for example, at least 98% by weight.

[0067] Heating of polymorph A or A' results in an endothermic event with an onset temperature of approximately 150°C, corresponding to a solid-solid transition of polymorph A or A' to polymorph B. Continued heating of the resulting solid, i.e., polymorph B, results in a second endothermic event corresponding to a melting point with an onset temperature of 205–220°C (see Figures 8a and 8b).

[0068] According to another independent aspect of the present invention, crystalline forms of psilocybin, hydrate A, are provided, characterized by one or more of the following: a. Peaks in the XRPD diffractogram at 8.9, 12.6, and 13.8°2θ±0.1°2θ; b.8.9, 12.6 and 13.8°2θ±0.1°2θ peaks in the XRPD diffractogram (further characterized by at least one additional peak at 6.5, 12.2, 19.4, 20.4 or 20.8°2θ±0.1°2θ); c. The XRPD diffractogram substantially illustrated in Figure 7d; or d. Endothermic events in a DSC thermogram with an onset temperature of 205–220°C, substantially illustrated in Figure 8d.

[0069] In one embodiment, psilocybin hydrate A exhibits an XRPD diffractogram characterized by the diffractogram summarized in Table 3. In one embodiment, the crystalline psilocybin hydrate A described herein contains (±0.1°²θ) of at least three peaks in Table 3. In one embodiment, the crystalline psilocybin hydrate A described herein contains (±0.1°²θ) of at least four peaks in Table 3. In one embodiment, the crystalline psilocybin hydrate A described herein contains (±0.1°²θ) of at least five peaks in Table 3. In one embodiment, the crystalline psilocybin hydrate A described herein contains (±0.1°²θ) of at least eight peaks in Table 3. In one embodiment, the crystalline psilocybin hydrate A described herein contains (±0.1°²θ) of at least ten peaks in Table 3. [Table 3]

[0070] In one embodiment, crystalline psilocybin hydrate A is characterized by XRPD diffractogram peaks at 8.9, 12.6, and 13.8°2θ±0.1°2θ. In another embodiment, crystalline psilocybin hydrate A is further characterized by at least one peak appearing at 6.5, 12.2, 19.4, 20.4, or 20.8°2θ±0.1°2θ. In yet another embodiment, crystalline psilocybin hydrate A is further characterized by at least two peaks appearing at 6.5, 12.2, 19.4, 20.4, or 20.8°2θ±0.1°2θ. In a further embodiment, crystalline psilocybin hydrate A exhibits an XRPD diffractogram substantially identical to the XRPD diffractogram shown in Figure 7d.

[0071] In one embodiment, crystalline psilocybin hydrate A is characterized by an endothermic event in a DSC thermogram having an onset temperature of 205-220°C, e.g., 210-220°C, e.g., 210-218°C, or e.g., 210-216°C. In another embodiment, crystalline psilocybin hydrate A is further characterized by an endothermic event in a DSC thermogram having an onset temperature of 85-105°C, or e.g., 90-100°C. In yet another embodiment, crystalline psilocybin hydrate A is characterized by an endothermic event in a DSC thermogram having an onset temperature of 205-220°C, e.g., 210-220°C, e.g., 210-218°C, or e.g., 210-216°C. It is characterized by endothermic events with an onset temperature of 85°C to 105°C, or 90°C to 100°C, etc. In yet another embodiment, crystalline psilocybin hydrate A exhibits a DSC thermogram substantially identical to the DSC thermogram in Figure 8d.

[0072] In another embodiment, crystalline psilocybin hydrate A is characterized by having a water content of 10–18%, for example 12–16%, or for example about 13%. Those skilled in the art will know methods for determining the water content of a compound, such as Karl Fischer titration. In one embodiment, crystalline psilocybin hydrate A is characterized by having a weight loss in the TGA thermogram of 10–18%, for example 12–16%, or for example about 13% at ambient temperatures of ~120°C, such as about 25°C.

[0073] In one embodiment, crystalline psilocybin hydrate A is an extremely pure crystalline form of hydrate A, for example, psilocybin contains at least 90% by weight, for example 95% by weight, for example 99% by weight, for example 99.5% by weight of hydrate A.

[0074] According to another independent aspect of the present invention, crystalline forms of psilocybin, polymorph B, are provided, characterized by one or more of the following: a. Peaks in the XRPD diffractogram at 11.1, 11.8, and 14.3°2θ±0.1°2θ; b.11.1, 11.8 and 14.3°2θ±0.1°2θ peaks in the XRPD diffractogram (further characterized by at least one additional peak at 14.9, 15.4, 19.3, 20.0 or 20.6°2θ±0.1°2θ); c. The XRPD diffractogram substantially illustrated in Figure 7c; or d. Endothermic events in a DSC thermogram with an onset temperature of 205–220°C, substantially illustrated in Figure 8c.

[0075] In one embodiment, psilocybin polymorph B exhibits an XRPD diffractogram characterized by the diffractogram summarized in Table 4. In one embodiment, the crystalline psilocybin polymorph B described herein includes (±0.1°²θ) at least three peaks in Table 4. In one embodiment, the crystalline psilocybin polymorph B described herein includes (±0.1°²θ) at least four peaks in Table 4. In one embodiment, the crystalline psilocybin polymorph B described herein includes (±0.1°²θ) at least five peaks in Table 4. In one embodiment, the crystalline psilocybin polymorph B described herein includes (±0.1°²θ) at least eight peaks in Table 4. In one embodiment, the crystalline psilocybin polymorph B described herein includes (±0.1°²θ) at least ten peaks in Table 4. [Table 4]

[0076] In one embodiment, crystalline psilocybin polymorph B is characterized by XRPD diffractogram peaks at 11.1, 11.8, and 14.3°2θ±0.1°2θ. In another embodiment, crystalline psilocybin polymorph B is further characterized by at least one peak appearing at 14.9, 15.4, 19.3, 20.0, or 20.6°2θ±0.1°2θ. In yet another embodiment, crystalline psilocybin polymorph B is further characterized by at least two peaks appearing at 14.9, 15.4, 19.3, 20.0, or 20.6°2θ±0.1°2θ. In a further embodiment, crystalline psilocybin polymorph B exhibits an XRPD diffractogram substantially identical to the XRPD diffractogram shown in Figure 7c.

[0077] In one embodiment, crystalline silybin polymorph B is characterized by an endothermic event in a DSC thermogram having a start temperature of 205-220 °C, such as 210-220 °C, such as 210-218 °C, or such as 210-216 °C. In yet another embodiment, crystalline silybin polymorph B exhibits a DSC thermogram substantially identical to the DSC thermogram of Figure 8c.

[0078] In another embodiment, crystalline silybin polymorph B is characterized by having a water content of less than 0.5% w / w, such as less than 0.4% w / w, such as less than 0.3% w / w, such as less than 0. % w / w, or such as less than 0.1% w / w. Those skilled in the art will know methods for measuring the water content of a compound, such as Karl Fischer titration. In one embodiment, crystalline silybin polymorph B is characterized by having a loss of less than 0.5% w / w, such as less than 0.4% w / w, such as less than 0.3% w / w, such as less than 0.2% w / w, such as less than 0.1% w / w in a TGA thermogram at ambient temperature ~200 °C, such as about 25 °C. In one embodiment, crystalline silybin polymorph B loses less than 2% by weight, such as less than 1% by weight, such as less than 0.5% by weight in a loss on drying test. The loss on drying test is carried out at 70 °C.

[0079] In one embodiment, crystalline silybin polymorph B is a highly pure crystalline form of polymorph B. For example, silybin contains at least 90% by weight, such as 95% by weight, such as 99% by weight, such as 99.5% by weight of polymorph B.

[0080] In another embodiment, crystalline silybin polymorph B is chemically pure. For example, silybin has a chemical purity of greater than 97%, such as greater than 98%, or such as greater than 99% by HPLC. In one embodiment, crystalline silybin polymorph B is 31It is free from any single impurity greater than 1%, more preferably less than 0.5%, including phosphoric acid as measured by 3P NMR and psilocine as measured by HPLC. In one embodiment, crystalline psilocybin polymorph B has a chemical purity greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph B is free from any single impurity greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph B does not contain psilocine at a level greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline psilocybin polymorph B is 31 When measured by 1P NMR, it does not contain phosphoric acid at a level greater than 1% by weight, more preferably less than 0.5% by weight. In one embodiment, crystalline psilocybin polymorph B has a chemical assay of at least 95% by weight, for example, at least 96% by weight, or for example, at least 98% by weight.

[0081] The psilocybin of the present invention in the form of polymorph A or A' has the general properties shown in Table 5 below. [Table 5]

[0082] The spectrum of psilocybin matches that shown in Table 6 below and in the spectra illustrated in Figures 10-13. [Table 6]

[0083] High purity is achieved through careful control of reaction conditions to ensure that potential organic impurities are significantly reduced.

[0084] Known and potential impurities in psilocybin are shown in Table 7 below. [Table 7-1] [Table 7-2]

[0085] Similarly, careful handling ensures that solvent levels always remain below the levels listed in Table 8. [Table 8]

[0086] Through careful selection of the operational methodology, the psilocybin drug substance of the present invention satisfies the acceptance criteria shown in Table 9 below. [Table 9] Abbreviations used in the table: NMT = below, NLT = above.

[0087] The methodology used to verify purity is described in the detailed explanation.

[0088] In fact, as shown in Table 10 below, criteria 6-13 are actually fully met. [Table 10] Abbreviations used in the table: NMT = less than or equal to, LT = less than or equal to.

[0089] Thus, crystalline psilocybin in the form of polymorph A or polymorph A' is protonated ( 1 H) and carbon ( 13 C) NMR, FT-infrared spectroscopy (FT-IR), and mass spectrometry (MS) - show spectra consistent with Figures 10-13.

[0090] Crystalline psilocybin in the form of polymorph A or polymorph A' meets either of the criteria specified in Table 9 or Table 10.

[0091] A batch of crystalline psilocybin is provided in the form of polymorph A or polymorph A' according to a second aspect of the present invention. In one embodiment, a batch of crystalline psilocybin, polymorph A or polymorph A' is provided, comprising at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, a batch of crystalline psilocybin, polymorph A or polymorph A' is provided, comprising at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, a batch of high-purity psilocybin is provided, comprising at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, a batch of high-purity psilocybin polymorph A is provided, comprising at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, a batch of high-purity psilocybin polymorph A' is provided, comprising at least 10 g, more preferably at least 100 g, and most preferably at least 250 g.

[0092] Alternatively, crystalline psilocybin may independently exist in the form of hydrate A or polymorph B.

[0093] A third aspect of the present invention provides a pharmaceutical preparation comprising crystalline psilocybin and one or more additives.

[0094] In one embodiment, a pharmaceutical preparation is provided comprising high-purity psilocybin and one or more additives. In another embodiment, a pharmaceutical preparation is provided comprising crystalline psilocybin polymorph A and one or more additives. In yet another embodiment, a pharmaceutical preparation is provided comprising crystalline psilocybin polymorph A' and one or more additives. In yet another embodiment, a pharmaceutical preparation is provided comprising high-purity crystalline psilocybin, polymorph A or polymorph A' and one or more additives. In yet another embodiment, a pharmaceutical preparation is provided comprising high-purity crystalline psilocybin polymorph A and one or more additives. In yet another embodiment, a pharmaceutical preparation is provided comprising high-purity crystalline psilocybin polymorph A' and one or more additives.

[0095] Alternatively, the crystalline psilocybin in the preparation may independently take the form of hydrate A or polymorph B.

[0096] Preferred pharmaceutical additives for oral formulations include diluents such as microcrystalline cellulose, starch, mannitol, anhydrous calcium hydrogen phosphate, or co-mixtures of silicon dioxide, calcium carbonate, microcrystalline cellulose, and talc; disintegrants such as sodium starch glycolate or croscarmellose sodium; binders such as povidone, copovidone, or hydroxypropyl cellulose; lubricants such as magnesium stearate or sodium stearyl fumarate; flow enhancers such as colloidal silicon dioxide; and film coatings such as Opadry II White or PVA-based Brown Opadry II.

[0097] Psilocybin is an active substance that is difficult to formulate for several reasons. Firstly, psilocybin has poor flow properties, and secondly, it is used in relatively low doses. This combination makes it difficult to ensure uniform content when forming tablets.

[0098] Excellent miscibility (meaning "mixture" or "composition"; the same applies hereafter) will have an AV value, or Acceptance Value, of less than 15, more preferably less than 10.

[0099] Excellent blending will also result in a label claim of greater than 90%, more preferably greater than 94%.

[0100] Within that context, these parameters indicate consistent dosing of psilocybin across tablets.

[0101] Standard excipients, particularly bulking agents, can be used for many pharmaceutical tablets. However, when compounding psilocybin tablets, the applicant found that non-standard bulking agents were preferable to achieve a satisfactory product.

[0102] In this regard, a functional bulking agent was selected. The functional bulking agent was a silicified bulking agent, preferably a silicified microcrystalline cellulose. A preferred form includes a high compressibility grade with a particle size range of about 45 to 150 microns.

[0103] In fact, a mixture of two functional extenders having different particle size ranges can be used in a weight percentage that prefers the larger particle size of the two.

[0104] In one embodiment, the silicified microcrystalline extender may comprise a first extender having a particle size range of about 45 to 80 microns in an amount of up to 30%, more preferably up to 20%, more preferably up to 15%, or less by weight, and a second extender having a particle size range of about 90 to 150 microns in an amount of up to 70%, more preferably up to 80%, more preferably up to 85%, or more than that.

[0105] The formulation may further contain, or consist of, a disintegrant, preferably sodium starch glycolate, a flow promoter, preferably colloidal silicon dioxide, and a lubricant, preferably sodium stearyl fumarate.

[0106] Further details of the formulation development are described in Example 12.

[0107] It should be noted that the formulation may contain any form of psilocybin, not just the preferred polymorphs disclosed.

[0108] Studerus et al., (2011) J Psychopharmacol 25(11)1434-1452, classified the oral doses of psilocybin as follows: extremely low dose at 0.045 mg / kg, low dose at 0.115–0.125 mg / kg, medium dose at 0.115–0.260 mg / kg, and high dose at 0.315 mg / kg.

[0109] Psilocybin will typically be present in formulated doses ranging from 0.01 mg / kg to 1 mg / kg. A typical human dose (for an adult weighing 60-80 kg) would be equal to a dose somewhere between 0.60 mg and 80 mg. In one embodiment, 2-50 mg of crystalline psilocybin, most preferably polymorph A or polymorph A', will be present in formulated doses such as 2-40 mg, 2-10 mg, 5 mg, 5-30 mg, 5-15 mg, 10 mg, 20-30 mg, or 25 mg. In one embodiment, 2-50 mg of crystalline psilocybin, particularly polymorph A, will be present in formulated doses such as 2-40 mg, 2-10 mg, 5 mg, 5-30 mg, 5-15 mg, 10 mg, 20-30 mg, or 25 mg. In one embodiment, 2 to 50 mg of crystalline psilocybin, particularly polymorph A', is present in compounded doses such as 2 to 40 mg, 2 to 10 mg, 5 mg, 5 to 30 mg, 5 to 15 mg, 10 mg, 20 to 30 mg, or 25 mg.

[0110] The preferred oral dose for adults is in the range of 1 mg to 40 mg, preferably 2 to 30 mg, more preferably 15 to 30 mg, for example, likely to be 5 mg, 10 mg, or 25 mg. Microdosing, typically about 1 / 10 of these doses, is also possible, and microdose formulations typically exist in the range of 0.05 mg to 2.5 mg.

[0111] The preferred pharmaceutical formulation is in oral dosage form.

[0112] The oral dosage form may be a tablet or a capsule.

[0113] For tablets, it is necessary to be able to accurately disperse the active substance. This is difficult due to the low dose and the hygroscopic and sticky nature of the active substance, which restricts its fluidity.

[0114] Psilocybin may be present with one or more additives. Preferred additives include microcrystalline cellulose and starch, more preferably silicified microcrystalline cellulose.

[0115] A crystalline psilocybin in the form of polymorph A or polymorph A' according to the first embodiment of the present invention is provided for use in pharmaceuticals. In one embodiment, crystalline psilocybin polymorph A is provided for use in pharmaceuticals. In one embodiment, crystalline psilocybin polymorph A' is provided for use in pharmaceuticals. In one embodiment, high-purity crystalline psilocybin polymorph A is provided for use in pharmaceuticals. In one embodiment, high-purity crystalline psilocybin polymorph A' is provided for use in pharmaceuticals.

[0116] Alternatively, crystalline psilocybin may independently exist in the form of hydrate A or polymorph B.

[0117] A crystalline psilocybin in the form of polymorph A or polymorph A' of the first aspect of the present invention is provided for use in treating central nervous system disorders.

[0118] Alternatively, crystalline psilocybin may independently exist in the form of hydrate A or polymorph B.

[0119] In one embodiment, crystalline psilocybin, polymorph A, or polymorph A' is provided for use in treating depression. In one embodiment, crystalline psilocybin, polymorph A, or polymorph A' is provided for use in treating drug-resistant depression. In one embodiment, crystalline psilocybin polymorph A is provided for use in treating drug-resistant depression. In one embodiment, crystalline psilocybin polymorph A' is provided for use in treating drug-resistant depression. In one embodiment, high-purity crystalline psilocybin polymorph A is provided for use in treating drug-resistant depression. In one embodiment, high-purity crystalline psilocybin polymorph A' is provided for use in treating drug-resistant depression.

[0120] Other conditions that can be treated include advanced-stage illnesses, such as anxiety disorders including anxiety and generalized anxiety disorder in cancer; depression including major depressive disorder; personality disorders including cluster headaches, obsessive-compulsive disorder, and conduct disorder; drug disorders including alcohol dependence, nicotine dependence, opioid dependence, and cocaine dependence; and other addictions including gambling addiction, eating disorders, and body dysmorphic disorder. Further treatment of symptoms is pain.

[0121] A method for treating central nervous system disorders is provided, in accordance with a sixth aspect of the present invention, comprising administering an effective dose of crystalline psilocybin in the form of polymorph A or polymorph A' according to the first aspect of the invention to a subject requiring treatment of central nervous system disorders.

