EA043831B1 - OBTAINING PSILOCYBIN, VARIOUS POLYMORPHIC FORMS, INTERMEDIATE COMPOUNDS, COMPOSITIONS AND THEIR APPLICATION - Google Patents

Description

This invention relates to the large-scale production of psilocybin for medical use.

By large scale we mean the production of batches of psilocybin weighing more than 10 grams, more preferably more than 100 grams, more preferably more than 250 grams, and up to and above kilograms.

It also relates to the production of intermediates, including, but not limited to, psilocin, various polymorphic forms of psilocybin, including isostructural variants, and composition thereof for medical use, particularly but not exclusively for the treatment of treatment-resistant depression, as defined in Diagnostic and Statistical Manual, 5th edition either alone or in combination with psychological support, which can be delivered digitally.

State of the art

Psilocybin was first synthesized in 1958 at Sandoz, see GB912714 and US3075992, and was widely available as a research chemical until the mid-1960s.

The plant-based psychedelic drug has been used as a psychotherapy adjuvant for the treatment of mood and alcohol use disorders, and 3 clinical studies recently reported its use for depressive symptoms: Griffiths et al., 2016, J. Psychopharmacol., 30(12) :1181-1197; Ross et al., 2016, J. Psychopharmacol., 30(12):1165-1180; and Carhart-Harris et al., 2016, Lancet Psychiatry, 3(7):619–627.

Methods for producing psilocybin are limited and include J. Nat. Prod., 2003, 66, p. 885-887; Helv. Chim. Acta., 1959, 42, 2073-2103; Experientia, 1958, 15, 397-399; and Synthesis, 1999, 935-938.

Based on this literature, applicants believe that the method described in J. Nat. Prod., 2003, 66, p. 885-887 (hereafter JNP) is the most suitable method for development into a commercial scale process.

The process described there yields quantities of the order of 10 g and involves 6 steps numbered (i)-(vi).

Similar to the claimed method, steps (ii) and (iii) are further discussed as one step (step 2) and the JNP method is reproduced as in FIG. 1 here.

Stage 1 (i) involves the reaction of 4-hydroxyindole (3) with acetic anhydride (Ac ₂ O) in pyridine and anhydrous dichloromethane (CH ₂ Cl ₂ ) at 0°C. Water is added, the mixture is evaporated and the resulting concentrate is dissolved in ethyl acetate, washed with water and saturated sodium chloride, the organic phase is dried over sodium sulfate and evaporated to give 4-acetylindole (4), which is collected by filtration and washed with water and ethyl acetate.

Step 2 (ii) and (iii), two-step acylation (ii) - amidation step (iii), involves the formation of 3-dimethylaminooxalyl-4-acetylindole (6) through (ii) reaction of 4-acetylindole (4) with oxalyl chloride ((COCl ) ₂ ) in anhydrous diethyl ether, stirring, adding n-hexane and maintaining at -20°C to obtain intermediate 3-(2-chloro-2-oxoacetyl)-1H-indol-4-yl acetate (5), which is separated filtering. The intermediate is dissolved in anhydrous tetrahydrofuran (THF) and reacted with dimethylamine ((CH ₃ ) ₂ NH) in tetrahydrofuran and pyridine. Anhydrous ether was added due to solidification, the reaction product was separated by filtration and washed with n-hexane, ethyl acetate and water to give 3-dimethoxyaminooxalyl-4acetylindole (6).

Step 3 (iv) involves the preparation of psilocin (1) by reacting 3-dimethylaminooxalyl-4acetylindole (6) with lithium aluminum hydride (LiAlH ₄ ) in anhydrous THF under argon. After refluxing and cooling, add anhydrous sodium sulfate, then sodium sulfate solution and then anhydrous sodium sulfate. The reaction mixture is diluted with ethyl acetate, quickly concentrated in vacuo, and the resulting psilocin crystals are quickly washed with methanol.

Step 4 (v) involves the preparation of benzyl[2-(4-hydroxyindol-3-yl)ethyl]dimethylammonio-4-obenzylphosphate (8) by reacting psilocin dissolved in anhydrous THF with n-butyllithium (n-BuLi) in n- hexane at -78°C and tetrabenzyl pyrophosphate [(BpO) ₂ PO] ₂ O, heating the reaction to 0°C and monitoring the production of intermediate phosphate dibenzyl 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl (7 ). When checking for its presence, aminopropyl silica gel is added, the mixture is diluted with ethyl acetate and filtered through a pad of celite by suction, the filtrate is concentrated in vacuo, redissolved in CH2Cl2 and the precipitate is collected by filtration.

Step 5 (vi) involves the preparation of psilocybin (2) by reacting (8) in methanol (MeOH) with hydrogen (H ₂ ) using a palladium activated carbon catalyst (Pd/C). Water is added due to product deposition and (8), its de-benzylated derivative is monitored along with the appearance of psilocybin, the reaction solution is filtered through a pad of celite. The product is collected by filtration and washed with ethanol to obtain a white needle-shaped crystalline form with a melting point of 190-198°C.

Unlike most methods, such as JNP, which use non-aqueous solvents such as methanol or ethanol, Experientia, 1958, 15, 397-399 uses a single recrystallization from water to produce psilocybin from a mushroom extract. The idea is to use boiling water to dissolve the starting material obtained by small scale chromatography, and the resulting high vacuum dried product is melted unclearly at 185-195°C, and mass loss is shown

25.4%, suggesting a clear difference from the purity and form obtained by the applicant.

During the development of the synthesis to produce psilocybin, the applicant conducted numerous hydrogenation reactions on the 5 g scale, which resulted in the production of various crystalline forms of psilocybin. The initial hydrogenation reaction produces hydrate A (JCCA2157E), which has the PXRD pattern shown in FIG. 7d, and the DSC and TGA thermograms shown in FIG. 8d. DSC shows an endotherm at ~97°C, which coincides with the mass decrease in TGA, indicating dehydration, and an endotherm with an onset temperature of ~216°C, which is considered melting. Another hydrogenation reaction produces an ethanol solvate (JCCA2158D), which is then analyzed by PRD (Figure 7e), DSC (Figure 8e), TGA (Figure 8e) and 1H NMR, showing 11% ethanol captured. The DSC thermogram shows an endotherm with an onset of ~154°C, which is considered melting along with ~13% mass loss in TGA. In another experiment conducted during development, the applicant crystallized psilocybin; Instead of remaining in solution in hot water, allowing a post-treatment stage on the filter, a precipitate precipitates at high temperatures (>90°C). The solids formed do not redissolve upon further heating or the addition of additional water. When cooling and isolating the solid substance (CB646E), PRD is carried out. The PXRD pattern (Fig. 7f) suggests a mixed phase of polymorph A' (JCCA2160-F-D4) and polymorph B (JCCA2160-F-TM2-C5). These discoveries highlight the importance of developing a process that will consistently produce the desired crystalline form, so the applicant began experiments to determine what these forms were so that they could produce chemically pure psilocybin in a controlled form suitable for medical use.

For clinical trials, any new active substance (NAS) must be capable of large-scale production (usually 100 g or more, more preferably more than 250 g, even more preferably more than 500 g, up to batches greater than a kilogram) depending on the amount of active substance, which should be administered to a person. It must also be chemically pure, well defined and shelf stable.

Moreover, any production method must be easily reproducible and ensure consistency from batch to batch.

A first object of the present invention is to provide psilocybin, a permanent polymorphic form, for administration to humans.

Another object of this invention is to obtain chemically pure psilocybin, a permanent polymorphic form, for administration to humans.

Another goal is to obtain chemically pure psilocybin in large quantities, since pure psilocybin must be produced on a large scale for commercial use.

Another object of the invention is to provide a method for crystallizing psilocybin in the desired polymorphic form.

Another object of the invention is to provide an adaptable method for the production of psilocybin from psilocin or 4-hydroxyindole.