[0122] In one embodiment, a method for treating depression is provided, comprising administering an effective dose of crystalline psilocybin in the form of polymorph A or polymorph A' to a subject who requires treatment for depression. In one embodiment, a method for treating drug-resistant depression is provided, comprising administering an effective dose of crystalline psilocybin in the form of polymorph A or polymorph A' to a subject who requires treatment for drug-resistant depression. In one embodiment, a method for treating drug-resistant depression is provided, comprising administering an effective dose of psilocybin polymorph A to a subject who requires treatment for drug-resistant depression. In one embodiment, a method for treating drug-resistant depression is provided, comprising administering an effective dose of psilocybin polymorph A' to a subject who requires treatment for drug-resistant depression. In one embodiment, a method for treating drug-resistant depression is provided, comprising administering an effective dose of high-purity crystalline psilocybin polymorph A to a subject who requires treatment for drug-resistant depression. In one embodiment, a method for treating drug-resistant depression is provided, comprising administering an effective dose of high-purity crystalline psilocybin polymorph A' to a subject who requires treatment for drug-resistant depression.

[0123] Alternatively, crystalline psilocybin may independently exist in the form of hydrate A or polymorph B.

[0124] To produce the psilocybin of the present invention, psilocybin was crystallized from water in a controlled manner.

[0125] A method for the large-scale production of psilocybin is provided according to a seventh aspect of the present invention, characterized in that the method comprises subjecting psilocybin to a water crystallization step, along with controlled drying, in order to produce crystalline psilocybin polymorph A according to the first aspect of the present invention.

[0126] In one embodiment, a method for the large-scale production of psilocybin is provided, characterized in that the method includes subjecting psilocybin to a water crystallization step, along with controlled drying, in order to produce crystalline psilocybin polymorph A having the XRPD diffractogram illustrated in Figure 7a and the DSC and TGA thermograph illustrated in Figure 8a. In one embodiment, a method for the large-scale production of psilocybin is provided, characterized in that the method includes subjecting psilocybin to a water crystallization step, along with controlled drying, in order to produce high-purity crystalline psilocybin-polymorph A having the XRPD diffractogram illustrated in Figure 7a and the DSC thermograph illustrated in Figure 8a.

[0127] Preferably, polymorph A is an isomorphic variant having the XRPD diffractogram shown in Figure 7a and the DSC thermograph shown in Figure 8a.

[0128] More preferably, psilocybin is recrystallized in water, typically about 10-20 volumes, heated with stirring to a temperature of at least 70°C, finished filtered with a suitable cutoff (typically less than 5 μm), seeded at a temperature of about 70°C, and cooled to about 5°C in a controlled manner over a period of more than 2 hours.

[0129] More preferably, this method includes controlled cooling that lowers the temperature by about 5°C to 15°C per hour, more preferably by about 10°C per hour.

[0130] Preferably, the final filtration step is performed through a filter of appropriate size, such as a 1.2 μm in-line filter.

[0131] Preferably, stirring is performed at approximately 400-500 rpm, typically at approximately 450 rpm.

[0132] Preferably, the crystalline species is psilocybin hydrate A. In one embodiment, 0.1% by weight or less of the crystalline species is added to the process.

[0133] Preferably, crystalline psilocybin is isolated by vacuum filtration.

[0134] In one embodiment, the isolated crystals are dried in a vacuum at a temperature of at least 30°C, for example 30-50°C, or for example 40-50°C. In one embodiment, the isolated crystals are dried in a vacuum for at least 10 hours, for example 12-18 hours, or for example about 30 hours. In one embodiment, the isolated crystals are dried in a vacuum at a temperature of at least 30°C, for example 30-50°C, or for example 40-50°C, for at least 10 hours, for example 12-18 hours, or for example about 30 hours. In one embodiment, the isolated crystals are dried until they lose less than 2% in weight in a drying loss test, for example less than 0.5%.

[0135] Preferably, the isolated crystals are washed several times in water and dried in a vacuum at about 50°C for at least 12 hours.

[0136] The resulting crystals are typically relatively large (ranging from 50 to 200 microns) and appear uniform under a 10x microscope, as illustrated in Figure 16a.

[0137] This differs from crystals obtained without controlled cooling, which are much smaller in size (typically 5–50 microns) when viewed under a 10x microscope, as illustrated in Figure 16b.

[0138] A psilocybin according to the first aspect of the present invention is provided, obtained by the crystallization method of the present invention, in accordance with the eighth aspect of the present invention.

[0139] A pharmaceutical preparation containing psilocybin according to the first aspect of the present invention is provided, obtained by the crystallization method of the present invention, in accordance with the ninth aspect of the present invention.

[0140] Psilocybin produced before crystallization can be produced by any synthetic or biological method, for example, by fermentation, or obtained by extraction from mushrooms.

[0141] A preferred manufacturing method involves using psilocine or 4-hydroxyindole as a starting material.

[0142] A method for the large-scale production of psilocybin from psilocine is provided in accordance with a tenth aspect of the present invention, comprising the following steps. i) Stage 4 - Reacting psilocine with tetrabenzyl pyrophosphate to form benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate; and ii) Stage 5 - Reacting benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate with hydrogen to form psilocybin.

[0143] According to an eleventh aspect of the present invention, a method for the large-scale production of psilocybin from 4-hydroxyindole is provided, comprising the following steps. i) Stage 1 - Reacting 4-hydroxyindole with acetic anhydride to form 1H-indole-4-yl acetate; ii) Stage 2 - Reacting 1H-indole-4-yl acetate with oxalyl chloride and dimethylamine to form 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4-yl acetate; iii) Stage 3 - Reacting 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4yl acetate with lithium aluminum hydride to form psilocine; iv) Stage 4 - Reacting psilocine with tetrabenzyl pyrophosphate to form benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate; and v) Stage 5 - Reacting benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate with hydrogen to form psilocybin.

[0144] A method for the large-scale production of psilocybin according to the 10th or 11th aspect of the present invention is provided, further comprising the following: vi) Stage 6 - A controlled drying aqueous crystallization step for producing crystalline psilocybin polymorph A according to a first aspect of the present invention.

[0145] In one embodiment, a method for the large-scale production of psilocybin according to a tenth or eleventh aspect of the present invention is provided, further comprising the following: vi) Stage 6 - A controlled drying, water crystallization step for producing crystalline psilocybin-polymorph A having an XRPD diffractogram substantially illustrated in Figure 7a and a DSC thermograph substantially illustrated in Figure 8a.

[0146] In one embodiment, a method for the large-scale production of psilocybin according to a tenth or eleventh aspect of the present invention is provided, further comprising the following: vi) Stage 6 - A controlled drying and water crystallization step for producing high-purity crystalline psilocybin-polymorph A having the XRPD diffractogram illustrated in Figure 7a and the DSC thermograph illustrated in Figure 8a.

[0147] Preferably, the crystalline psilocybin is polymorph A.

[0148] In developing a methodology for the large-scale production of psilocine or psilocybin, the applicant overcomes one or more significant problems in each of stages 1 to 5, and these problems are considered in relation to each step of the large-scale production of psilocine or psilocybin. More precisely, the methods by which each problem is overcome are considered separate and independent inventions, as they have applications in the production of other active substances, such as other derivatives, salts, esters, etc., that yield intermediates or prodrugs to psilocine or psilocybin.

[0149] Preferably, the stage 4(i) reaction involves the use of sodium hexamethyldisilazane (NaHMDS).

[0150] This has advantages over using butyllithium in that it is i) easier to handle and ii) does not introduce lithium into the reaction, which can cause problems in downstream processing.

[0151] Preferably, the reaction uses THF as the solvent.

[0152] This has the advantage of yielding products of significantly higher purity.

[0153] Preferably, in (i), the reaction is initiated below -50°C.

[0154] This has the advantage of reducing the level of impurities (m / z 295.2 observed by LCMS) that could subsequently affect purity downstream.

[0155] More preferably, the step in stage 4(ii) uses THF as the solvent.

[0156] This has the advantage of ensuring that concentration is avoided and facilitates a simple stirring process to obtain the product.

[0157] Preferably, the Stage 4(ii) step includes a stirring step to obtain benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate.

[0158] The stirring process has the advantage of simplifying the process and improving the yield.

[0159] To ensure that the Stage 4(ii) reaction is carried out to completion, the level of intermediate 4A is monitored, and as soon as it is complete, benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate is filtered and dried in an oven.

[0160] This has the advantage of minimizing impurities and yielding a purer product.

[0161] Preferably, the Stage 5 reaction is monitored by HPLC using relative retention time (RRT) for the level of the intermediate, and completion is determined when the intermediate is present at a level of less than 0.2%.

[0162] Crude psilocybin (stage 5 product, (12)) contains major stage 5 impurities with relative retention times (RRTs) of approximately 1.89 and 2.45, respectively, as well as psilocine (RRT 1.66). These impurities are listed in Table 7. Typically, crude psilocybin (stage 5 product (12)) contains 0.24 area% of RRT 1.89 impurity, 0.03 area% of RRT 2.45 impurity, and 1.86 area% of psilocine. In addition, pyrophosphate impurities (RRT 0.31) are present in the crude psilocybin at levels of approximately 2-6 area%, for example, according to HPLC.

[0163] At this level, a subsequent crystallization process can be carried out to give substantially pure psilocybin, for example, psilocybin with a purity of at least 95 area%, e.g., at least 98 area%, or e.g., at least 99 area%, as determined by HPLC. In one embodiment, pyrophosphate impurities (RRT0.31) are present in substantially pure psilocybin at levels of less than 0.3 area%, e.g., less than 0.2 area%, or e.g., less than 0.1 area%, as determined by HPLC.

[0164] Furthermore, during this stage, water is added to the reactants to maintain psilocybin in solution.

[0165] Preferably, the catalyst is recovered by filtration.

[0166] Preferably, in Stage 1, the reaction is carried out in DCM and pyridine.

[0167] This has the advantage of avoiding flammable solvents.

[0168] Preferably, the reaction mixture is washed with citric acid to give a pH of about 2-3 to remove excess pyridine, and the acid phase is separated from the DCM phase.

[0169] This has the advantage of allowing the isolation of intermediate 2A, and enables purification that does not contain excess oxalyl chloride.

[0170] More preferably, the DCM phase is further washed with sodium bicarbonate at approximately pH 8.

[0171] This has the advantage of being a purer process.

[0172] Preferably, 1H-indole-4-ylacetate is precipitated in heptane.

[0173] This aids in precipitation and overcomes the problem of partial solubility.

[0174] Preferably, magnesium sulfate is used as a desiccant.

[0175] Preferably, tert-butyl methyl ether (TBME) and tetrahydrofuran (THF) are used as solvents.

[0176] Preferably, the reaction with oxalyl chloride is carried out at approximately 30°C to 40°C.

[0177] This has the advantage of ensuring a high reaction rate and providing an improved level of completion.

[0178] Preferably, intermediate 2A is isolated by filtration.

[0179] This has the advantage of purifying the intermediate without containing excess oxalyl chloride.

[0180] Preferably, in stage 2, step i, intermediate 2A is also washed to remove excess oxalyl chloride.

[0181] Preferably, intermediate 2A is washed with TBME.

[0182] Preferably, heptane is added to precipitate further intermediate 2A.

[0183] Preferably, in stage 2, step ii, dimethylamine is used in excess.

[0184] This has the advantage of yielding an even more improved impurity profile and yield.

[0185] Preferably, the pH is maintained at approximately pH 7 or above pH 7.

[0186] Preferably, the reaction is carried out in TBME.

[0187] Preferably, this stage further includes a purification step to remove the dimethylamine salt.

[0188] This has the advantage of improving purity.

[0189] Preferably, this stage includes slurrying and filtration steps.

[0190] This has the advantage of improved handling and purity.

[0191] More preferably, this stage includes slurring with water and / or IPA, filtering, and drying the isolated 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4yl acetate.

[0192] This has the advantages of improved purity and yield, and reduced hydrolysis.

[0193] Preferably, in stage 3, the reaction is carried out in the solvent THF.

[0194] This has the advantage of forming a suspension / emulsion without thickening.

[0195] Preferably, 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4yl acetate is added to a solution of LiAlH4 in THF.

[0196] Preferably, the reaction is stopped with acetone, followed by citric acid, to ensure that the mixture remains strongly basic (pH 11 or higher).

[0197] This has the advantage of yielding a high yield.

[0198] Preferably, the psilocine is filtered, washed in THF, slurryed in PrOAc:TBME, filtered, washed in TBME, and dried.

[0199] This has the advantage of yielding a highly pure product, for example, a product that is at least 95% pure by HPLC, at least 98% pure by HPLC, or at least 99% pure by HPLC.

[0200] A preferred production method includes each of stages 1 through 6, but it will be understood that each feature of each stage can exist independently or can be used in combination with any other features from the same or different steps of the reaction.

[0201] Psilocybin in the specified forms, polymorph A or polymorph A', and such high-purity psilocybin have not been obtained previously, and to the best of the applicant's knowledge, the production of polymorph A and polymorph A' (as illustrated in Figures 7a and 7b and 8a and 8b) is novel. In fact, the production of large batch quantities of polymorph A is novel. The crystallization methodology and, in part, the manufacturing method of the present invention make it possible to obtain crystalline psilocybin of such high chemical purity.

[0202] Furthermore, considering the unstable nature of this compound, a crystalline form was obtained that was shown to be stable for at least 12 months (as described below) under accelerated conditions.

[0203] Polymorphs A and A' (Figures 7a and 7b) are distinct from polymorph B (Figure 7c), hydrate A (Figure 7d), ethanol solvate (Figure 7e), and mixture (Figure 7f (top)), as is evident from their XRPD diffractograms and DSC thermographs, as described below.

[0204] The relationships between different polymorphs are shown in Figure 9.

[0205] In fact, the size and shape of the crystals are determined by crystallization methods, which can then affect their stability and ability to formulate products.

[0206] In a particularly preferred embodiment, psilocybin is produced through a six-step process, as outlined below.

[0207] A method for producing crystalline psilocybin according to a first aspect of the present invention is provided, characterized in that the method comprises subjecting psilocybin to a water crystallization step, along with controlled drying, in order to produce crystalline psilocybin polymorph A or polymorph A' according to a first aspect of the present invention. In one embodiment, a method for producing crystalline psilocybin according to a first aspect of the present invention is provided, characterized in that the method comprises a water crystallization step, along with controlled drying, in order to produce crystalline psilocybin polymorph A or polymorph A' having an XRPD diffractogram substantially illustrated in Figure 7a or Figure 7b and a DSC thermograph substantially illustrated in Figure 8a or Figure 8b. In one embodiment, a method for producing psilocybin according to a first aspect of the present invention is provided, characterized in that the method includes a water crystallization step, along with controlled drying, for producing high-purity crystalline psilocybin-polymorph A or polymorph A' having an XRPD diffractogram illustrated in Figure 7a or 7b and a DSC thermograph illustrated in Figure 8a or 8b.

[0208] Preferably, polymorphs A and A' are isomorphic variants having XRPD diffractograms substantially illustrated in Figures 7a and 7b and DSC thermographs substantially illustrated in Figures 8a and 8b.

[0209] More preferably, psilocybin is recrystallized in about 10-20 volumes of water, heated with stirring to a temperature of at least 70°C, finished filtered with a suitable cutoff (typically less than 5 μm), seeded at a temperature of about 70°C, and cooled to about 5°C in a controlled manner over a period of more than 2 hours.

[0210] More preferably, this method includes controlled cooling that lowers the temperature by about 5°C to 15°C per hour, more preferably by about 10°C per hour.

[0211] Preferably, the final filtration step is performed through a filter of appropriate size, such as a 1.2 μm or 0.45 μm in-line filter.

[0212] Preferably, stirring is performed at approximately 400-500 rpm, typically at approximately 450 rpm.

[0213] Preferably, the crystalline species is psilocybin hydrate A. In one embodiment, 0.1% by weight or less of the crystalline species is added to the process.

[0214] Preferably, crystalline psilocybin is isolated by vacuum filtration.

[0215] In one embodiment, the isolated crystals are dried in a vacuum at a temperature of at least 30°C, for example 30-50°C, or for example 40-50°C. In one embodiment, the isolated crystals are dried in a vacuum for at least 10 hours, for example 12-18 hours, or for example about 30 hours. In one embodiment, the isolated crystals are dried in a vacuum at a temperature of at least 30°C, for example 30-50°C, or for example 40-50°C, for at least 10 hours, for example 12-18 hours, or for example about 30 hours. In one embodiment, the isolated crystals are dried until they lose less than 2% in weight in a drying loss test, for example less than 0.5%.

[0216] Preferably, the isolated crystals are washed several times in water and dried in a vacuum at about 50°C for at least 12 hours.

[0217] The resulting crystals are typically relatively large (ranging from 50 to 200 microns) and appear uniform under a 10x microscope, as illustrated in Figure 16a.

[0218] This differs from crystals obtained without controlled cooling, which are much smaller in size (typically 5–50 microns) when viewed under a 10x microscope, as illustrated in Figure 16b. Stage 1: Synthesis of 1H-indole-4-ylacetate (3)

[0219] The core reaction is the reaction of 4-hydroxyindole (1) with acetic anhydride (2) to form 1H-indole-4-ylacetate (3) (Figure 2).

[0220] Most preferably, Stage 1 is as follows:

[0221] 4-hydroxyindole (1), DCM (12), and pyridine (13) are added to a vessel and cooled to approximately 0-5°C. Acetic anhydride (2) is added dropwise, the mixture is heated to approximately 20-25°C, and stirred until HPLC analysis is complete. The reactants are washed with aqueous citric acid (14) and aqueous NaHCO3 (15), dried over MgSO4 (16), filtered, and evaporated to approximately half the volume. Heptane (17) is added, and distillation is continued to remove most of the DCM. The mixture is cooled to approximately 5-25°C, filtered, washed with heptane, and dried overnight in a vacuum oven to isolate 1H-indole-4-ylacetate (3) as a solid suitable for use in the following stages. Stage 2: Synthesis of 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4yl acetate (6)

[0222] The core reaction is the reaction of 1H-indole-4-ylacetate (3) with oxalyl chloride (4) and dimethylamine (5) to form 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4-ylacetate (6) (Figure 3).