In developing a suitable technique, the applicant was faced with numerous problems and difficulties which he had to overcome, and this is a separate, independent object: to overcome the problems identified at each stage and to use the inventions separately or in combination.

It is a further object of the invention to formulate psilocybin in accordance with the present invention in a form suitable for administration to humans and for use in medicine, in particular in the treatment of disorders of the central nervous system (CNS) and more particularly but not exclusively in the treatment of depression, in particular resistant to depression medications, alone or in combination with a digital health product or digital solution.

The essence of the invention

According to a first aspect of the present invention, there is provided crystalline psilocybin in the form of polymorph A or polymorph A', characterized by one or more of

a) peaks in the SRD diffractogram at 11.5, 12.0 and 14.5° 2θ±0.1° 2θ;

b) peaks in the PRD diffraction pattern at 11.5, 12.0 and 14.5° 2θ±0.1° 2θ, additionally characterized by at least one additional peak at 19.7, 20.4, 22.2, 24, 3 or 25.7°2θ±0.1°2θ;

c) PXRD diffraction pattern, which is essentially illustrated in FIG. 7a or 7b; or

d) an endothermic event in the DSC thermogram having an onset temperature of 205-220° C., which is essentially illustrated in FIG. 8a or 8b.

Polymorph A.

In accordance with a preferred embodiment of the present invention there is provided crystalline psilocybin in the form of polymorph A, characterized by one or more of

a) peaks in the SRD diffractogram at 11.5, 12.0, 14.5 and 17.5° 2θ±0.1° 2θ;

b) peaks in the SRD diffractogram at 11.5, 12.0, 14.5 and 17.5° 2θ±0.1° 2θ, additionally characterizing

- 2 043831 has at least one additional peak at 19.7, 20.4, 22.2, 24.3 or 25.7° 2θ±0.1°2θ;

c) PXRD diffraction pattern which is essentially illustrated in FIG. 7a; or

d) an endothermic event in the DSC thermogram having an onset temperature of 205-220° C., which is essentially illustrated in FIG. 8a.

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

In one embodiment, polymorph A of psilocybin exhibits a PRD diffraction pattern that differs from the diffraction pattern summarized in Table 1. 1. In one embodiment described herein, crystal polymorph A of psilocybin has at least 3 peaks (±0.1°2θ) from the table. 1. In the specific embodiment described herein, crystal polymorph A of psilocybin contains at least 4 peaks (±0.1° 2θ) from table. 1. In the specific embodiment described herein, crystal polymorph A of psilocybin contains at least 5 peaks (±0.1°2θ) from the table. 1. In the specific embodiment described herein, crystal polymorph A of psilocybin contains at least 6 peaks (±0.1° 2θ) from table. 1. In the specific embodiment described herein, crystal polymorph A of psilocybin contains at least 8 peaks (±0.1° 2θ) from table. 1. In the specific embodiment described herein, crystal polymorph A of psilocybin contains at least 10 peaks (±0.1° 2θ) from table. 1. In the specific embodiment described herein, crystal polymorph A of psilocybin contains at least 15 peaks (±0.1° 2θ) from table. 1. The peak at about 17.5° 2θ±0.1° 2θ distinguishes polymorph A of psilocybin from polymorph A', which has no or essentially no peak (i.e., has a relative intensity compared to the peak 14.5° 2θ± 0.1° 2θ less than 2%, more preferably less than 1%).

Table 1

PDP peak positions for polymorph A

Position [°2Τ] Relative intensity [%] 5.6 8.42 P,5 13.05 12.0 26.45 14.5 100 17.5 10.71 19.7 37.29 20.4 20.06 22.2 17.83 23.2 6.99 24.3 17.93 25.7 16.4 26.8 3.15 27.8 4.54 29.7 9.53 31.2 6.51 32.6 2.45 33.7 1.75

In one embodiment, crystalline polymorph A of psilocybin is characterized by PRD diffractogram peaks of 11.5, 12.0, 14.5 and 17.5° 2θ±0.1° 2θ. In another embodiment, psilocybin crystal polymorph A is further characterized by at least one additional peak occurring at 19.7, 20.4, 22.2, 24.3 or 25.7° 2θ ± 0.1° 2θ. In another embodiment, psilocybin crystal polymorph A is further characterized by at least two additional peaks occurring at 19.7, 20.4, 22.2, 24.3 or 25.7° 2θ ± 0.1° 2θ. In another embodiment, psilocybin crystal polymorph A is further characterized by at least three additional peaks occurring at 19.7, 20.4, 22.2, 24.3 or 25.7° 2θ ± 0.1° 2θ. In yet another embodiment, crystal polymorph A of psilocybin exhibits a PXD diffraction pattern substantially the same as the PXD diffractogram shown in FIG. 7a.

In one embodiment, crystal polymorph A of psilocybin is characterized by PRD diffraction peaks at 14.5 and 17.5° 2θ ± 0.1° 2θ with a peak at 17.5° 2θ having an intensity that is at least 5% of the intensity of the peak at 14 .5° 2θ, more preferably at least

- 3 043831 from 6% to at least 7%, at least 8%, from at least 9% to at least 10%.

In one embodiment, crystalline polymorph A of psilocybin has no or substantially no PRD diffraction peaks at 10.1. By essentially absent is meant that any peaks in the PRD diffraction pattern at 10.1 are less than 2% of the intensity of the peak at 14.5° 2θ, for example less than 1%, or are not detected in the PRD diffraction pattern.

In one embodiment, crystal polymorph A of psilocybin is characterized by an endothermic event in the DSC thermogram having an onset temperature of 205-220°C, eg 210-220°C, eg 210-218°C, or eg 210-216°C. In another embodiment, crystal polymorph A of psilocybin is further characterized by an endothermic event in the DSC thermogram having an onset temperature of 145-165°C, for example 145-160°C, or for example 145-155°C. In another embodiment, crystal polymorph A of psilocybin is characterized by an endothermic event having an onset temperature of 205-220°C, for example 210-220°C, for example 210-218°C, or for example 210-216°C, and an endothermic event having an onset temperature of 145-210°C. 165°C, for example 145-160°C or for example 145-155°C, in a DSC thermogram. In yet another embodiment, crystal polymorph A of psilocybin exhibits a DSC thermogram substantially the same as the DSC thermogram in FIG. 8a.

In another embodiment, psilocybin crystalline polymorph A is characterized by a water content of <0.5% w/w, such as <0.4% w/w, such as <0.3% w/w, such as < 0.2% w/w or such as <0.1% w/w. One skilled in the art will know methods for determining the water content of a compound, for example by Karl Fischer titration. In one embodiment, psilocybin crystalline polymorph A is characterized by a loss of <0.5% w/w, such as <0.4% w/w, such as <0.3% w/w, such as <0 .2% w/w, such as <0.1% w/w, in a TGA thermogram at ambient temperature, such as about 25 and 200°C. In one embodiment, crystalline polymorph A of psilocybin loses less than 2% by weight when tested for loss on drying, such as less than 1% by weight, such as less than 0.5% by weight. Loss on drying testing is carried out at 70°C.

In one embodiment, psilocybin crystal polymorph A is a highly pure crystalline form of polymorph A, for example, psilocybin contains at least 90% by weight, such as 95%, such as 99%, such as 99.5%, polymorph A.

In one embodiment, crystalline polymorph A of psilocybin is a white or off-white solid.

In another embodiment, crystalline polymorph A of psilocybin is chemically pure, for example, psilocybin 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 polymorph A of psilocybin is free of individual impurities greater than 1%, more preferably less than 0.5%, including phosphoric acid as measured by ³¹ P NMR and psilocin as measured by HPLC. In one embodiment, crystalline polymorph A of psilocybin has a chemical purity of greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area% by HPLC. In one embodiment, crystalline polymorph A of psilocybin is free of individual impurities greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline polymorph A of psilocybin does not contain psilocin at a level of more than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, psilocybin crystalline polymorph A is free of phosphoric acid at levels greater than 1 wt%, more preferably less than 0.5 wt%, as measured by ³¹ P NMR. In one embodiment, psilocybin crystal polymorph A has a chemical assay of at least 95 wt%, such as at least 96 wt%, or such as at least 98 wt%.