[0223] Most preferably, Stage 2 is as follows:

[0224] 1H-indole-4-ylacetate (3) is dissolved in a mixture of THF (19) and TBME (18) at room temperature. Oxalyl chloride (4) is added dropwise, and the reaction is exothermic at approximately 35-40°C. The temperature range is maintained through the remaining additions. The reaction mixture is then stirred at approximately 40°C until completion by HPLC. The reaction mixture is cooled to room temperature, and heptane (17) is added to precipitate further solids. The slurry is stirred, then allowed to settle, and then most of the solvent (18 / 19) is removed by decantation. The solids are washed twice in the container with heptane (17). TBME (18) is added to obtain a yellow slurry, and the mixture is cooled to approximately -20°C. Dimethylamine solution (5) is added while maintaining the temperature at -20°C to -10°C. The reaction mixture is then warmed to room temperature and stirred until completion, adding further dimethylamine if necessary. The reaction was filtered, washed with heptane (17), and dried in a vacuum oven. The slurry was then rinsed in water (20), and then in IPA (21). Crude 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4yl acetate (6) was further purified by chemical reaction, and then dried in a vacuum oven to obtain (6) as a solid suitable for use in the following stages. Stage 3: Synthesis of 3-(2-(dimethylamino)ethyl)-1H-indole-4-ol(psilosine)(8)

[0225] The core reaction is the reaction of 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4yl acetate (6) with lithium aluminum hydride (7) to form psilocine (8) (Figure 4).

[0226] Most preferably, Stage 3 is as follows:

[0227] 3[(dimethylcarbamoyl)carbonyl]-1H-indole-4-yl acetate (6) was slurryed in THF (19) and cooled to approximately 0°C. While maintaining the temperature at approximately 0-20°C, a THF solution of LiAlH4 (7) was added dropwise. The reaction was then refluxed until completion by HPLC. The reaction was cooled to 0°C, and excess LiAlH4 was quenched by adding acetone (22), followed by aqueous citric acid (14). The batch was filtered to remove lithium and aluminum salts. The filtrate was dried with MgSO4 (16), filtered, concentrated, and mounted on a silica pad (23). The pad was eluted with THF (19), and the fraction containing the product was evaporated. The resulting solid was slurryed in an iPrOAc:TBME (24 / 18) mixture, filtered, and washed with TBME. The solid was dried in an oven to obtain high-purity psilocine (8) as a grayish-white solid. Stage 4: Synthesis of benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate (10)

[0228] The core reaction is the reaction of psilocine (8) with tetrabenzyl pyrophosphate (9) to form benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate (10) (Figure 5).

[0229] Most preferably, Stage 4 is as follows:

[0230] Psilosine (8) was added to a container, followed by THF (19). The reaction mixture was cooled to -50°C to -70°C, and NaHMDS (25) was added dropwise at approximately -45°C to -70°C. The temperature was adjusted to approximately -45°C to -60°C, and tetrabenzyl pyrophosphate in THF was added. The batch was heated to 0°C, and then the solid byproduct was removed by filtration, and the filtrate was concentrated under vacuum. The concentrated mixture was then heated to approximately 40°C and stirred under controlled monitoring and HPLC until the intermediate was converted to the stage 4 product (10). The batch was cooled to approximately 0-5°C, and the resulting solid was isolated by filtration and dried under vacuum to give benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate (10) as a solid. Stage 5: Synthesis of intermediate grade hydrogen phosphate 3-[2-(dimethylazaniumyl)ethyl]-1H-indole-4-yl (crude psilocybin) (12)

[0231] The core reaction involves reacting benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate (10) with hydrogen (11) to form psilocybin (12) (Figure 6).

[0232] Most preferably, Stage 5 is as follows:

[0233] Pd / C(26), methanol(24), and 3-[2-(benzyldimethylazanium) [ethyl yl]-1H-indole-4-yl phosphate (10) was added to the vessel, and hydrogen (11) was injected into the resulting mixture until completion by HPLC. To keep the product in solution, purified water (20) was added during this process. The mixture was heated to about 35°C to 45°C, then filtered through a Celite (27) bed and washed with methanol (24) and purified water (20). The filtrate was evaporated under vacuum and azeotropically mixed with ethanol (28) to obtain intermediate grade psilocybin (12). Stage 6: Synthesis of 3-[2-(dimethylazaniumyl)ethyl]-1H-indol-4-yl hydrogen phosphate (Sirobin)

[0234] The nucleus purification / polymorph determination step is a water crystallization step, followed by a controlled cooling and drying step to produce high-purity crystalline sirobin, polymorph A or polymorph A'.

[0235] Most preferably, Stage 6 is as follows.

[0236] Intermediate-grade sirobin (12) (Stage 5) was added to a container containing purified water (20), and the mixture was heated until sirobin (12) dissolved. Then, the resulting bulk solution was finish-filtered into a pre-warmed container. Preferably, the temperature was adjusted to about 68°C to 70°C, and sirobin hydrate crystal seeds (i.e., hydrate A) were added to the reactants. Then, the batch was cooled to about 0 to 10°C in a controlled manner, stirred, the solid was collected by filtration, and washed with purified water. Then, the isolated solid was dried in vacuo to obtain high-purity crystalline sirobin, polymorph A or A' as a grayish-white solid.

Brief Description of the Drawings

[0237] Hereinafter, embodiments of the present invention will be further described with reference to the accompanying drawings. [Figure 1] Figure 1 is a schematic diagram of the reaction taught in JNP. [Figure 2] Figure 2 is a schematic diagram of the Stage 1 reaction of one aspect of the present invention. [Figure 3] Figure 3 is a schematic diagram of the Stage 2 reaction of one aspect of the present invention. [Figure 4] Figure 4 is a schematic diagram of the Stage 3 reaction of one aspect of the present invention. [Figure 5] Figure 5 is a schematic diagram of the Stage 4 reaction of one aspect of the present invention. [Figure 6] Figure 6 is a schematic diagram of the Stage 5 reaction of one aspect of the present invention. [Figure 7A]Figure 7A is the XRPD diffractogram of polymorph A (GM764B). [Figure 7B] Figure 7B is the XRPD diffractogram of polymorph A' (JCCA2160F). [Figure 7C] Figure 7C is the XRPD diffractogram of polymorph B (JCCA2160-F-TM2). [Figure 7D] Figure 7D is the XRPD diffractogram of hydrate A (JCCA2157E). [Figure 7E] Figure 7E is the XRPD diffractogram of ethanol solvate (JCCA2158D). [Figure 7F] Figure 7F shows the XRPD diffractogram (top) of the product (CB646-E) obtained during the development of this process, compared with the diffractogram polymorph A' (JCCA2160F) (center) and polymorph B (JCCA2160-TM2) (bottom). [Figure 8A] Figure 8A shows the DSC and TGA thermographs of polymorph A (GM764B). [Figure 8B] Figure 8B shows the DSC and TGA thermographs of polymorph A' (JCCA2160F). [Figure 8C] Figure 8C is a DSC thermograph of polymorph B (GM748A). [Figure 8D] Figure 8D shows the DSC and TGA thermographs of hydrate A (JCCA2157E). [Figure 8E] Figure 8E shows the DSC and TGA thermographs of ethanol solvate (JCCA2158D). [Figure 9] Figure 9 is a morphological diagram showing the interrelationships of forms in a water-based system. [Figure 10] Figure 10 shows the 1H NMR spectrum of psilocybin (please read this in conjunction with Attribution Example 7). [Figure 11] Figure 11 shows the 13C NMR spectrum of psilocybin (please read this in conjunction with Attribution Example 7). [Figure 12] Figure 12 shows the FT-IR spectrum of psilocybin. [Figure 13] Figure 13 shows the mass spectrum of psilocybin. [Figure 14] Figure 14 shows the numbered structural formula of psilocybin. [Figure 15] Figure 15 shows the temperature solubility curve for psilocybin in water. [Figure 16A] Figure 16A is a micrograph showing crystals obtained by controlled cooling. [Figure 16B] Figure 16B is a micrograph showing crystals obtained by uncontrolled cooling and drying. [Figure 17] Figure 17 is a morphological phase diagram showing the interrelationships of morphologies in different solvent systems. [Figure 18] Figure 18 shows the XRPD diffractogram-pattern C for solids isolated at 25 and 50°C. [Figure 19] Figure 19 shows XRPD diffractograms—patterns D, E, and F—for solids isolated at 25 and 50°C. [Figure 20] Figure 20 shows a comparison of XRPD diffractograms obtained for solids isolated from the equilibrium state of amorphous psilocybin in solvents A-H. [Figure 21] Figure 21 shows a comparison of XRPD diffractograms obtained for solids isolated from the equilibrium state of amorphous psilocybin in solvents I-P. [Figure 22] Figure 22 shows a comparison of XRPD diffractograms obtained for solids isolated from the equilibrium state of amorphous psilocybin in solvents R~Y. [Modes for carrying out the invention]

[0238] In contrast to the prior art, the present invention aims to produce psilocybin on a commercial scale in quantities or batches at least 100 g, more preferably at least 250 g, which are one-logarithmic or two-logarithmic levels higher than the levels described in JNP (which describes a "large" scale method for producing gram quantities on a 10 g scale). To demonstrate many important development processes from JNP, the following description details the experiments and studies conducted at each process stage, and illustrates the choices made to overcome numerous technical challenges encountered in the large-scale production of GMP-compliant shiloshivin (7) (including various intermediates (2-6)) starting from 4-hydroxyindole (1).

[0239] References to specific numerical values include at least that specific value if the context does not clearly indicate the contrary meaning. When a range of values is expressed, other embodiments include from one specific value and / or to another specific value. Further, references to values expressed in ranges include all respective values within that range. All ranges include both endpoints and can be combined.

[0240] It will be understood that when a value is represented as approximate by use of the antecedent "about", that specific value forms another embodiment.

[0241] As used herein, the singular forms "a", "an" and "the" include the plural.

[0242] When used in reference to a numerical range, cut-off or specific value, the term "about" is used to indicate that the indicated value can vary by up to 10% from the recited value. Since many of the numerical values used herein are experimentally determined, it should be understood by those skilled in the art that such determinations can often vary from one experiment to another. Due to this inherent variability, the values used herein should not be considered overly limiting. For this reason, the term "about" is used to encompass variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less or ±0.1% or less from the stated value.

[0243] ​As used herein, “to treat” and similar terms mean to reduce the severity and / or frequency of a symptom, to eliminate a symptom and / or the underlying cause of such symptom, to reduce the frequency or likelihood of a symptom and / or its underlying cause, to delay, prevent and / or slow the progression of a disease and / or disorder, and to improve or correct damage directly or indirectly caused by a disease and / or disorder.

[0244] The following abbreviations are used herein. DSC (Differential Scanning Calorimetry) RT-room temperature TBME - Methyl tert-butyl ether TGA-Thermogravimetric analysis THF-tetrahydrofuran Regarding wrt- XRPD-X-ray Powder Diffraction [Examples]

[0245] [Example 1] Stage 6: Crystallization process and resulting polymorphs Experiment to produce morphology A': 1.0 g of crude psilocybin was added to a 25 mL flask. Water (12.8 mL / 16 volume based on the activity of the added material) was added. The mixture was stirred and heated to 80°C. A dark brown solution with visible undissolved solids was obtained. The mixture was filtered through a heated 0.45 μm filter into a hot 25 mL flask. The undissolved solids were removed to obtain a dark brown solution. The solution was re-equilibrated at 75°C and then slowly cooled to ambient temperature (10°C / hour). The resulting light brown solution was allowed to equilibrate at ambient temperature for 16 hours. The suspension was cooled to 5°C before isolation of the solids by vacuum filtration. The filter cake was washed with water (0.8 mL / 1 volume) and dried in vacuum at 50°C for 16 hours. 75% yield, 99% chemical purity, NMR assay >98%.

[0246] The above procedure was repeated using 14 volumes (11.2 mL) of water. 69% yield, 99% chemical purity, NMR assay >98%.

[0247] In both cases, dissolution of crude psilocybin was achieved at approximately 75°C. Upon gradual cooling, precipitation was observed at approximately 60°C.

[0248] In both cases, psilocybin polymorph A' was produced and confirmed by XRPD (diffractogram consistent with Figure 7b) and DSC (thermogram consistent with Figure 8b). Experiment to produce morphology A

[0249] 94 g of crude psilocybin obtained from the Stage 5 process (containing approximately 4% pyrophosphate impurities and approximately 93% pure according to HPLC) was subjected to aqueous recrystallization as described below. did.

[0250] The protocol used sufficient water (12 liters), a rapid stirring speed (450 rpm), and a controlled cooling profile (10°C / hour).

[0251] Psilocybin (94.0 g) (CB650E) was added to a 2 L flask. Water (902 ml, 12 volumes based on the activity of the added material) was added. The mixture was stirred and heated to approximately 78°C. A dark brown solution with visible undissolved solids was obtained. The mixture was finished filtered through a 1.2 μm inline filter into a hot 5 L flask fitted with an overhead stirrer (450 rpm). Undissolved solids were removed to obtain a clarified dark brown solution. The solution was re-equilibrated at approximately 75°C for 15 minutes, and then slowly cooled to ambient temperature (10°C / hour). After maturation in water at 68-70°C, psilocybin hydrate A (GM758A - XRPD diffractogram matching Figure 7d) was seeded into the solution. The resulting light brown suspension was equilibrated at ambient temperature for approximately 16 hours. The suspension was cooled to 5°C for 1 hour before isolation of the solids by vacuum filtration. The filtered cake was washed with water (282 mL, 3 volumes) and dried in a vacuum at approximately 50°C for 30 hours.

[0252] This process was successfully completed with a yield of 75%. The chemical purity of the solid was confirmed to be 99.3%. Analysis of the solid by XRPD after 30 hours of drying showed polymorph A (Figure 7a). Characteristic perturbations were observed at approximately 17°2θ, such as 17.5°2θ, and were prominent in the bulk material. Determination of the properties of the solid states of polymorph A and polymorph A'

[0253] The DSC and TGA thermograms obtained for polymorph A (Figure 8a) were equivalent to those obtained for polymorph A' (Figure 8b). The TGA thermograms obtained for polymorphs A and A' (Figure 8a) did not show weight loss before decomposition. This suggests that the difference between the XRPD obtained for polymorph A (Figure 7a, with a perturbation at approximately 17°2θ) and the XRPD obtained for polymorph A' (Figure 7b, with no perturbation) obtained on a small scale was not due to excessive hydration.

[0254] Microscopy of the solid (Figure 16a) reveals rod-shaped crystals with good uniformity and a size range between 50 and 200 microns.

[0255] The XRPD diffractogram obtained for polymorph A' does not show the same degree of perturbation at approximately 17°2θ as for polymorph A. The perturbation in the XRPD diffractogram at approximately 17°2θ was more pronounced for psilocybin produced on a large scale (compared to that obtained on a small scale), which was unexpected. The applicant demonstrated that hydrate A is the only polymorph that exists over a wide temperature range without having a diffraction peak in the 17°2theta region (see Figure 7d). This strongly suggests that dehydration causes hydrate A to disintegrate, yielding polymorphs A or A' with scale-dependent variation, and that polymorph A is the true form, while polymorph A', formed on a small scale, is atypical.

[0256] To investigate the validity of this theory and to demonstrate the recovery to polymorph A, a small amount of bulk was re-dried after being immersed again in water (to regenerate hydrate A). A small amount of sample (250 mg - psilocybin polymorph A) was equilibrated in water (10 vol) for 1 hour. The suspension was filtered, and analysis of the wet solid confirmed that hydrate A was produced (Figure 7d) and there was no perturbation at 17° 2 theta. This material was dried in vacuum for 16 hours, and the solid was re-evaluated by XRPD. Polymorph A' material was confirmed by XRPD (Figure 7b), and a decrease in XRPD perturbation was observed. Further drying and maturation of the original bulk solid at ambient temperature did not change the XRPD diffractogram of the solid. Two solid forms were obtained, polymorph A and polymorph A'. The XRPD diffractogram for ' is virtually identical except for a 17.5° 2-theta peak. The thermal properties are also identical. The difference between the XRPD diffractograms of polymorph A and polymorph A' is slight, and both polymorphs transform very rapidly and kinetically into the hydrated state.

[0257] Further experiments were conducted to determine whether the difference in the XRPD diffractograms of polymorph A and polymorph A' was due to a larger crystallization process that subsequently resulted in a larger-grained solid that did not dry as effectively and caused the change, or whether it was due to the crystal habit and size difference of the crystalline solid. Psilocybin polymorph A (the polymorph form identified before the experiment) was ground using a mortar and pestle and evaluated by XRPD. No change in polymorphism was observed. Water (<1 mL) was added to another portion (51 mg) and the moisture content was evaluated to confirm the formation of a hydrate. Both lots were dried in vacuum at 50°C for approximately 18 hours and re-evaluated by XRPD. The ground sample remained as polymorph A. The dehydrated hydrated sample was shown to be polymorph A' (i.e., no reflection at approximately 17.5°2θ). This suggested that size / crystal habit alone was not the sole reason for the original reflection peak.

[0258] TGA evaluation revealed that the input lot showed low mass loss (0.139% by weight) up to approximately 70°C. Solids that were degraded in particle size and subsequently dried showed a greater mass loss of 0.343% by weight up to approximately 75°C, while hydrated and dried solids showed the smallest mass loss of 0.069% by weight up to approximately 80°C. Solids that were degraded in particle size and subsequently dried were maintained at 80°C for 10 minutes (past the point of mass loss according to TGA), but XRPD evaluation revealed no change from the input, meaning that low levels of hydration and partial swelling of the crystal lattice were not the cause of the variation.

[0259] It is possible to generate polymorph A' through hydration of polymorph A and subsequent small-scale drying of the isolated solid.