Polymorph A'.

According to another embodiment of the invention, there is provided a crystalline polymorph A' of psilocybin characterized by one or more of

a) peaks in the SRD diffractogram at 11.5, 12.0 and 14.5° 2θ±0.1° 2θ, but no or substantially absent peak at 17.5° 2θ±0.1° 2θ;

b) peaks in the PRD diffraction pattern at 11.5, 12.0 and 14.5° 2θ±0.1° 2θ, but the absence or essentially absence of a peak at 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) SRD X-ray diffraction pattern, which is essentially illustrated in FIG. 7b; or

d) an endothermic event in the DSC thermogram having an onset temperature of 205-220° C., which is essentially illustrated in FIG. 8b.

By essentially absent a peak at 17.5° 2θ±0.1° 2θ we mean, if present, a peak having a relative intensity compared to the peak at 14.5° 2θ±0.1° 2θ, less than 5%, more preferably less than 4%, less than 3 to 2%, 1% or less.

In one embodiment, polymorph A' of psilocybin exhibits a PRD diffraction pattern characterized by the diffraction pattern summarized in table. 2. In one embodiment described herein, the crystal polymorph A' of psilocybin contains at least 3 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 4 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 5 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 6 peaks (±0.1°2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 8 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 10 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 15 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 20 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ. In the specific embodiment described herein, the crystal polymorph A' of psilocybin contains at least 25 peaks (±0.1° 2θ) from the table. 2, but has no or essentially no peak at 17.5° 2θ±0.1° 2θ.

table 2

PDP peak positions for polymorph A'

Position [°2T] Relative intensity [%] 5.5 4.89 10.1 4.09 11.5 22.05 12.0 22.77 14.5 100 14.9 11.29 17.5 1.08 18.7 2.44 19.4 23.02 19.6 33.7 20.3 17.01 21.1 12.08 21.6 8.51 22.2 15.54 22.6 8.78 23.1 10.11 24.3 21.83 25.1 4.36 25.8 15.4 26.3 4.28 26.8 2.86 27.8 5.96 28.6 1.91 29.7 10.56 31.1 7.35 32.6 3.72 33.8 1.54

In one embodiment, crystal polymorph A' of psilocybin has PRD diffraction peaks at 11.5, 12.0, and 14.5°2θ±0.1°2θ, but has essentially no peak at 17.5°2θ±0.1 °2θ. In another embodiment, the psilocybin crystal polymorph A' is further characterized by at least

- 5 043831 at least one additional peak appearing at 19.7, 20.4, 22.2, 24.3 or 25.7° 2θ±0.1° 2θ. In another embodiment, the psilocybin crystal 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, the crystal polymorph A' of psilocybin is further characterized by the presence of a peak appearing at 10.1° 2θ±0.1° 2θ. In yet another embodiment, crystalline polymorph A' of psilocybin exhibits a PXD diffraction pattern substantially the same as the PXD diffractogram shown in FIG. 7b.

In one embodiment, crystal polymorph A' of psilocybin is characterized by PRD 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 , for example less than 4%, for example less than 3%, for example less than 2%, for example less than 1% or for example about 1%.

In one embodiment, crystal polymorph A' of psilocybin is characterized by PRD 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% of the intensity of the peak at 14. 5°2θ, for example at least 2%, for example at least 3% or for example about 4%.