[0260] To obtain hydrate A from both lots, approximately 60 mg each of psilocybin polymorph A and polymorph A' were added to 0.2 ml of water. Half of each hydrate A was dried in vacuum at 25°C for approximately 17 and 1 / 4 hours, and the remainder of each hydrate A was dried at ambient temperature under N2 flow for approximately 17 and 1 / 4 hours. After drying, the solids were isolated and evaluated by XRPD. XRPD evaluation of the solid isolated from the polymorph A input confirmed that hydrate A was successfully produced and that the solid dried to yield polymorph A' from both drying methods. XRPD evaluation of the solid isolated from the polymorph A' input confirmed that hydrate A was successfully produced and that the solid dried to remain as polymorph A' from both drying methods.

[0261] On the small scales studied, polymorphs A and A' would dry and, via conversion to hydrate A, give polymorph A'.

[0262] The particle size of psilocybin polymorph A (100 mg) was reduced using a mortar and pestle. To evaluate whether reducing the particle size affected the dehydration of the sample, the ground lot was subjected to two different drying programs. The first sample was maintained at 80°C for 10 minutes, and the second sample was maintained at 110°C for 10 minutes. Both solids were evaluated by XRPD, revealing that polymorph A was retained. To investigate whether the ground lot, which had been subjected to prior isothermal stress, was maintained at 110°C for a length of time sufficient to affect its morphology, a portion of the ground lot was dried in vacuum at 110°C for approximately 24 hours. XRPD evaluation revealed a slight morphological change, with a slightly reduced intensity, but polymorph A reflection still present at approximately 17.

[0263] It was concluded that polymorph A would not readily convert to polymorph A' through particle size reduction and / or drying at high temperatures. methodology

[0264] The stability assessment of psilocybin free from pyrophosphate impurities showed that the level of stage 3 intermediate impurities (psilocine) produced by the hydrolysis of psilocybin at temperatures above 80°C was of interest. For example, when an 83 mg / mL aqueous solution of psilocybin was heated to 90°C and analyzed by HPLC after 1, 2, and 4 hours, the levels of stage 3 impurities were measured at 0.28, 1.82, and 7.79 area%, respectively. In comparison, when 50 mg of psilocybin was dissolved in water (1.2–1.8 ml; a sufficient volume to maintain the solution) and heated to 70, 75, and 80°C for 4 hours, the levels of stage 3 impurities were measured by HPLC at 0.53, 0.74, and 2.30 area%, respectively. To achieve dissolution and final filtration, recrystallization was performed by heating crude psilocybin to 75°C–80°C. Immediate cooling of the solution limits the level of psilocybin hydrolysis by reducing the residence time of the material at excessive temperatures.

[0265] Further experimental recrystallization of psilocybin was performed by introducing the following changes.

[0266] Vary the amount of water used;

[0267] Vary the stirring;

[0268] It has a controlled cooling profile;

[0269] It has a rapid (uncontrolled) cooling profile.

[0270] Using a smaller volume of water (only 12 volts) did not interfere with the recrystallization process, and psilocybin dissolution was achieved at a temperature that allowed for the final filtration step. Different cooling rates were shown to result in different crystal size distributions; slow, controlled cooling at approximately 10°C / hour produced relatively larger, more uniform average crystal sizes (Figure 16a), while rapid cooling profiles yielded smaller crystals (Figure 16b). The controlled cooling profile was preferred, which was reflected in the improved purity compared to controlled cooling.

[0271] Using this process yielded psilocybin with 99.3% chemical purity in 75% yield. The thermal properties of the solid were consistent with the desired properties. Differences in the XRPD diffractograms of the dry solid suggested that the dry profile may be important in determining how hydrate A disintegrates to give the preferred solid form. Polymorph A was demonstrated to be stable for 12 months under accelerated stability testing conditions.

[0272] experiment

[0273] Under N2 conditions, Stage 5 was added to the container, followed by water (approximately 12-15 volumes based on the amount of active Stage 5). To achieve dissolution, the mixture was heated to approximately 80°C and filtered through a 1.2 μm inline filter into a clean, new flask heated to 80°C. The stirring speed was set to a high level (450 rpm), and the solution was equilibrated at 70-75°C. The solution was cooled to ambient temperature at approximately 10°C / hour, and psilocybin hydrate A (0.001 × amount of Stage 5 added) was seeded at 68-70°C. The suspension was maintained at ambient temperature overnight, then cooled to approximately 5°C and maintained for 1 hour. The suspension was filtered and washed with water (2-3 volumes based on the amount of active Stage 5 added). The pure psilocybin was dried in vacuum at 50°C. Crystalline material psilocybin (polymorph A or polymorph A' depending on the scale) was obtained, for example, Using 94g of psilocybin yielded polymorph A, while using 1g of psilocybin yielded polymorph A'. Typically, batch sizes larger than 5g yielded polymorph A, while batch sizes smaller than 5g yielded polymorph A'.

[0274] The differences and advantages from JNP can be summarized as follows: i) This additional crystallization step results in a defined crystalline form—polymorph A (or A'). ii) Short-term heating to approximately 80°C has the advantage of maximizing solubility (and avoiding hydrolysis), which ensures a good yield. iii) At approximately 70-80°C, a final filtration can be used to remove insoluble impurities. This is best achieved using an in-line filter, typically of about 1.2 μm. This ensures good chemical purity. iv) By using a high stirring speed (typically around 450 rpm), rapid dissolution is ensured, and the time the solution is maintained at 80°C is minimized, thus avoiding increased levels of stage 3 intermediate impurities formed by the hydrolysis of psilocybin. v) Controlled cooling, typically provided at around 10°C / hour, results in a more uniform crystal size and maintains its morphology as crystalline hydrate A. vi) Seeding psilocybin hydrate A in solution at approximately 70°C promotes crystallization of hydrate A. vii) To maximize purity and yield polymorph A or A' depending on the scale, the crystals are washed in water and dried at approximately 50°C. Examples 2-6 Stages 1-5: Psilocybin Production

[0275] As previously stated herein, the following examples represent significant developments from the process described in JNP and illustrated in Figure 1. [Example 2] Stage 1 (Figure 2)

[0276] Stage 1 conditions in JNP involved using 1.1 equivalents of Ac2O and 1.2 equivalents of pyridine in solvent DCM. After stirring overnight, the reaction was found to be complete (99% product, 0% starting material (SM)). The reaction mixture was washed with water and concentrated under vacuum to obtain a brown oily substance. In the literature, the oily substance was taken up in RINKAN and concentrated by evaporation to give a solid precipitate at low volume. the study

[0277] However, no solid precipitation from ƒ was observed in the applicant's possession. Solid precipitation was promoted by grinding with heptane, but this would not form an expandable process. The solid was collected to obtain a high-purity Stage 1 product (75% yield, 95% ultrapure according to NMR).

[0278] The reaction functioned well, but the isolation technique needed further improvement to obtain a solid that was easy to handle. Isolation of the solid by filtration was then expected to provide a means of purification.

[0279] To determine whether solid precipitation could be promoted and whether it would be possible to directly isolate the solid from the reaction mixture, the reaction was first tested in RINKAN. However, the reaction profile in RINKAN proved to be less favorable than that in DCM, and therefore this reaction was abandoned.

[0280] Since it was thought that pyridine might be inhibiting the recrystallization of the product, the applicant washed pyridine from the DCM reaction mixture. The reaction was repeated (0.4% starting material, 98.7% product completion by HPLC), and the reaction mixture was washed with 20% citric acid to achieve pH 2 / 3, removing pyridine and then saturated NaHCO3 (aqueous solution) to avoid low pH during the evaporation step. The organic matter was dried, and the solvent was switched to heptane to obtain the Stage 1 precipitate. The solid was collected by filtration and dried in vacuum to obtain pure Stage 1 (87% yield, over 95% purity by NMR).

[0281] Stability tests were conducted, and it was confirmed that the reaction mixture was stable overnight when stirred with 20% citric acid and saturated NaHCO3. The product was found to be stable when oven-dried at 40°C and 60°C. Scale expansion

[0282] The Stage 1 reaction was successfully scaled up to process over 100 g of 4-hydroxyindole. The reaction proceeded as expected and was work-treated to yield the Stage 1 product (93% yield, approximately 98% NMR purity). GMP raw material synthesis

[0283] A large-scale Stage 1 reaction was performed to supply GMP starting material (processing of over 500 g of 4-hydroxyindole). The reaction proceeded as expected, yielding consumption of the starting material by HPLC (99.2% product, less than 0.1% starting material). The reaction was work-processed using established methods, and the Stage 1 product was obtained after drying (94% yield, 99.1% by HPLC, 99% by NMR assay).

[0284] During development, it was also noted that the Stage 1 method is effective in removing trace impurities present in several batches of 4-hydroxyindole. Low levels of impurities present in 4-hydroxyindole were completely removed after the Stage 1 reaction, yielding clean material with high yield (89%) and purity (99% by HPLC and 99% by NMR assay).

[0285] experiment

[0286] Under N2 conditions, 4-hydroxyindole (1 equivalent limiting reagent) was added to the container, followed by DCM (dichloromethane; 6 volumes based on the amount of 4-hydroxyindole added). The reaction mixture was cooled to 0-5°C, and pyridine was added dropwise at 0-5°C (1.2 equivalents). Acetic anhydride (1.1 equivalents) was added dropwise at 0-5°C, and the reaction mixture was heated to 20-25°C for 1-1.5 hours, followed by stirring at 20-25°C for a further 3 hours. Samples of the reaction mixture were taken and analyzed for completion. The reaction mixture was then washed three times with 20% citric acid aqueous solution (3 × 3 volumes based on the amount of 4-hydroxyindole added) and once with saturated NaHCO3 (3 volumes based on the amount of 4-hydroxyindole added). The DCM solution was dried with MgSO4, filtered, and the DCM layer was concentrated to half its volume by distillation. Heptane (6 volumes based on the amount of 4-hydroxyindole added) was added, and further DCM was removed by distillation until complete precipitation of Stage 1 occurred. The reaction mixture was cooled to 15-25°C, the solid was collected by filtration, washed with heptane (1 volume based on the amount of 4-hydroxyindole added), and dried overnight at 60°C under vacuum.

[0287] The differences and advantages from JNP can be summarized as follows: i) The applicant washed away the pyridine using citric acid at a pH of approximately 2-3. This promoted improved isolation and crystallization. In fact, the DCM phase was separated, and the aqueous citric acid phase was discarded. ii) Further washing in sodium bicarbonate resulted in further improvement. iii) By replacing the solvent with heptane, solid precipitation was improved, the yield was maximized, and a reproducible high-purity Stage 1 was obtained. [Example 3] Stage 2 (Figure 3) Process i - Formation of acid chlorides

[0288] To determine whether it is a viable alternative to the volatile and highly flammable Et2O used in the literature, we first attempted to form reactive intermediate 2A by reacting the Stage 1 product with oxalyl chloride (1.5 equivalents) in a mixture of TBME and THF (6 vol / 1 vol). The reaction was completed after approximately 18 hours with a solubility profile similar to that of Et2O (precipitation of Stage 1 and Stage 2A in solution).

[0289] Because the acid chloride intermediate is easily hydrolyzed, leading to inconsistent analytical results, we developed a method to prepare and analyze more robust samples by quenching the reaction in THF / NMe2 (to give Stage 2) and then analyzing them by HPLC.

[0290] To obtain the highest purity and yield intermediate, the ratio of TBME to THF was optimized, and a preferred ratio of 6:1 TBME:THF was selected for scalability. Other ratios of TBME:THF may be used.

[0291] The large-scale reaction was carried out using a preferred solvent mixture (1 volt THF, 6 volt TBME), with the addition of oxalyl chloride performed at 30–35°C. The resulting solution was then heated at 40°C for 2.5 hours, completing the reaction with approximately 1% of the Stage 1 product remaining. Performing the addition while the solution was hot ensured a high reaction rate and resulted in an improved level of completion with a much shorter reaction time (2.5 hours vs. overnight). The product was still observed to precipitate at approximately 15 minutes after heating, and no adverse effects on the reaction profile were observed by HPLC.

[0292] Since the stability of intermediate 2A was unknown, we attempted to isolate the intermediate and telescope the material to stage 2 rather than risk decomposition (hydrolysis). The reaction profile was complex, with multiple components present at low levels. TBME was added, and the precipitate was collected. However, HPLC / NMR revealed that this was also a complex mixture.

[0293] Due to the poor reaction profile, it was deemed necessary to isolate intermediate 2A to allow for purification that would avoid the presence of excess oxalyl chloride. The reaction was repeated, and a yellow precipitate was collected by filtration and washed with TBME to remove excess oxalyl chloride (80% yield). NMR analysis confirmed that the product was of sufficient purity (approximately 95% according to NMR). However, despite storage under nitrogen, some degradation was observed over the following days, giving rise to partial hydrolysis including deprotection of the acetate group.

[0294] To investigate and mitigate the possibility of hydrolysis of the intermediate acid chloride during isolation, further research was conducted on telescoping techniques. It was found that the TBME liquid could be easily decanted by allowing the reaction mixture to precipitate, and then, in a similar manner, the remaining solid could be washed away using a further portion of the TBME. This made it possible to purify intermediate 2A without excess oxalyl chloride while minimizing exposure to water.

[0295] Some of the intermediate dissolves in the THF / TBME mixture, resulting in a loss of some reaction yield. It appeared that a solution could be obtained. This was confirmed by the addition of heptane to the decantation liquid, which yielded a further solid precipitate. To constrain this solubility, heptane (8 volumes) was added before decantation. Rather than washing the solid with TBME, heptane was also used for washing (3 × 6 volumes), which maximized the yield while maintaining high purity of the intermediate. This methodology has been successfully scaled up and is a preferred process. Step ii - Reaction with dimethylamine

[0296] The literature (Synthesis, 1999, 6, 935-938; DENichols) suggested that HNMe2 gas is effective for this conversion. However, to simplify large-scale processing, this was replaced with either solid HNMe2.HCl with additional excess base, or a solution of HNMe2 in THF. JNP uses HNMe2 in the presence of excess base (pyridine).

[0297] To optimize the reaction with dimethylamine through a series of test reactions, the initially isolated intermediate 2A was used (see Table 11).

[0298] [Table 11]

[0299] The conditions using pyridine (#1), as provided in the literature, were tested, along with similar reactions in Et2O (#3), a biphasic reaction using Me2NH.HCl and aqueous K2CO3 (#2), and a reaction using excess 2M Me2NH in THF (#4). In all cases, the main component by HPLC was the desired product, and the conditions using aqueous base and excess Me2NH were generally much cleaner than those using pyridine. Significant hydrolysis products were observed in all cases, which were thought to be the result of unreacted intermediate 2A being quenched during sample preparation for HPLC analysis. The reactions were work-treated by adding water, and then the organic solvent was removed under vacuum to precipitate the solid.

[0300] Reaction with excess amine resulted in a much improved impurity profile, higher yield (72% vs. 63%) and purity (98% vs. 90%). This approach limited the water content in the reaction and thus minimized the opportunity for hydrolysis to occur. It was also anticipated that purification would be easier due to the absence of pyridine in the isolated solid. For these reasons, conditions using excess HNMe2 as the base were chosen for scaling up.

[0301] The reaction in Et2O yielded a clean (#4) profile. However, it became clear that switching to a less volatile solvent such as TBME would be advantageous for facilitating large-scale processing. This would facilitate telescoping this acid chloride into the reaction. For these reasons, the reaction was chosen to be carried out in TBME using excess 2M Me2NH in THF.

[0302] The addition of water was thought to aid in workup by solubilizing the HNMe2.HCl salt, which was present and resulted in a very concentrated mixture and slow filtration. This was attempted. However, adding water to the reaction in TBME and THF resulted in poor recovery rates, and analysis of the liquid revealed further impurities and extensive acetate deprotection (phenol products). Therefore, further development of purification methods was needed. Purification development

[0303] It was desirable to develop a purification strategy to remove hydrolysis products and other observed impurities. It was also desirable to include water in the crystallization process to reduce the salt content of the isolated material (assuming HNMe2.HCl). For this purpose, a series of 15 solvents and solvent mixtures were screened (100 mg scale, 10 volumes of solvent, heating and cycling up to 60°C).

[0304] [Table 12]

[0305] * Because it was a concentrated suspension and solid particles adhered to the glass vial, the recovery rate is not representative.

[0306] From the solvents screened, acetone / water yielded recrystallized material with almost no solubility observed at room temperature. Since this was an aqueous system, it had the advantage of being useful for purging Me2NH.HCl from the solid.

[0307] The acetone / water recrystallization process was scaled up. A solution was obtained at the temperature before the addition of further water (4 volumes) (5 volumes acetone, 1 volume water), and the mixture was cooled to room temperature to allow crystallization (62% recovery, over 99% HPLC purity). Subsequently, this process was further scaled up by adding more water to aid recovery (total 5 volumes acetone, 10 volumes water, 78% recovery).

[0308] This process was further scaled up (30g), and the crude solid was obtained through recrystallization. The purity of the product was high, but there was a decrease in yield (56% yield, 99% by NMR assay, and 99.4% by HPLC).

[0309] To improve the recovery rate, the amount of water added was further increased from 10 to 15 volumes. This maintained a product purity above 99% and, at a small scale, yielded a higher recovery rate (90% recovery rate, compared to the previously observed 56-70%). However, scaling up this modified method again resulted in a low recovery rate (58% yield). Therefore, due to the problems encountered when scaling up recrystallization, we explored alternative purification methods based on the initial slurry screening performed (Table 12 above).

[0310] Using materials isolated from a large-scale Stage 2 reaction, a redevelopment of the purification strategy was carried out. The reaction proceeded as expected, yielding a crude product after oven drying (70% and 79% active material yields according to NMR assay). To remove a significant salt component (estimated to be HNMe2.HCl), a portion was slurryed with water at room temperature. After drying, this yielded a 75% recovery (95% according to NMR assay), demonstrating that this is an effective means of reducing salt content. HPLC purity remained unchanged at approximately 93%. Methods for increasing the chemical purity of the solid were then explored.

[0311] From the initial screening, EtOH:H2O, IPA, and acetone:H2O all appeared to yield high-purity products with excellent recovery rates, so these solvent systems were selected for further study. The input purity was 92.7%, with the main impurities at levels of 1.4%, 1.0%, and 0.8%, respectively.

[0312] [Table 13]

[0313] * The reaction was initially carried out in 5 volumes of 1:1 IPA:H2O. However, it became too concentrated to stir, so an additional 2 volumes of IPA were added.