In one embodiment, crystalline polymorph A' of psilocybin is characterized by an endothermic event in a DSC thermogram having an onset temperature of 205-220°C, such as 210-220°C, such as 210-218°C, or such as 210-216°C. In another embodiment, the crystal polymorph A' of psilocybin is further characterized by an endothermic event in the DSC thermogram having an onset temperature of 145-165°C, such as 145-160°C, or such as 145-155°C. In another embodiment, crystal polymorph A' of psilocybin is characterized by an endothermic event having an onset temperature of 205-220°C, such as 210-220°C, such as 210-218°C, or such as 210-216°C, and an endothermic event having an onset temperature of 145-165°C, such as 145-160°C or such as 145-155°C, in a DSC thermogram. In yet another embodiment, crystal polymorph A' of psilocybin exhibits a DSC thermogram substantially the same as the DSC thermogram in FIG. 8b.

In another embodiment, the psilocybin crystalline polymorph A' is characterized by a water content of <0.5% w/w, such as <0.4% w/w, such as <0.3% w/w, such as <0.2% w/w or such as <0.1% w/w. One skilled in the art will know methods for determining the water content of a compound, for example by Karl Fischer titration. In one embodiment, the psilocybin crystalline polymorph A' is characterized by a loss of <0.5% w/w, such as <0.4% w/w, such as <0.3% w/w, such as < 0.2% w/w, such as <0.1% w/w, in a TGA thermogram from ambient temperature, such as 25 to 200°C. In one embodiment, the crystalline polymorph A' of psilocybin loses less than 2% by weight when tested for loss on drying, such as less than 1% by weight, such as less than 0.5% by weight. Loss on drying testing is carried out at 70°C.

In one embodiment, the crystalline polymorph A' of psilocybin is a highly pure crystalline form of the polymorph A', for example, psilocybin contains at least 90% by weight, such as 95%, such as 99%, such as 99.5%, polymorph A'.

In one embodiment, crystalline polymorph A' of psilocybin is a white or off-white solid.

In another embodiment, crystalline polymorph A' of psilocybin 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% by HPLC. In one embodiment, crystalline polymorph A' of psilocybin is free of individual impurities greater than 1%, more preferably less than 0.5%, including phosphoric acid as measured by ³¹ P NMR and psilocin as measured by HPLC. In one embodiment, crystalline polymorph A' of psilocybin has a chemical purity of greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area% by HPLC. In one embodiment, the psilocybin crystalline polymorph A' is free of individual impurities greater than 1 area %, more preferably less than 0.5 area %, as measured by HPLC. In one embodiment, the psilocybin crystalline polymorph A' does not contain psilocin at a level of more than 1 area %, more preferably less than 0.5 area %, as measured by HPLC. In one embodiment, the crystalline polymorph A' of psilocybin does not contain phosphoric acid at a level of more than 1 wt.%, more preferably less than 0.5 wt.%, as measured by ³¹ P NMR. In one embodiment, the psilocybin crystal polymorph A' has a chemical assay of at least 95 wt%, such as at least 96 wt%, or such as at least 98 wt%.

PRD diffraction patterns and PRD peak positions are obtained using Cu Ka radiation.

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

In one embodiment, a high purity crystalline psilocybin, polymorph A, or polymorph A' (12A or 12A') is provided exhibiting a PXRD pattern substantially as shown in FIG. 7a or 7b, and a DSC thermogram such as essentially shown in FIG. 8a or 8b, or mixtures thereof.

Preferably, crystalline polymorph A of psilocybin (12A) exhibits a PRD diffraction pattern as shown in FIG. 7a, and a DSC thermogram as shown in FIG. 8a.

Preferably, the crystalline polymorph A' of psilocybin (12A') exhibits a PRD diffraction pattern substantially as shown in FIG. 7b, and a DSC thermogram essentially the same as shown in FIG. 8b.

Preferably, the high purity crystalline polymorph A of psilocybin (12A) is characterized by a PRD diffractogram that is substantially shown in FIG. 7a, and a DSC thermogram, which is essentially shown in FIG. 8a.

Preferably, the high purity crystalline polymorph A' of psilocybin (12A') is characterized by a PRD diffractogram, which is shown in FIG. 7b, and the DSC thermogram shown in FIG. 8b.