[0314] ** The mixture was concentrated, and the present solids were extremely fine, so some of the solids passed through the filter, making filtration difficult.

[0315] The results of these tests suggested that good recovery was possible from these systems, particularly the IPA-based system. EtOH:H2O gave a slightly better impurity profile than IPA alone, but the recovery rate was not as good (79% vs. 90%). The impurity profile with IPA was significantly enhanced by the presence of water (98.7% vs. approximately 97.5%), but this resulted in a lower recovery rate (79% vs. 90%). This suggested a certain level of water solubility for this compound. To determine if a balance could be found that yielded high purity and approximately 90% recovery, final tests were conducted with reduced water volume using IPA and EtOH:H2O in water. This system improved the yield, but filtration was slow, and therefore, further solvent mixtures were also evaluated.

[0316] [Table 14]

[0317] The 5-volume EtOH / water slurry was extremely concentrated and difficult to handle. Since the purity of the solids was consistent across all tests (slight variations were likely due to the quality of filtration and washing), the 100% IPA condition was scaled up because it yielded a high recovery rate and the resulting suspension was easy to handle.

[0318] The initial scaling up of the preferred slurry yielded 96.4% HPLC purity (impurity levels of 1.2%, 0.7%, and 0.4%) (92% recovery). Liquid analysis showed that they were rich in all major impurities – 72% by HPLC (8.6%, 3.8%, and 3.4%). This was considered a suitable purification method giving high recovery, so the use of this material was tested in the following stages to ensure that impurity tracking and removal were achieved downstream (over 99% in Stage 3, with no single impurities exceeding 0.5%).

[0319] It was found that the slurry could be expanded when the remaining crude Stage 2 material (70% assay) was slurryed with water to remove inorganic substances, and then slurryed in IPA to give material of improved purity (97% by NMR assay, 76% yield compared to Stage 2, 96.8% by HPLC, with impurities at 1.1%, 0.8%, and 0.4%). GMP raw material synthesis

[0320] To provide the GMP campaign, a large-scale, Stage 2 reaction was performed. The reaction proceeded as expected, yielding a crude product, which was slurryed with water and filtered to give Stage 2, which was 93% pure by HPLC. This was further slurryed in 8 volumes of IPA and filtered to give the Stage 2 product (93.7% by HPLC, 92% assay, 66% active substance yield). Since the purity obtained was lower than that observed during the development campaign, a usage test was performed to confirm that the obtained high-purity Stage 3 was suitable for subsequent processing (GMP raw material).

[0321] Under identical conditions, a second batch was performed to obtain the crude product, which was 90% pure by HPLC after aqueous slurrying. This material was subsequently slurryed with water and purified by IPA slurrying to give 384 g of Stage 2 product (93.0% by HPLC, 91% by NMR assay, 60% active substance yield).

[0322] A third batch was performed to supply GMP synthesis again. The crude product was successfully purified by water, followed by IPA slurrying, giving Stage 2 (79% yield) with increased purity (97.3% by HPLC, 96% by NMR assay) compared to the previous batch.

[0323] experiment

[0324] After adding Stage 1 (1 equivalent limiting reagent) to a container under N2 conditions, THF (1 volume relative to the amount added in Stage 1) and TBME (6 volumes relative to the amount added in Stage 1) were added. Then, oxalyl chloride was added dropwise to the container (1.5 equivalents), generating heat to initially raise the temperature to 35-40°C, and then cooled as needed to maintain the temperature at 35-40°C. Immediately after addition, the reaction mixture was heated to 40°C and stirred for 2-6 hours. A sample of the reaction mixture was taken and analyzed for completion, then cooled to room temperature, and heptane (8 volumes relative to the amount added in Stage 1) was added, resulting in further solid precipitation. The reaction mixture was stirred for 10 minutes, and then the solid was allowed to settle. Most of the solvent was removed from the solid by decantation, and then the solid was washed twice with heptane (6 volumes × 2 relative to the amount added in Stage 1), with decantation performed similarly after each wash. A sample of the solid was then taken and analyzed. TBME was added to a container (4 volumes relative to the amount added in Stage 1) to obtain a yellow slurry, which was cooled to -20°C using a dry ice / acetone bath. While maintaining the temperature at -20°C to -10°C, a 2M solution of Me2NH in THF (2 equivalents) was added dropwise to the container over approximately 15 minutes. The reaction mixture was slowly warmed to room temperature and stirred overnight. If necessary, further Me2NH could be added at this point. Samples of the reaction mixture were taken and analyzed for completion. The reaction mixture was filtered and washed with heptane (2 volumes × 2 relative to the amount added in Stage 1), and the isolated solid was dried under vacuum at 60°C. Crude Stage 2 was slurryed in water (8 volumes relative to the amount added in Stage 1) for 2 to 18 hours, then filtered and washed with water (2 volumes relative to the amount added in Stage 1). The solid was dried under vacuum at 60°C to obtain crude Stage 2 containing less than 2% w / w water (measured by Karl Fischer titration (KF)). The crude Stage 2 was slurryed in IPA (10 volumes) for 2 to 18 hours, then filtered, washed with IPA (1 volume relative to the mass of crude Stage 2), and oven-dried at 60°C under vacuum.

[0325] The differences and advantages from JNP can be summarized as follows: Process 1 i) Firstly, the use of the THF / TBME solvent system as an alternative to diethyl ether resulted in lower volatility and flammability. ii) Secondly, the addition of oxalyl chloride is carried out at an elevated temperature, and the mixture is heated to 40°C. Improved solubility resulted in suppression of the capture of Stage 1 product during precipitation. This also led to a higher reaction rate, improved completion levels, and shorter reaction times. iii) Thirdly, intermediate 2A was isolated to enable purification that would eliminate excess oxalyl chloride. iv) Fourthly, heptane was added to aid in the precipitation of intermediate 2A. Process 2 v) By ensuring that the amine is used in excess, a minimum amount of water is present, and therefore hydrolysis is reduced, resulting in significantly improved purity and yield. vi) Finally, the use of water and IPA slurry yielded a Stage 2 product of good purity. [Example 4] Stage 3

[0326] The first experimental Stage 3 reaction was carried out using the purified Stage 2 material (over 99% by HPLC) and the provided reaction conditions. Since the Stage 2 material was found to be almost insoluble in THF, back-addition was performed rather than adding the Stage 2 solution to LiAlH4. Four equivalents of LiAlH4 were used as a 1M solution in THF and added at 20-25°C for approximately 2 hours. At this point, 10% product was observed, and several intermediate species were present. The reaction was heated under reflux for approximately 7 hours to give a 93% product conversion (by HPLC). The reaction was work-up to give the crude Stage 3 product (approximately 90% by HPLC, approximately 90% by NMR, and a corrected yield of approximately 87%).

[0327] A test reaction was carried out, and the amount of LiAlH4 added was successfully reduced (3 equivalents vs. 4 equivalents). This was expected to be helpful for workup by reducing the amount of Li and Al salts produced. After heating under reflux for a long time (10-18 hours), most of the reaction intermediate was consumed (2-3% remaining), and by HPLC, approximately 95% was the product. Development of post-processing

[0328] The first experimental reaction was successfully work-finished using Rochelle salt, but the volume used was extremely large (approximately 100 volumes), and this method would not be a viable process for scaling up. Various alternative work-finishing methods were investigated to test and reduce the required volume, and to assist in the removal of the Li / Al salt.

[0329] Quenching was attempted with a reduced volume using ethyl acetate, followed by Rochelle salt. A gray solid was present as a thick paste that settled at the bottom of the flask. Filtration failed, but the liquid could be decanted, and the solid was slurred again in THF / ethyl acetate to extract the product. The product was then isolated by aqueous workup and concentration. This yielded a product of good purity (90-95% according to NMR) in good yield (94% without correction for purity). However, this process was not immediately suitable for large-scale production.

[0330] The reaction was quenched by adding siRNA, followed by saturated Na2SO4, in the presence of anhydrous Na2SO4 to act as a binder. The reaction yielded a granular solid that could be easily filtered. The product was then isolated by aqueous workup and concentration. A good yield was obtained (approximately 94% without correction for purity), but the product contained higher levels of major impurities according to NMR (10% vs. the commonly observed 2-4%).

[0331] The reaction was quenched with 20% AcOH at 0°C, resulting in the formation of an unfilterable gel. This reaction was abandoned.

[0332] The reaction was then quenched with Â, followed by 20% citric acid, yielding a solid that could be separated by filtration. The liquid was concentrated to obtain the product. This method yielded a slightly lower yield (approximately 77% without correction for purity), but the product was of very high purity (>95%).

[0333] The reaction was further quenched by adding HCl, followed by water (3 mL per 1 g of LiAlH4 in THF). A gel that could not be easily filtered formed, and this reaction was abandoned.

[0334] Finally, the reaction was quenched by the Feiser method. Water (1 mL per 1 g of LiAlH4), then 15% NaOH (1 mL per 1 g of LiAlH4), and finally water (3 mL per 1 g of LiAlH4) were added. This gave a solid that could be filtered from the reaction mixture. The liquid was then partitioned and concentrated under vacuum (87% yield, 90-95% according to NMR).

[0335] These experiments are summarized in Table 15 below.

[0336] [Table 15]

[0337] Quenching with both citric acid and NaOH allowed for easy filtration of the reaction mixture, yielding a solid requiring minimal solvent volume. The NaOH-based condition yielded a higher yield (approximately 10%), but the product obtained was of lower purity and may require further purification before use in subsequent stages. The lower yield with citric acid was likely due to some precipitation of the citrate product, which had a purifying effect, yielding a clean product immediately after concentration. Selecting these conditions for scaling up and further optimizing the amount of citric acid added was expected to enable the isolation of a clean product in high yield from this process.

[0338] To attempt recovery and maximize it, the reaction was repeated using a slightly reduced amount of added citrate. This reaction yielded the product in 57% yield, and a further 20% yield was obtained by restrush of the filtered cake in THF (97.7% for both samples according to HPLC).

[0339] This reaction was scaled up. However, during the HCl quench, where the reactants had previously been observed to become concentrated, the reactants became gummy in the flask, forming a thick clump that restricted mixing. The addition of citric acid subsequently resulted in a normal slurry / gel, but this did not constitute a viable process. The reaction was work-treated to maximize recovery by slurring the filtered cake again in THF, giving a 76% active material yield and 95.0% by HPLC.

[0340] The reaction was repeated to develop a better quench and avoid the formation of the gum-like substance observed with siRNA. Partial quenching was performed by adding acetone, which resulted in a readily agitated suspension / emulsion with no signs of thickening. Citric acid treatment was then carried out to obtain a filterable mixture. This quenching was successfully performed on the remainder of the reaction and post-treatment yielded the crude product in good yield (71% assay, 82% corrected yield, 98.0% by HPLC).

[0341] After quenching, the reaction mixture was generally found to be at pH 8 / 9. As part of the workup optimization process, different pH levels were investigated. The reactants were divided for workup; half received a slightly reduced amount of citrate (to obtain pH 11 / 12 after quenching), and the other half was brought to pH 7 by the addition of further citrate. The pH 11 reaction was workup to obtain material for an 85% NMR assay (73% yield), and the pH 7 reaction gave a 60% NMR assay (62% yield). It was clear from these results that obtaining the correct pH after quenching was crucial for yielding above 70%. By slightly reducing the amount of citrate added (approximately 2 volumes of 20% citrate), an increase of approximately 8% in yield was obtained. With this information, the pH of future reactions was monitored during quenching to ensure that the mixture remained strongly basic. Purification development

[0342] Purification screening was performed using 100 mg of the crude psilocine product, which had been slurryed in 10 volumes of solvent under a heating cycle of 60°C. The slurry was cooled to room temperature over the weekend, and all solids were collected by filtration. Stability to acids and bases was also tested for the purpose of performing acid / base posttreatment. The screening results are shown in Table 16 below.

[0343] [Table 16]

[0344] The first of the three highlighted impurities coincided with the most stable reaction intermediate (requiring reflux for conversion to the product), observed at approximately 70% when the addition of LiAlH4 was complete. The third impurity was not present in the inputs and appeared to be generated during the slurrying procedure. Of the solvent remaining in the slurry, iPrOAc provided the highest purity. Although some recrystallization was observed, MeCN had the potential to remove impurities during crystallization and had a recovery rate that could be improved during development. Some decomposition was observed with both acid and base, and the KOH sample rapidly turned black.

[0345] Two of the most promising solvents (MeCN and i The scale of purification of crude stage 3 material was increased using PrOAc. To improve recovery rates, the solvent volume was reduced to a minimum. The results of these trials are shown in Table 17 below.

[0346] [Table 17]

[0347] Recrystallization was achieved from 5 volumes of MeCN, and thermal slurry formation was achieved in 3 volumes of iPrOAc. The results were obtained (all at 75°C). Regardless of the reduced volume, the recovery rate from MeCN was similarly poor, but the product was of extremely high purity (>95% according to NMR). The recovery rate from iPrOAc was better, and analysis by NMR showed a significant increase in product purity (approximately 95%).

[0348] Although the HPLC and NMR purity of the material from the iPrOAc slurry was high, low assay values ​​(85% by NMR assay) were observed. To improve the assay values ​​of the material and to remove the color (all materials obtained so far were strongly purple, green, or brown), purification with silica pads was investigated.

[0349] Crude psilocin (71% assay, 98.0% by HPLC) was passed through 4 equivalents of silica and eluted with THF. A grayish-white solid with 80% recovery was obtained, having slightly improved HPLC purity (98.4%) and assay value (approximately 82% assay). This proved to be an effective means of increasing the assay value of the product and was therefore included as part of the post-reaction treatment.

[0350] Next, in order to attempt and improve the recovery rate while maintaining chemical purity (input purity 98.4%), a series of tests were conducted using silica-treated input material (100 mg / slurry). i The PrOAc / reverse solvent slurry (Table 18) was used.

[0351] [Table 18]

[0352] Since all purity values ​​were equivalent, two solvent systems were selected for scaling based on the best recovery rate obtained. To better evaluate the recovery rate, the scale of two preferred slurries (TBME and toluene as reverse solvents) was increased (1.0 g per slurry).

[0353] [Table 19]

[0354] It provides material with over 99% HPLC purity with a recovery rate of approximately 80%, and is combined with a silica pad. All of these options presented appear to offer effective means of purification of the psilocine product. During slurrying, further color migrated into the liquid, and psilocine was obtained as a white solid. All impurities were effectively removed to less than 0.5%. Since a non-toxic ICH class 3 solvent was used, for scaling up, i I selected PrOAc:TBME slurry. Scale expansion

[0355] The developed Stage 3 conditions were scaled up, and the reaction proceeded. After refluxing overnight, a 94.4% product was obtained with 2.9% reaction intermediates present, as measured by HPLC (typical for this process). Following the silica pad, psilocine was obtained with 83% purity, 66% active material yield, and 97.0% purity, as measured by NMR assay. This material was further purified by slurrying in iPrOAc / TBME to obtain a 62% yield, with 100% purity by NMR assay and 99.7% purity by HPLC.

[0356] Because the crude yield obtained from this reaction was lower than expected (66% vs. approximately 75%), the filtered cake and silica pad were tested again to try and recover more material. However, this was unsuccessful.

[0357] The lower-than-expected yield may have been due to product degradation during workup, but pre-treatment stress tests indicated the material was stable under the conditions used. To investigate this further, the reaction was repeated. To confirm that no product degradation occurred during workup, the crude product was isolated before the silica pad and samples were taken for further stress testing.

[0358] The reaction proceeded as expected and completed (93.7% product, 2.9% intermediate), was concentrated to give crude material (77% NMR assay, 66% active material yield). The filtered cake was again slurred in THF / MeOH, but no significant psilocine was isolated. Further citrate was added to lower the pH from 8 to 4 (to try and replace all products coordinated to the aluminum salt), and the cake was again slurred in THF, but again no significant psilocine was isolated. No mass balance was obtained from the reaction, and the 66% active material yield was in good agreement with previously obtained results. Silica pad and i This batch was purified by slurring in PrOAc / TBME to obtain a high-purity material with a 62% yield (99.8% according to HPLC).

[0359] Despite the relatively large volume of solvent used and the need for silica pads to remove aluminum and lithium species, this process remained highly suitable for the required scale.

[0360] The Stage 3 reaction was further scaled up for processing. The reaction proceeded as expected and was completed after 18 hours (approximately 91% product, approximately 3% reaction intermediate remaining). Workup with silica pads and slurry yielded high-purity psilocin in 57% yield (over 99% by HPLC, 99% by NMR assay, 0.35% w / w water Karl Fischer).

[0361] experiment

[0362] Stage 2 (1 equivalent limiting reagent), followed by THF (5 volumes relative to the amount of Stage 2 added), was added to the container. The mixture was cooled to 0°C, and while maintaining the temperature at 0–20°C, a 1 M THF solution of LiAlH4 (3 equivalents) was added dropwise over 30–45 minutes. After the addition, the reaction mixture was stirred at 10–20°C for 30 minutes, then heated under reflux and stirred for approximately 16 hours. A sample of the reaction mixture was taken, analyzed for completion, cooled to 0°C, and quenched by dropwise addition of acetone (9.3 equivalents) at 0–30°C, followed by 20% citric acid aqueous solution (1.9 volumes relative to the amount of Stage 2 added) at 0–30°C. The pH of the addition was monitored to ensure that the pH was maintained > 11, and the addition was stopped early if necessary. The resulting suspension was stirred for 1 hour, filtered, and washed with THF (2 volumes relative to the amount of Stage 2 added) to remove Li and Al salts. For approximately 1 hour, the filtration cake was slurried in THF (12.5 volumes relative to the amount added in Stage 2), filtered, washed with THF (5 volumes relative to the amount added in Stage 2), and the products were recovered from the Li and Al salts. The combined organic matter was dried with MgSO4 and filtered. The filtrate was evaporated under vacuum until approximately 10 volumes remained (relative to the amount added in Stage 2), and this solution was applied to a silica pad (3 equivalents relative to the amount added in Stage 2). The silica pad was eluted with THF, the product fractions were combined, and evaporated under vacuum until dry. For 2 to 18 hours, the crude Stage 3 (psilocine) was slurried in 1:1 iPrOAc:TBME (5 volumes relative to the mass in Step 18), filtered, washed with TBME (2.5 volumes relative to the mass in Step 18), and dried under vacuum at 40°C to isolate pure psilocine.

[0363] The differences and advantages from JNP can be summarized as follows: i) Firstly, the applicant used THF as the solvent, but quenched the reaching with acetone. This resulted in a suspension / emulsion without concentration. ii) Secondly, the applicant quenched with citrate while maintaining a basic pH, typically around 11. pH adjustment ensured that high yields could be obtained. iii) Thirdly, after purification with a silica pad to remove the remaining Li / Al salt, elution with THF and slurrying with iPrOAc:TBME then yield a highly purified product that is subsequently dried. [Example 5] Stage 4

[0364] To process a 2.58 g sample, analysis was first performed by HPLC using literature conditions, yielding approximately 88% conversion to intermediate 4A. The product was purified by adding aminopropyl silica and filtration through Celite. The resulting green oily substance (5.5 g) was slurryed in DCM, yielding a precipitate of benzyl transition and zwitterionic stage 4 (4.1 g, 70% yield, approximately 95% according to NMR). Process i

[0365] The initial development at this stage is to make it easier to handle and, ideally, to avoid introducing additional lithium during synthesis. n The focus was on finding a substitute for BuLi. The following base: Li t BuO, K t Initial screening was performed under different conditions including BuO, NaH, NaHMDS, and NaNH2. All reactions yielded products, and NaHMDS did not. n It functioned just as well as BuLi. All of the reactants became extremely concentrated, gelation was observed, and overhead stirring was recommended for efficient mixing.

[0366] The initial screening was for NaHMDS n This suggests that it is a suitable substitute for BuLi (81% conversion to product / intermediate 4A). These conditions indicate that n The reaction was scaled up to 1.5 g in parallel with the reference reaction using BuLi. Overhead stirring was used in both cases.

[0367] [Table 20] Abbreviations used in the table: St 3 = Stage 3, Int 4A = Intermediate 4A, St 4 = Stage 4

[0368] The reaction profiles obtained in both cases were remarkably similar to those of the NaHMDS reaction consuming Stage 3. Both reactants were filtered through Celite to remove the white precipitate and then concentrated. According to NMR, ( n In the example using BuLi, in particular, excess benzyl protons were present in both cases, and the isolated yield was greater than 100%. The NaHMDS conditions gave a favorable reaction profile and proved successful, so they were selected for further scaling up. However, workup and purification development were necessary. Process ii

[0369] HPLC data were obtained from materials isolated from the above trial using NaHMDS and n We demonstrated that the rearrangement of BuLi and subsequent concentration yielded a zwitterionic stage 4. Purification to remove benzyl phosphate by-products and other impurities from this material was attempted by slurring in numerous solvents.

[0370] [Table 21] Abbreviations used in the table: St 4 = Stage 4

[0371] * Poor filtration

[0372] White solids were obtained from several solvents, but the solids obtained from DCM and TBME transformed into a pale purple, gum-like substance when stored over the weekend. The white solids obtained from IPA and acetone remained free-flowing white solids during storage, suggesting that these solids may be more stable and easier to handle.

[0373] The slurry in IPA and acetone was scaled up to 1 g. However, gumming was observed immediately upon addition of the solvent. Vigorous stirring slowly dispersed the gumming substance, eventually showing signs of crystallization, and a white slurry was formed after overnight stirring. However, this process was not suitable for scaling up. The solid was isolated in good yield using IPA, yielding the highest purity.

[0374] THF was also investigated because it had the advantage of being a reaction solvent. However, when this was tried, the initial gum formation was observed again (isolated in approximately 80% yield, approximately 92% by HPLC). To attempt to avoid gum-like substance formation and give more controlled crystallization, crude Stage 4 was first solubilized in a small volume of DMSO (2 volumes). Then, THF (10 volumes) was added to this, and the solution was stirred over the weekend. This slowly gave a precipitate of the product, which was collected by filtration and washed with THF to give Stage 4 (86% yield) in 96% HPLC purity (over 95% by NMR).

[0375] Since THF crystallization was successful and complete conversion to zwitterionic stage 4 occurred during concentration of the reaction mixture (THF / siRNA) at 40°C, it was expected that solvent changes could be avoided and the product could be directly crystallized by stirring the reaction mixture at 40°C.

[0376] Using both reactions, two 4g NaHMDS reactions were carried out, completing with approximately 80% conversion to intermediate 4A. One reactant was diluted with RINKAN and the other with THF, and both were filtered to remove phosphate byproducts. Brine washing was performed to further reduce phosphate impurity levels, the organic matter was dried, and the solutions were concentrated to 10 volumes. These solutions were stirred overnight at 40°C to yield conversion to stage 4 and precipitation (approximately 1% stage 3, approximately 0.2% intermediate 4A, approximately 82% stage 4). Solids were collected by filtration, yielding 8.03g (88% yield) from RINKAN / THF and 5.65g (62% yield) from THF. The brown / gray solid obtained from RINKAN / THF was less pure (approximately 90% and 78% assay according to HPLC) compared to the white solid obtained from THF (97% and 88% assay according to HPLC). Analysis of the aqueous layer from the THF reaction indicated the presence of the product, and further loss occurred in the final THF filtrate.

[0377] Due to the higher purity obtained from THF, this solvent was further investigated to optimize recovery. Due to product loss into the aqueous layer, brine washing was omitted, and the reaction mixture was further concentrated after the reaction to minimize loss during the final filtration step. This new method was tested on a 75g scale by concentrating portions of the reaction mixture to 8 and 6 volumes. Upon filtration, no difference in yield was observed between the two portions, with a total yield of 140.4g (90% and 74% active product yield according to NMR assay, and 90% according to HPLC). Tracking impurities

[0378] Three main impurities were observed in the isolated product, and the identities of two of these species were proposed based on MS data.

[0379] Debenzylated impurities (typically about 2–5% by HPLC) were shown to yield psilocybin during subsequent hydrogenation and are therefore acceptable at higher levels. The main observed impurities in isolated Stage 4 (typically about 5–8% by HPLC) were anhydrous impurities. These were tracked through subsequent hydrogenation and were shown to be readily removed by recrystallization from water as highly soluble pyrophosphorate impurities resulting from debenzylation. Other major observed impurities (m / z 295.2 observed by LCMS) were found to be controlled to less than 2% by restricting the reaction temperature (below -50°C) and were not observed in psilocybin after hydrogenation.

[0380] The impurity profile of the 140g batch produced above showed 90.0% Stage 4, 6.4% anhydrous impurities, 0.2% N-debenzylated impurities, and 1.2% m / z 295.2 impurities. GMP synthesis

[0381] Using established methods, the first large-scale Stage 3 batch (544g input) was completed, yielding 213.5g (53% yield, 99% by HPLC). The second batch (628.2g input) was also successfully processed, yielding 295.2g (66% yield, 99% by HPLC).

[0382] Some variation in yield at this stage was observed across three large batches (57%, 53%, and 66%). This is likely a result of minor differences in post-treatment and quenching methods.

[0383] experiment

[0384] Stage 3 was added to a container, followed by THF (15 volumes relative to the amount of Stage 3 added), and the mixture was cooled to below -50°C using a dry ice / acetone bath. While maintaining a temperature below -45°C, and targeting below -50°C, 1.13 equivalents of 1 M NaHMDS solution in THF were added. The reaction mixture was stirred at -60 to -50°C for 30 minutes. While maintaining the reaction temperature below -30°C, tetrabenzyl pyrophosphate (2.26 equivalents) was added all at once, followed by an additional 20 volumes of THF. The reaction mixture was heated to 0°C for 1.5 to 2 hours, and a sample was taken to confirm completion. The reaction mixture was filtered to remove the phosphate salt while washing with THF (8 volumes). The filtrate was concentrated until 6 to 8 volumes remained, and stirred overnight at 40°C to convert intermediate 4A to the Stage 4 product. A sample of the reaction mixture was taken to confirm completion, then filtered, and the solid was washed with THF (2 volumes). The Stage 4 product was dried in a vacuum oven at 40°C.

[0385] The differences and advantages from JNP can be summarized as follows: Process i i) Firstly, sodium hexamethyldisilazane was introduced to assist in deprotonation. This proved to be a more manageable alternative to butyllithium, as it does not introduce further lithium during the reaction. ii) Secondly, by diluting the reactants with THF, a much higher purity intermediate 4A was obtained. iii) Thirdly, by controlling the reaction temperature to below -50°C, the undesirable mz295.2 observed by LCMS was controlled to a level of less than 2%. Process ii iv) Fourthly, by monitoring the levels of Stage 4A impurities, particularly N-debenzylated Stage 4 (Table 7) and anhydrous Stage 4 (Table 7), pure products can be reproducibly produced. v) The conversion of intermediate stage 4A to stage 4 can be carried out in the reaction solvent, avoiding the need for time-consuming solvent exchange. vi) Finally, the obtained solid is washed with THF and oven-dried to obtain Stage 4. [Example 6] Stage 5

[0386] If catalyst poisoning is observed during the development of this stage and it is necessary to include an activated carbon treatment (charcoal treatment) step in the process to suppress incomplete hydrogenation, This is possible. However, activated carbon treatment is not always required in the standard procedure.

[0387] After 3 hours of hydrogen spraying, the typical reaction showed a high level of completion (over 90% of the product, with 3-5% of the starting material remaining). A small amount of water was added to aid solubility, and after another hour of hydrogen spraying, stage 4 consumption was achieved.

[0388] The successful reaction was post-treated by filtration, followed by evaporation to remove methanol, leaving the product as a concentrated suspension in water. Ethanol was added, and the solid was filtered to obtain psilocybin in 69% yield. 1 1H NMR confirmed the identity of the product but indicated the presence of trace amounts of related impurities. LCMS analysis showed a purity of 95.2%, with the main impurity (4.1%) identified as pyrophosphate impurities (Table 7), originating from anhydrous impurities in Stage 4. This impurity was later shown to be effectively purged during the recrystallization of the final product (Stage 6).

[0389] HPLC analysis revealed that the Stage 4 material obtained from the final THF workup was 88.0% pure, contained 7.3% N-debenzylation Stage 4 (converted to the product), and free of anhydrous impurities. The reaction was then carried out further using this Stage 4 material. Completion was again confirmed, and the reaction was workup as described above to obtain psilocybin in 46% yield. The low yield was thought to be due to precipitation of the product during the catalytic filtration step. 1 1H NMR confirmed the identity of the product, and HPLC showed a purity of 98.9%.

[0390] Further development of the reaction conditions was carried out to optimize the amount of water used and minimize product loss during the filtration process. After the reaction, the solution was obtained by adding 10 volumes of water while heating to 40°C. This made it possible to remove the catalyst by filtration without causing product loss on the filter.

[0391] During the reaction and workup, some Stage 3 was generated by hydrolysis, with levels of approximately 1–2.5% appearing typical for this process. A decrease in Stage 3 levels was demonstrated during the final recrystallization of the product. Scale expansion

[0392] A large Stage 4 batch (non-GMP) was processed as a single batch (148 g of active substance input). Stage 4 consumption was achieved, along with 88% product and 0.9% Stage 3 due to hydrolysis. Anhydrous impurities (6.4%) were completely converted to the corresponding pyrophosphate impurities (5.2%).

[0393] The large hydrogen adduct was filtered and concentrated, and after evaporation from ethanol to reduce the water content, 109 g of crude product was obtained (approximately 71% and 86% yield according to NMR assay). experiment

[0394] 10% Pd / C (approximately 50% water-moistened, type 87L, 0.1 × Stage 4 addition) was added to the container under N2, followed by methanol (20 volumes for Stage 4) and Stage 4. N2 was replaced with H2, and the reaction was stirred under H2 (at atmospheric pressure) for 1-2 hours. A sample of the reaction was taken to confirm completion, and then water (10 volumes for Stage 4) was added while maintaining a temperature below 25°C. The mixture was stirred for a further 1-2 hours under H2 (at atmospheric pressure). A sample of the reaction was taken to confirm completion.

[0395] If the reaction was incomplete, H2 was added again and the reaction was continued for another 1 to 12 hours until completion was observed. Then, the reactants were placed under N2 and heated to 40°C for 15 to 12 hours. The reaction was maintained for 45 minutes. The reaction mixture was filtered through Celite to remove the catalyst and washed with methanol (13.3 volumes relative to the Stage 4 addition amount) and water (6.7 volumes relative to the Stage 4 addition amount). The filtrate was concentrated under vacuum and azeotropically heated with ethanol to remove water until a solid was obtained. The differences and advantages from JNP can be summarized as follows:

[0396] The reaction is primarily monitored by HPLC using relative retention time (RRT) for intermediate levels, with completion controlled by the presence of less than 0.2% intermediate. Stage 5 pyrophosphate impurities are also carefully monitored to ensure control during final recrystallization.

[0397] The final Stage 6 process is as described in Example 1. [Example 7] Test methodology and protocol

[0398] The following methodology / protocol was used to test for purity and other properties. 7.1 NMR

[0399] Using a 400MHz spectrometer, the psilocybin in D2O was analyzed. 1 H and 13 A 13C NMR spectrum was obtained. The chemical shifts were: 1 In 1H NMR, the ppm value is relative to D2O (□=4.75 ppm). 13 The 13C NMR spectrum is reported in ppm relative to MeOH (□=49.5 ppm) added as a reference. Literature values ​​for psilocybin are reported in JNP. NMR analysis of psilocybin was consistent with the structure and the literature reports, yielding data with only slight variations in the chemical shift relative to the proton near the ionizable group. However, this variation is expected, as the zwitterionic nature of the compound makes this material extremely sensitive to small changes in pH.

[0400] 1 1H NMR and 13The 13C NMR data is summarized below, and the spectra are shown in Figures 10 and 11.

[0401] 1 H NMR data (400MHz, D2O): 2.79 (s, 3H), 3.18 (t, J=7.4Hz, 2H), 3.31 (t, J=7.4Hz, 2H), 6.97 (d, J=8.0Hz, 1H), 7.08 (s, 1H), 7.10 (t, J=8.0Hz, 1H), 7.19 (d, 8.2Hz, 1H).

[0402] 13 ¹³C NMR data (400MHz, D2O (+ trace amounts of MeOH): 22.3 (1 x CH2), 43.4 (2 x CH3), 59.6 (1 x xCH2), 108.4 (1 x CH), 108.6 (1 x C), 109.5 (1 x CH), 119.1 (d, 3 J P-H =6.7Hz,1 x C), 123.3(1 x CH2), 124.8(1 x CH), 139.3(1 x C), 146.3(d, 2 J P-H =6.7Hz, 1 x C) 7.2 FT-IR

[0403] Data were collected using a Perkin Elmer Spectrum Two® Spectrometer equipped with the UATR Two accessory. Analysis of psilocybin (batch: AR755) by FT-IR spectroscopy yielded a spectrum (Figure 12) consistent with the proposed structure. 3244 cm⁻¹ -1 The broad peak is typical of amine salts. The rest of the peaks are within the fingerprint region and therefore cannot be attributed individually. 7.3.Mass spectrometry

[0404] The mass spectrum of psilocybin (AR755) was obtained using a Bruker Esquire 3000 plus Ion Trap Mass Spectrometer and was in agreement with the structure. The mass spectrum (Figure 13) shows the (M+H) of psilocybin. + and (2M+H) + The corresponding main peaks are shown at m / z = 284.8 and 568.1. This indicates that this molecular ion has the molecular formula (C) of psilocybin. 12 H 17 This suggests that it has an m / z 284 corresponding to N2O4P (Figure 14). 7.4 Ignition residue

[0405] The ignition residue method followed the pharmacopoeia method with one adjustment. Inconsistent results were obtained when the crucible was heated to 500°C. This is thought to be due to the low volatility of the phosphate residue produced. Therefore, for psilocybin, the temperature was increased to 800°C, and consistent and accurate results were obtained. 7.5 HPLC-Assay and Purity Measurement

[0406] The HPLC method used to quantify psilocybin, its chemical purity, and impurities was gradient HPLC-UV, and the conditions are summarized in Table 22. External standards were used for quantification. Approximately 1 mg / mL of psilocybin was dissolved in purified water:MeOH(95:5). Sonication was performed to ensure complete dissolution.

[0407] Purity obtained by HPLC is calculated based on area percentage and correlated to a known retention time standard.

[0408] HPLC assays are calculated on an anhydrous basis, based on weight % relative to standards of known purity and composition.

[0409] [Table 22] 7.6 Residual solvent content by HRGC

[0410] The HRGC method for quantifying residual solvent is a headspace method and is described in Table 23 below.

[0411] [Table 23]

[0412] The levels of the following solvents and reagents will be measured: methanol, ethanol, THF, and toluene. 7.7 Melting point according to DSC

[0413] DSC data were collected using a PerkinElmer Pyris 6000 DSC (or similar). The instrument was validated for energy and temperature calibration using certified indium. Samples were weighed (typically 0.5–3.0 mg) into pinhole aluminum sample pans. The pans were compressed using an aluminum pan cover. The pans were heated from 30–300°C at 20°C / min while purging with dry nitrogen at 20 mL / min. During the melting point method, each batch of psilocybin polymorph A or A' exhibited two endothermic events. The first endothermic event was attributed to the solid-solid transition of polymorph A or A' to polymorph B, and the second endothermic event was attributed to the melting of polymorph B. 7.8 Polymorphism using XRPD

[0414] The solid-state morphology of psilocybin is determined by XRPD. XRPD diffractograms were collected under ambient conditions using a diffractometer (PANalytical X'Pert PRO or equivalent) with Cu Kα radiation (45kV, 40mA), a θ-θ angle meter, a focusing mirror, a divergence slit (1 / 2 inch), and a solar slit (4mm) for both the incident and divergence beams. The acquisition range was 0.2°s. -1 The continuous scan speed was 3–35°²θ. The obtained diffractograms were compared to those of the reference diffractogram of polymorph A or A' to ensure agreement (Figure 7a or 7b, respectively). 7.9 Thermogravimetric analysis (TGA)

[0415] TGA data were collected using a PerkinElmer Pyris 1 TGA (or similar). The instrument was calibrated for temperature using certified weights and certified Alumel and Perkalloy equipment. A predetermined amount of sample (typically about 5 mg) was placed in an aluminum crucible and heated from ambient temperature to 400°C at 20°C / min. A nitrogen purge of 20 mL / min was maintained over the sample. 7.10 Loss on drying

[0416] Using a precisely weighed 1g portion, the sample is dried under vacuum at 70°C until it reaches a certain weight, and the weight loss during drying is measured in pairs.