Polymorph A (including its isostructural variant, polymorph A') (Figs. 7a and 7b) differs from polymorph B (Fig. 7c), hydrate A (Fig. 7d) and ethanol solvate (Fig. 7e; solvate A), and the relationship between some of the different shapes are shown in Figs. 9.

Crystal polymorph A of psilocybin or polymorph A' is a white or off-white solid and/or has a chemical purity of greater than 97%, more preferably greater than 98%, and most preferably greater than 99% by HPLC, and has no individual impurities greater than 1%, more preferably less 0.5%, including phosphoric acid measured by ³¹ P NMR and psilocin measured by HPLC. In one embodiment, high purity crystalline psilocybin, polymorph A or polymorph A' is provided. In one embodiment, the crystalline psilocybin, polymorph A, or polymorph A' has a chemical purity of greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area% by HPLC. In one embodiment, the crystalline psilocybin, polymorph A, or polymorph A' has no individual impurities greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, the crystalline psilocybin, polymorph A, or polymorph A' does not contain psilocin at a level of more than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, the crystalline psilocybin, polymorph A, or polymorph A' does not contain phosphoric acid at a level of more than 1 wt.%, more preferably less than 0.5 wt.%, as measured by ³¹ P NMR. In one embodiment, the crystalline psilocybin, polymorph A, or polymorph A' has a chemical assay of at least 95 wt%, such as at least 96 wt%, or such as at least 98 wt%.

Heating of polymorph A or A' causes an endothermic event having an onset temperature of about 150°C, corresponding to the solid-to-solid transition of polymorph A or polymorph A' to polymorph B. Continued heating of the resulting solid, i.e. polymorph B, causes a second endothermic event corresponding to the melting point, having an onset temperature of 205-220°C (see Fig. 8a and 8b).

According to another independent aspect of the present invention there is provided a crystalline form of psilocybin, hydrate A, characterized by one or more of

a) peaks in the SRD diffraction pattern at 8.9, 12.6 and 13.8° 2θ±0.1° 2θ;

b) peaks in the PRD diffraction pattern at 8.9, 12.6 and 13.8° 2θ±0.1° 2θ, additionally characterized by at least one additional peak at 6.5, 12.2, 19.4, 20, 4 or 20.8° 2θ±0.1°2θ;

c) PXRD diffraction pattern, which is essentially illustrated in FIG. 7d; or

d) an endothermic event in the DSC thermogram having an onset temperature of 205-220° C., which is essentially shown in FIG. 8d.

In one embodiment, psilocybin hydrate A exhibits a PRD diffraction pattern characterized by the diffraction pattern summarized in Table. 3. In one embodiment described herein, crystalline psilocybin hydrate A contains at least 3 peaks (±0.1° 2θ) from the table. 3. In the specific embodiment described herein, crystalline psilocybin hydrate A contains at least 4 peaks (±0.1° 2θ) from the table. 3. In the specific embodiment described herein, crystalline psilocybin hydrate A contains at least 5 peaks (±0.1° 2θ) from the table. 3. In the specific embodiment described herein, crystalline psilocybin hydrate A contains at least 8 peaks (±0.1° 2θ) from the table. 3. In the specific embodiment described herein, crystalline psilocybin hydrate A contains at least 10 peaks (±0.1° 2θ) from the table. 3.

Table 3

Positions of PDP peaks for hydrate A

Position [°2T] Relative intensity [%] 5.6 14.4 6.5 18.84 8.9 100 12.2 11.51 12.6 18.65

- 7 043831

In one embodiment, crystalline psilocybin hydrate A exhibits XRD 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 occurring at 6.5, 12.2, 19.4, 20.4 or 20.8° 2θ ± 0.1° 2θ. In another embodiment, crystalline psilocybin hydrate A is further characterized by at least two peaks occurring at 6.5, 12.2, 19.4, 20.4 or 20.8° 2θ ± 0.1° 2θ. In yet another embodiment, crystalline psilocybin hydrate A exhibits a PXRD pattern substantially the same as the PXD pattern shown in FIG. 7d.