[0417] Calculation:

number

[0418] To provide samples for evaluating analytical method selectivity, psilocybin drug substances were subjected to stress under various conditions in both solution and solid states.

[0419] A forced degradation test was performed on psilocybin in accordance with the requirements of ICH Q1A(R2). The test under stress conditions provided information regarding the possible degradation pathways and intrinsic stability of psilocybin. The optimized analytical method used demonstrated specificity for psilocybin. This method was demonstrated to be appropriate and can be used to detect changes in the identity, purity, and potency of the product. The method used was also shown to be compliant with ICH Q2(R1) (validation of analytical methods) in terms of specificity and free from interference from potential impurities and degradation products. Therefore, the HPLC method is considered appropriate for measuring the purity of psilocybin and associated impurities.

[0420] The control sample of psilocybin remained stable in solution throughout the study period (7 days for non-photostable samples). When heated in solution, psilocybin slowly decomposed, producing psilocine as the main impurity. Psilocybin was also stable at room temperature under acidic conditions. However, at 60°C, slow and steady decomposition was observed, producing psilocine as the main impurity. In the presence of a base, psilocybin was slightly unstable at room temperature and slowly decomposed into a variety of impurities over the study period. Under peroxide conditions, only very low levels of impurities were formed, and the overall purity decreased by approximately 0.5%. In the solid state, slow chemical decomposition was observed (3 days at 150°C), producing mainly psilocine (stage 3) as an impurity. Psilocybin was stable under photostable conditions, both in solid and in solution. Stability testing

[0421] As shown in Table 24, stability studies were initiated using two batches of psilocybin.

[0422] [Table 24]

[0423] The samples were double-bagged in food-grade polyethylene bags, sealed in an outer polyethylene container, and stored at 2-8°C, 25°C / 60%RH, and 45°C / 75%RH. A desiccant bag was placed between the inner polyethylene bags to prevent moisture absorption. Appearance, water content, purity, and assay tests were performed.

[0424] The protocols for the two trials are shown in Tables 25 and 26. The 1-month and 3-month stability data for batch GM764B are detailed in Tables 27 and 28 below. The 1-month, 3-month, 6-month, 9-month, and 12-month stability data for GMP batch 170231 are given in Tables 29, 30, 31, 32, and 33 below, respectively.

[0425] [Table 25]

[0426] [Table 26]

[0427] [Table 27]

[0428] [Table 28]

[0429] [Table 29]

[0430] [Table 30]

[0431] [Table 31]

[0432] [Table 32]

[0433] [Table 33]

[0434] Over the first 12 months of the ICH stability test, psilocybin proved to be chemically stable under low temperature (2–8°C), ambient (25°C / 60%RH), and accelerated (40°C / 75%RH) conditions. There were no changes in appearance, and HPLC analysis remained consistent. Water content varied in all samples, initially due to the initial effects, and subsequently due to the degradation of the desiccant bags used in the test. [Example 9] - Experiment for forming hydrate A

[0435] Psilocybin (200 mg), followed by deionized water (4 ml), was added to the crystallization tube. After equilibrating the mixture at 25°C for 2 hours, the solid was isolated by vacuum filtration. The material was divided into two equal parts. One part was not subjected to further drying to give hydrate A, lot GK2, by XRPD and DSC (diffractogram and thermogram corresponding to Figures 7d and 8d, respectively). [Example 10] - Experiment to form polymorph B

[0436] Psilocybin polymorph A (250 mg) was added to a round-bottom flask and heated to 173°C using an oil bath, maintaining this temperature for 5 minutes. The solid was cooled to ambient temperature and isolated to obtain lot GK3 with a 93% recovery rate. XRPD and DSC analysis (diffractogram and thermogram corresponding to Figures 7c and 8c, respectively) revealed that lot GK3 was polymorph B. [Example 11] - Solid State Inspection

[0437] Numerous polymorphism tests were completed. A summary of the solid forms found is shown in Figure 17. The majority of the forms found originated from solvent perturbations. In some cases, stoichiometric solvates were isolated, while in others, non-stoichiometric solvates were isolated. Slurry of polymorph A

[0438] As a primary pathway to modifying the solid form, and to visually evaluate the solubility of the material in 24 diverse solvents at 25–50°C, solvent-mediated equilibration of psilocybin pattern A was performed.

[0439] At room temperature, psilocybin pattern A (40 mg) was added to a tube, and the solvents listed in Table 34 were added in 0.4 ml (10 volts) increments until the total volume reached 1.2 ml (30 volts), and observations were recorded. The mixture was constantly stirred. The thermal cycle was performed as follows: 50°C for 18 hours, cooled to 20°C over 2 hours, aged for 4 hours, heated to 50°C for 4 hours, cooled to 20°C over 2 hours, aged for 18 hours. Repeated 50°C-20°C cycles over 24 hours were performed, with the following applied: If isolated after heating to 50°C and sufficient solid material was obtained, then it is the A series. If isolated after cooling to 20°C and sufficient solid material was obtained, then it is a B-series compound. All isolated solids were dried in a vacuum at 50°C for 24 hours and analyzed by XRPD. The observations are given in Table 34.

[0440] In 30 volumes of solvents and solvent mixtures tested at 50°C, the API was mostly insoluble, yielding heavy suspensions. Water solubilized psilocybin at 50°C.

[0441] [Table 34]

[0442] result:

[0443] In the figures (Figures 18 and 19), "25C" represents the isolation of the solid at 25°C, and "50C" represents the isolation of the solid at 50°C. For example, GM832-20_50_A9 represents GM832 entry 20 (MeCN) isolated at 50°C.

[0444] 50℃ slurry

[0445] Entries 1, 2, 3, 4, 5, 7, 8, 10, 11, 13, 14, 15, 16, 22, 23, 24: The XRPD diffractograms broadly matched polymorph A, but had an additional peak of intensity varying at 18°²θ.

[0446] Entries 6, 9, 12, 20: The XRPD diffractograms obtained for isolated solids were broadly matched (see Figure 18), with additional peaks observed at 10°2θ and 13.2°2θ for some samples. This XRPD diffractogram was denoted as pattern C. No species correlations were observed between solvents (CH3NO2, TBME, iPrOAc, and CH3CN).

[0447] Entry 17: The obtained XRPD diffractogram had multiple diffraction peaks (Figure 19). The XRPD diffractogram was labeled as pattern D.

[0448] Entry 18: The obtained XRPD diffractogram had multiple diffraction peaks (Figure 19). The XRPD diffractogram was denoted as pattern E.

[0449] Entry 19: The obtained XRPD diffractogram had multiple diffraction peaks (Figure 19). The XRPD diffractogram was denoted as pattern F.

[0450] 25℃ slurry:

[0451] Entries 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 20, 21, 22: All XRPD diffractograms are similar to those obtained for polymorph A.

[0452] Entries 6, 12, and 24: XRPD diffractograms obtained for isolated solids broadly matched pattern C (see Figure 18).

[0453] Entry 23: XRPD analysis showed that hydrate A was formed.

[0454] Entry 17: The obtained XRPD diffractogram had multiple diffraction peaks (Figure 19). The XRPD diffractogram was labeled as pattern D.

[0455] Entry 18: The obtained XRPD diffractogram had multiple diffraction peaks (Figure 19). The XRPD diffractogram was denoted as pattern E.

[0456] Entry 19: The obtained XRPD diffractogram had multiple diffraction peaks (Figure 19). The XRPD diffractogram was denoted as pattern F.

[0457] Analysis of results:

[0458] The XRPD diffractogram for solids isolated at 25°C is broadly identical to the XRPD diffractogram obtained for solids isolated at 50°C.

[0459] Patterns D, E, and F originated from alcohols (MeOH, EtOH, and IPA). During development, a solvated state was assumed, considering that the ethanol solvate had been previously isolated. Although the XRPD diffractograms for the ethanol solvate are not identical, given that precise variations in solvent levels can lead to fluctuating ordered states within the lattice, a comparison between these XRPD diffractograms provides a strong hypothesis that these more pronounced phase variations are caused by solvent entrainment.

[0460] XRPD patterns D, E, and F (Figure 19) are all dissimilar to the XRPD diffractograms for the hydrate (Figure 7d).

[0461] A direct comparison of XRPD diffractograms obtained for solids isolated at two different temperatures from MeOH, EtOH, and IPA (patterns D, E, and F - Figure 19) shows that the diffractograms are identical. The two MeOH diffractograms are similar, while EtOH and IPA are directly comparable.

[0462] DSC analysis was performed on isolated solids, and TGA was also performed where sufficient sample was available. Solids yielding patterns D, E, and F all featured endothermic activity around 170–180°C, while others exhibited distinct thermal profiles. TGA analysis of solids isolated from MeOH slurry showed one long mass loss (3.1%) between approximately 25–190°C. Stoichiometric methanol solvate would require 10.3% by weight of solvent. TGA analysis of solids isolated from EtOH slurry showed two distinct mass loss stages. The first mass loss (0.3% by weight) occurring before 100°C is thought to be due to water, and the second, larger loss (11.5% by weight) is thought to be due to the solvent. Stoichiometric ethanol solvate would require 13.9% by weight of solvent. TGA analysis of solids isolated from IPA slurry featured two distinct mass loss stages. The first mass loss (0.4% by weight) before 100°C is thought to be due to water, while the second, larger mass loss (13.9% by weight) is thought to be due to the remaining solvent. Stoichiometric IPA solvation requires 17.5% by weight of solvent.

[0463] Amorphous psilocybin slurry

[0464] To produce an amorphous material, a small sample of psilocybin (0.5 g) was dissolved in water (0.5 L, 1000 volts), filtered, and freeze-dried. The psilocybin was recovered as a grayish-white fibrous material (Lot MC1368A; 412 mg, 82%, XRPD amorphous).

[0465] To visually evaluate the solubility of amorphous APIs and to induce morphological modification, a series of slurry maturation processes were performed as follows. Psilocybin (15 mg) was added to the tube. Then, solvent was added at ambient temperature (20°C, 0.3 ml, 20 volumes), and the suspension was stirred. Observations were made. After stirring for 1 hour, the sample was heated at 45°C for 18 hours and observed. The sample was heated at 50°C for 8 hours and observed. The sample was stirred at 25°C for 72 hours and subjected to a final thermal cycle before isolation. The observations are shown in Table 35. [Table 35]

[0466] result

[0467] Most solvents returned solids that were considered semi-crystalline (primarily amorphous, exhibiting significant reflection at approximately 18°2θ).

[0468] Equilibrium formation in MeCN restored true amorphous material.

[0469] Polymorph B was converted from chlorobutane, nitromethane, and IPA (Figures 20 and 22).

[0470] In the polymorph A slurry experiment described above, pattern D isolated from MeOH was returned from EtOH equilibration, whereas in this study, MeOH returned a semi-crystalline solid.

[0471] Solids similar to pattern A were recovered from water, THF:water, and IPA:water (4:1).

[0472] A solid similar to pattern D was recovered from EtOH:water (4:1), supporting the finding that pattern D can be isolated from EtOH alone.

[0473] Solids similar to pattern E were recovered from chloroform.

[0474] From the solvents investigated, true polymorphs A or A' could not be isolated after long-term equilibration and thermal aging of amorphous psilocybin. [Example 12] - Development of formulations.

[0475] The initial series of experiments were conducted using formulations as described in Table 36 below. The objective was to identify suitable single or combination extenders for large-scale formulations. [Table 36]

[0476] The results regarding the main physiological and chemical properties—specifically, the fluidity, miscibility, and content uniformity of the substance—are described in Table 37 below. [Table 37]

[0477] Batch (APL-117-6085-03) exhibited excellent miscibility (highest, intermediate, and lowest) and extremely good content uniformity across the different samples analyzed; however, its flowability (based on Carr's index) approached its upper limit, and the formulation was predicted not to accommodate higher drug loads.

[0478] For this reason, numerous alternative formulations were tested. The objective was to explore combinations of other fillers with the aim of improving powder flowability and achieving excellent miscibility and content uniformity.

[0479] Fewer Compactcels compared to batch 3 (APL-117-6085-03) We also tested formulations containing MAB and larger amounts of flow promoters.

[0480] These further formulations are listed in Table 38 below. [Table 38]

[0481] The results for these batches are shown in Table 39 below. [Table 39]

[0482] APL-117-6085-05 failed to achieve excellent miscibility and also did not meet the content uniformity standards.

[0483] Both APL-117-6085-06 and APL-117-6085-07 showed improved powder flowability, but their mixing uniformity with the two formulations was inferior to that of APL-117-6085-03.

[0484] As a result, the applicant has developed a modified additive, more specifically, a silicified additive having different particle sizes. We focused on the bulking agents. These formulations are listed in Table 40 below. [Table 40]

[0485] Prosolv is a silicified microcrystalline cellulose, and two variants were selected to determine whether particle size affects the results. Compared to standard microcrystalline cellulose (typical size range is 80–350 microns, depending on sieving), Prosolv has a finer particle size distribution and gives an increased surface area. It was hypothesized that the increased surface area could give better flowability and increased compaction, along with improved content uniformity and stability in the formulation. The ratio of Prosolv50 to Prosolv90 was intended to produce a particle size distribution spanning both finer and coarser particles.

[0486] These results are shown in Table 41 below. [Table 41]

[0487] Similar to the consistency of miscibility (over 95% after accounting for error), we can acknowledge the excellent performance of the main parameters of content uniformity (over 90%, actually over 94%) and AV (less than 10, actually less than 7).

[0488] According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) A crystalline psilocybin in the form of polymorph A or polymorph A', a. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ; b. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ [further characterized by at least one additional peak at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ]; c. The XRPD diffractogram substantially illustrated in Figure 7a or 7b; or d. Endothermic events in DSC thermograms with a starting temperature of 205-220°C, substantially illustrated in Figure 8a or Figure 8b. Crystalline psilocybin in the form of polymorph A or polymorph A', characterized by one or more of the following. (Section 2) Crystalline psilocybin in the form of polymorph A or polymorph A' as described in item 1 above, further characterized by an endothermic event in a DSC thermogram having an onset temperature of 210-215°C. (Section 3) a. Peaks in the XRPD diffractogram at 11.5, 12.0, 14.5, and 17.5°2θ±0.1°2θ; b. Peaks in the XRPD diffractogram at 11.5, 12.0, 14.5 and 17.5°2θ±0.1°2θ [further characterized by at least one additional peak at 19.7, 20.4, 22.2, 24.3 or 25.7°2θ±0.1°2θ]; c. The XRPD diffractogram substantially illustrated in Figure 7a; or d. Endothermic events in a DSC thermogram with an onset temperature of 205–220°C, substantially illustrated in Figure 8a. Crystalline psilocybin in the form of polymorph A described in item 1 or 2 above, characterized by one or more of the above. (Section 4) A crystalline psilocybin in the form of polymorph A as described in item 3 above, wherein the peak at 17.5°2θ±0.1°2θ has a relative intensity of at least 5% compared to the peak at 14.5°2θ±0.1°2θ. (Section 5) a. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ [wherein the peak at 17.5°2θ±0.1°2θ is absent or substantially absent]; b. Peaks in the XRPD diffractogram at 11.5, 12.0, and 14.5°2θ±0.1°2θ [wherein the peak at 17.5°2θ±0.1°2θ is absent or substantially absent, and further characterized by at least one additional peak at 19.7, 20.4, 22.2, 24.3, or 25.7°2θ±0.1°2θ]; c. The XRPD diffractogram substantially illustrated in Figure 7b; or d. Endothermic events in a DSC thermogram with an onset temperature of 205–220°C, substantially illustrated in Figure 8b. Crystalline psilocybin in the form of polymorph A' described in item 1 or 2 above, characterized by one or more of the above. (Section 6) Crystalline psilocybin in the form of polymorph A' described in item 5 above, wherein each peak at 17.5°2θ±0.1°2θ has a relative intensity of less than 5% compared to the peak at 14.5°2θ±0.1°2θ. (Section 7) ii) Water content less than 0.5% w / w or iii) Loss of less than 0.5% w / w in a TGA thermogram at ambient temperatures of 25°C to 200°C. Crystalline psilocybin in the form of polymorph A or polymorph A' as described in any of items 1 to 6 above, further characterized by having one of the above. (Section 8) Crystalline psilocybin in the form of polymorph A or polymorph A' as described in any of the preceding items, having a chemical purity greater than 97 area% by HPLC and free from any single impurity greater than 1% by weight and 1 area%, including phosphoric acid as measured by 31P NMR and psilocine as measured by HPLC. (Section 9) The crystalline psilocybin is a white or grayish-white solid, and is a crystalline psilocybin in the form of polymorph A or polymorph A' as described in any of the preceding items. (Section 10) Crystalline psilocybin in the form of polymorph A or polymorph A' described in any of the preceding sections, including a spectrum that matches one or more of Figures 10 to 13. (Section 11) Crystalline psilocybin in the form of polymorph A or polymorph A' as described in any of the preceding items, including the acceptance criteria for one or more quality characteristics as shown in points 6 to 13 of Table 9. (Section 12) A batch of crystalline psilocybin polymorph A as described in items 1-4 or 7-11 above, comprising at least 100 g. (Section 13) A pharmaceutical preparation comprising crystalline psilocybin in the form of polymorph A or polymorph A' as described in any of the preceding items. (Section 14) A pharmaceutical preparation as described in item 13 above, in oral dosage form. (Section 15) The pharmaceutical preparation according to item 13 or 14, wherein crystalline psilocybin in the form of polymorph A or polymorph A' is present in an amount that gives a dose of 0.01 mg / kg to 1 mg / kg. (Section 16) A pharmaceutical preparation according to any of items 13 to 15 above, comprising one or more additives. (Section 17) The pharmaceutical preparation described in item 16 above, wherein the aforementioned additive 1 or more comprises microcrystalline cellulose or starch. (Section 18) The pharmaceutical preparation according to item 16, wherein the aforementioned additive 1 or more comprises silicified microcrystalline cellulose. (Section 19) Crystalline psilocybin in the form of polymorph A or polymorph A' as described in items 1 to 11 above, for use in pharmaceuticals. (Section 20) Crystalline psilocybin in the form of polymorph A or polymorph A' as described in items 1 to 11 above, for use in treating central nervous system disorders. (Section 21) Crystalline psilocybin in the form of polymorph A or polymorph A' as described in items 1 to 11 above, for use in treating drug-resistant depression. (Section 22) A method for the large-scale production of psilocybin (12) in the form of polymorph A, wherein the method comprises subjecting psilocybin (12) to a water crystallization step along with controlled drying in order to produce the crystalline psilocybin polymorph A described in any of items 1 to 4 or 7 to 11 above. A characteristic method. (Section 23) Crystalline psilocybin in polymorph A(12A) form for use in pharmaceuticals. (Section 24) Crystalline psilocybin in polymorph A(12A) form for use in treating drug-resistant depression. (Section 25) A method for treating drug-resistant depression, comprising administering an effective dose of crystalline psilocybin in the form of polymorph A(12A) to a subject who requires treatment for drug-resistant depression. (Section 26) a. Peaks in the XRPD diffractogram at 11.5, 12.0, 14.5, and 17.5°2θ±0.1°2θ; b. Peaks in the XRPD diffractogram at 11.5, 12.0, 14.5 and 17.5°2θ±0.1°2θ [further characterized by at least one additional peak at 19.7, 20.4, 22.2, 24.3 or 25.7°2θ±0.1°2θ]; c. The XRPD diffractogram substantially illustrated in Figure 7a; or d. Endothermic events in a DSC thermogram with an onset temperature of 205–220°C, substantially illustrated in Figure 8a. A method for the large-scale production of psilocybin (12) in the form of polymorph A(12A), characterized by comprising subjecting psilocybin (12) to a water crystallization step, along with controlled drying, in order to produce crystals characterized by one or more of the above. (Section 27) The method according to item 26, wherein the psilocybin is recrystallized in about 10 to 20 volumes of water, heated with stirring to a temperature of at least 70°C, filtered to finish, seeded at a temperature of about 70°C, and cooled to about 5°C over a period of more than 2 hours. (Aspect 1) A composition comprising crystalline psilocybin characterized by X-ray diffraction (XRPD) peaks at 11.5±0.1, 12.0±0.1, 14.5±0.1, 17.5±0.1 and 19.7±0.1°2θ, wherein the composition is free of a single impurity in an area percentage greater than 1% as determined by HPLC analysis, and wherein the impurity is psilocybin, phosphoric acid, [ka] That is, composition. (Aspect 2) The composition according to embodiment 1, having less than 1 area percent of psilocine as determined by HPLC analysis. (Aspect 3) The composition according to embodiment 1, having less than 1% by weight of phosphoric acid as determined by 31P NMR. (Aspect 4) Less than 1% [ka] [ka] The composition according to embodiment 1, comprising: (Aspect 5) The composition according to embodiment 1, comprising crystals having a size in the range of 50 to 200 microns. (Aspect 6) The composition according to embodiment 1, wherein the crystalline psilocybin is further characterized by a water content of less than 0.5% w / w. (Aspect 7) The composition according to embodiment 6, wherein the water content is less than 0.4% w / w. (Pattern 8) The composition according to embodiment 6, wherein the water content is less than 0.3% w / w. (Aspect 9) The composition according to embodiment 6, wherein the water content is less than 0.2% w / w. (Aspect 10) The composition according to embodiment 6, wherein the water content is less than 0.1% w / w. (Aspect 11) The composition according to Embodiment 1, wherein the crystalline psilocybin is further: a) Drying loss of 2% w / w or less; b) Ignition residue of 0.5% w / w or less; c) Residual solvent content of methanol (3000 ppm or less), ethanol (5000 ppm or less), THF (720 ppm or less), and toluene (890 ppm or less) as measured by HRGC; and d) Inductively coupled plasma mass spectrometry (ICP-MS) elemental analysis as follows: i. 1.5ppm or less Cd; ii. 1.5ppm or less Pb; iii. 4.5ppm or less As; iv. 9.0ppm or less Hg; v. 15ppm or less Co; vi. 30ppm or less V; vii. 60ppm or less Ni; viii. Li; and 165 ppm or less ix. 30ppm or less Pd A composition characterized by one or more of the following. (Aspect 12) The composition according to embodiment 1, wherein the crystalline psilocybin is further characterized by at least one peak selected from the group consisting of 20.4±0.1, 22.2±0.1, 24.3±0.1, and 25.7±0.1°2θ. (Aspect 13) The composition according to embodiment 1, wherein the crystalline psilocybin is further characterized by a loss of less than 0.5% w / w in a TGA thermogram between 25°C and 200°C. (Aspect 14) The composition according to embodiment 1, wherein the crystalline psilocybin is further characterized by an endothermic event in a DSC thermogram having an onset temperature between 205°C and 220°C. (Aspect 15) The composition according to embodiment 1, wherein the crystalline psilocybin is further characterized by an endothermic event in a DSC thermogram having an onset temperature between 145°C and 155°C. (Aspect 16) A pharmaceutical composition comprising crystalline psilocybin according to embodiment 1 in a therapeutically effective amount and at least one pharmaceutically acceptable excipient. (Aspect 17) The pharmaceutical composition according to embodiment 16, wherein the at least one pharmaceutically acceptable additive is a diluent. (Aspect 18) The pharmaceutical composition according to embodiment 17, wherein the diluent is microcrystalline cellulose, starch, mannitol, anhydrous calcium hydrogen phosphate, silicon dioxide, calcium carbonate, talc, or a combination thereof. (Aspect 19) The pharmaceutical composition according to embodiment 18, wherein the diluent contains microcrystalline cellulose. (Aspect 20) The pharmaceutical composition according to embodiment 19, wherein the microcrystalline cellulose comprises silicified microcrystalline cellulose. (Aspect 21) The pharmaceutical composition according to embodiment 19, wherein the microcrystalline cellulose comprises a mixture of two silicified microcrystalline cellulose variants. (Aspect 22) The pharmaceutical composition according to embodiment 21, wherein the first silicified microcrystalline cellulose variant has a particle size of approximately 45 to 80 microns, and the second variant has a particle size of approximately 90 to 150 microns. (Aspect 23) The pharmaceutical composition according to embodiment 22, wherein about 30% or less of the microcrystalline cellulose is a first variant having a particle size of about 45 to 80 microns, and about 70% or more of the microcrystalline cellulose is a second variant having a particle size of about 90 to 150 microns. (Aspect 24) The pharmaceutical composition according to embodiment 23, wherein about 20% or less of the microcrystalline cellulose is a first variant having a particle size of about 45 to 80 microns, and about 80% or more of the microcrystalline cellulose is a second variant having a particle size of about 90 to 150 microns. (Aspect 25) The pharmaceutical composition according to embodiment 23, wherein about 15% or less of the microcrystalline cellulose is a first variant having a particle size of about 45 to 80 microns, and about 85% or more of the microcrystalline cellulose is a second variant having a particle size of about 90 to 150 microns. (Aspect 26) The pharmaceutical composition according to embodiment 16, wherein the at least one additive comprises a disintegrant. (Aspect 27) The pharmaceutical composition according to embodiment 26, wherein the disintegrant comprises sodium starch glycolate or croscarmellose sodium. (Aspect 28) The pharmaceutical composition according to embodiment 16, wherein the at least one additive comprises a binder. (Aspect 29) The pharmaceutical composition according to embodiment 28, wherein the binder comprises povidone, copovidone, or hydroxypropylcellulose. (Aspect 30) The pharmaceutical composition according to embodiment 16, wherein the at least one additive comprises a lubricant. (Aspect 31) The pharmaceutical composition according to embodiment 30, wherein the lubricant is magnesium stearate or sodium stearyl fumarate. (Aspect 32) The pharmaceutical composition according to embodiment 16, wherein the at least one additive comprises a flow promoter. (Aspect 33) The pharmaceutical composition according to embodiment 32, wherein the flow promoter is colloidal silicon dioxide. (Aspect 34) The pharmaceutical composition according to embodiment 16, wherein the pharmaceutical composition is a capsule. (Aspect 35) The pharmaceutical composition according to embodiment 16, wherein the pharmaceutical composition is a tablet. (Aspect 36) The pharmaceutical composition according to embodiment 16, comprising approximately 1 mg to approximately 40 mg of the crystalline psilocybin. (Aspect 37) The pharmaceutical composition according to embodiment 16, comprising approximately 1 mg of the crystalline psilocybin. (Aspect 38) The pharmaceutical composition according to embodiment 16, comprising approximately 25 mg of the crystalline psilocybin. (Aspect 39) The pharmaceutical composition according to embodiment 16, wherein the crystalline psilocybin has a chemical purity of more than 99 area percent as determined by HPLC. (Pattern 40) The pharmaceutical composition according to embodiment 16, wherein the crystalline psilocybin has a water content of less than 0.5% w / w. (Aspect 41) The pharmaceutical composition according to embodiment 16, having less than 1 area % of psilocine as determined by HPLC analysis. (Aspect 42) The pharmaceutical composition according to embodiment 16, having less than 1% by weight of phosphoric acid as determined by 31P NMR. (Aspect 43) The pharmaceutical composition according to embodiment 37, wherein the crystalline psilocybin has a chemical purity of more than 98 area% as determined by HPLC analysis. (Aspect 44) The pharmaceutical composition according to embodiment 37, wherein the crystalline psilocybin has a water content of less than 0.5% w / w. (Aspect 45) The pharmaceutical composition according to embodiment 37, having less than 1 area percent of psilocine as determined by HPLC analysis. (Aspect 46) The pharmaceutical composition according to embodiment 37, having less than 1% by weight of phosphoric acid as determined by 31P NMR. (Aspect 47) The pharmaceutical composition according to embodiment 38, wherein the crystalline psilocybin has a chemical purity of more than 98 area percent as determined by HPLC. (Aspect 48) The pharmaceutical composition according to embodiment 38, wherein the crystalline psilocybin has a water content of less than 0.5% w / w. (Aspect 49) The pharmaceutical composition according to embodiment 38, having less than 1 area percent of psilocine as determined by HPLC analysis. (Appearance 50) The pharmaceutical composition according to embodiment 38, having less than 1% by weight of phosphoric acid as determined by 31P NMR. (Aspect 51) A composition comprising crystalline psilocybin according to any one of embodiments 1 to 15 or a pharmaceutical composition according to any one of embodiments 16 to 50 for use in the treatment of subjects requiring treatment for generalized anxiety disorder, personality disorder, drug disorder, gambling addiction, eating disorder, body dysmorphic disorder, pain or depression. (Appearance 52) The composition for use or pharmaceutical composition according to embodiment 51, wherein the generalized anxiety disorder is major depressive disorder (MDD), cluster headache, or obsessive-compulsive disorder (OCD). (Aspect 53) The composition for use or pharmaceutical composition according to embodiment 51, wherein the personality disorder is conduct disorder. (Aspect 54) The composition for use or pharmaceutical composition according to embodiment 51, wherein the drug disorder is alcohol dependence, nicotine dependence, opioid dependence, or cocaine dependence. (Aspect 55) The composition for use or pharmaceutical composition according to embodiment 51, wherein the depression is drug-resistant depression. (Aspect 56) A method for preparing a pharmaceutical composition, (1) To provide a crystalline psilocybin composition characterized by powder X-ray diffraction (XRPD) peaks at 11.5±0.1, 12.0±0.1, 14.5±0.1, 17.5±0.1 and 19.7±0.1°2θ; and (2) Formulating the composition with at least one pharmaceutically acceptable additive. A method for providing the pharmaceutical composition comprising the above. (Aspect 57) The method according to embodiment 56, wherein the psilocybin has a chemical purity of more than 97 area% as determined by HPLC analysis. (Pattern 58) The method according to embodiment 56, wherein the psilocybin does not contain a single impurity in an area percentage of more than 1 percent as determined by HPLC analysis. (Aspect 59) A batch comprising approximately 100 g or more of crystalline psilocybin, wherein the crystalline psilocybin is characterized by powder X-ray diffraction (XRPD) peaks at 11.5±0.1, 12.0±0.1, 14.5±0.1, 17.5±0.1, and 19.7±0.1°2θ. (Appendix 60) The batch according to embodiment 59, wherein the crystalline psilocybin has a chemical purity of more than 97 area% as determined by HPLC analysis. (Aspect 61) The batch according to embodiment 59, wherein the crystalline psilocybin does not contain a single impurity in an area percentage of more than 1 percent as determined by HPLC analysis. (Aspect 62) Crystalline psilocybin, and the following: (1) Reacting psilocine with tetrabenzyl pyrophosphate to form benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate; (2) Reacting benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate with hydrogen in the presence of a catalyst to form at least about 100 g or more of psilocybin; and (3) Crystallizing the crystalline psilocybin from water. Crystalline psilocybin, prepared by a process that provides the crystalline psilocybin, comprising the above. (Aspect 63) The crystalline psilocybin according to embodiment 62, having a chemical purity of more than 97 area% as determined by HPLC analysis. (Personal aspect 64) The crystalline psilocybin according to embodiment 62, which does not contain a single impurity in an area percentage of more than 1 percent as determined by HPLC analysis. (Patent 65) Crystalline psilocybin according to embodiment 62, characterized by powder X-ray diffraction (XRPD) peaks at 11.5±0.1, 12.0±0.1, 14.5±0.1, 17.5±0.1 and 19.7±0.1°2θ.

Claims

1. A method for producing a psilocybin drug substance using psilocine as a starting material or intermediate, The psilocybin drug substance includes crystalline psilocybin characterized by peaks in the XRPD diffractogram at 11.5±0.1, 12.0±0.1, 14.5±0.1, 17.5±0.1 and 19.7±0.1°2θ, wherein its form is polymorph A. The psilocybin drug substance contains the crystalline psilocybin such that its chemical purity, as measured by HPLC, exceeds 97 area percent. ３１ It does not contain phosphoric acid as an individual impurity at a level higher than 1% by weight, as measured by NMR, and does not contain psilon as an individual impurity at a level higher than 1% per area, as measured by HPLC. The aforementioned method, A method for producing a psilocybin drug substance, comprising a recrystallization step in which crude psilocybin is dissolved in water, the solution is heated to a temperature not exceeding 80°C, and the crystalline psilocybin is recrystallized from the water, thereby providing the psilocybin drug substance.

2. moreover, The process includes a step of reacting psilocine with tetrabenzyl pyrophosphate using hexamethyldisilazane sodium (NaHMDS) to produce benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate, The method for producing a psilocybin drug substance according to claim 1, wherein the generated benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate is used to produce the crude psilocybin.

3. moreover, The process includes a step of reacting the generated benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indole-4-yl phosphate with hydrogen in the presence of a catalyst, thereby producing the crude psilocybin as a solid material. The method for producing a psilocybin drug substance according to claim 2, wherein the crude psilocybin produced is used to carry out the recrystallization in the recrystallization step.

4. The method for producing a psilocybin drug substance according to claim 1, wherein the psilocybin drug substance is contained in a drug composition that is orally administered in the form of a tablet to a subject who needs to be treated for drug-resistant depression.

5. The method for producing a psilocybin drug substance according to claim 4, wherein the peak at 17.5 ± 0.1°²θ has a relative intensity of at least 5% compared to the peak at 14.5 ± 0.1°²θ.

6. The method for producing a psilocybin drug substance according to claim 4, wherein the crystalline psilocybin has an endothermic event in a DSC thermogram having a first onset temperature in the range of 145°C to 155°C and a second onset temperature in the range of 205°C to 220°C.

7. The crystalline psilocybin is a) Drying weight loss of 2% w / w or less; b) Ignition residue of 0.5% w / w or less; c) Residual solvent content of methanol (3000 ppm or less), ethanol (5000 ppm or less), THF (720 ppm or less), and toluene (890 ppm or less), as measured by HRGC; d) ３１ Phosphate content of 1% w / w or less as measured by P NMR; and e) Inductively coupled plasma mass spectrometry (ICP-MS) elemental analysis as follows: i. Cd less than 1.5 ppm; ii. Pb less than or equal to 1.5 ppm; iii. As below 4.5 ppm; iv. Hg below 9.0 ppm; v. Co below 15 ppm; vi. V below 30 ppm; vii. Ni below 60 ppm; viiii. Li less than or equal to 165 ppm; and ix. Pd below 30 ppm A method for producing a psilocybin drug substance according to claim 4, further characterized by one or more of the following.

8. The method for producing a psilocybin drug substance according to claim 4, wherein the crystalline psilocybin does not have or substantially has a peak at 10.1 ± 0.1° 2θ in the XRPD diffractogram.

9. The method for producing a psilocybin drug substance according to claim 8, wherein the peak at 10.1 ± 0.1°²θ has a relative intensity of less than 2% compared to the peak at 14.5 ± 0.1°²θ.

10. The method for producing a psilocybin drug substance according to claim 8, wherein the peak at 10.1 ± 0.1°²θ has a relative intensity of less than 1% compared to the peak at 14.5 ± 0.1°²θ.

11. The method for producing a psilocybin drug substance according to claim 8, wherein the peak at 10.1 ± 0.1°²θ is undetectable in the XRPD diffractogram.

12. The recrystallization step includes a step of recrystallizing crystalline psilocybin by using crystalline psilocybin hydrate A as the crystal species, The method for producing a psilocybin drug substance according to claim 1, wherein the crystalline psilocybin hydrate A is characterized by XRPD diffractogram peaks at 8.9, 12.6, and 13.8°2θ±0.1°2θ, or further characterized by at least one peak appearing at at least one of 6.5, 12.2, 19.4, 20.4, and 20.8°2θ±0.1°2θ, or further characterized by at least two peaks appearing at at least two of 6.5, 12.2, 19.4, 20.4, and 20.8°2θ±0.1°2θ.

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