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, such as 210-220°C, such as 210-218°C, or such as 210-216°C. In another embodiment, crystalline psilocybin hydrate A is further characterized by an endothermic event in the DSC thermogram having an onset temperature of 85-105°C or such as 90-100°C. In another embodiment, crystalline psilocybin hydrate A is characterized by an endothermic event having an onset temperature of 205-220°C, such as 210-220°C, such as 210-218°C, or such as 210-216°C, and an endothermic event having a temperature beginning 85-105°C or such as 90-100°C, in a DSC thermogram. In yet another embodiment, crystalline psilocybin hydrate A exhibits a DSC thermogram substantially the same as the DSC thermogram in FIG. 8d.

In another embodiment, crystalline psilocybin hydrate A is characterized by a water content of 10-18%, such as 12-16%, or such as about 13%. One 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 has a TGA thermogram mass loss of 10-18%, such as 12-16%, or such as about 13%, at ambient temperature, such as about 25 to 120°C.

In one embodiment, psilocybin crystalline hydrate A is a highly pure crystalline form of hydrate A, for example, psilocybin contains at least 90% by weight, such as 95% by weight, such as 99% by weight, such as 99.5% by weight, hydrate A.

According to another independent aspect of the present invention there is provided a crystalline form of psilocybin, polymorph B, characterized by one or more of

a) peaks in the SRD diffractogram at 11.1, 11.8 and 14.3° 2θ±0.1° 2θ;

b) peaks in the SRD diffraction pattern at 11.1, 11.8 and 14.3° 2θ±0.1° 2θ, additionally characterized by

- 8 043831 with at least one additional peak at 14.9, 15.4, 19.3, 20.0 or 20.6° 2θ±0.1°2θ;

c) PXRD diffraction pattern, which is essentially illustrated in FIG. 7s; or

d) an endothermic event in the DSC thermogram having an onset temperature of 205-220° C., which is essentially illustrated in FIG. 8s.

In one embodiment, polymorph B of psilocybin exhibits a PRD diffraction pattern characterized by the diffraction pattern summarized in table. 4. In one embodiment described herein, crystal polymorph B of psilocybin contains at least 3 peaks (±0.1° 2θ) from table. 4. In the specific embodiment described herein, crystal polymorph B of psilocybin contains at least 4 peaks (±0.1° 2θ) from table. 4. In the specific embodiment described herein, crystal polymorph B of psilocybin contains at least 5 peaks (±0.1° 2θ) from table. 4. In the specific embodiment described herein, crystal polymorph B of psilocybin contains at least 8 peaks (±0.1° 2θ) from table. 4. In the specific embodiment described herein, crystal polymorph B of psilocybin contains at least 10 peaks (±0.1° 2θ) from table. 4.

Table 4

In one embodiment, crystalline polymorph B of psilocybin is characterized by PRD diffractogram peaks at 11.1, 11.8 and 14.3° 2θ±0.1° 2θ. In another embodiment, psilocybin crystal polymorph B is further characterized by at least one peak occurring at 14.9, 15.4, 19.3, 20.0 or 20.6° 2θ±0.1° 2θ. In another embodiment, psilocybin crystal 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 yet another embodiment, crystalline polymorph B of psilocybin exhibits a PXD diffraction pattern substantially the same as the PXD diffractogram shown in FIG. 7s.

In one embodiment, crystal polymorph B of psilocybin is characterized by an endothermic event in a DSC thermogram having an onset 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 psilocybin polymorph B exhibits a DSC thermogram substantially the same as the DSC thermogram in FIG. 8s.

- 9 043831

In another embodiment, crystalline polymorph B of psilocybin is characterized by a water content of <0.5% w/w, such as <0.4% w/w, such as <0.3% w/w, such as < 0.2% w/w or such as <0.1% w/w. One skilled in the art will know methods for determining the water content of a compound, such as Karl Fischer titration. In one embodiment, psilocybin crystalline polymorph B is characterized by a loss of <0.5% w/w, such as <0.4% w/w, such as <0.3% w/w, such as <0 .2% w/w, such as <0.1% w/w, in a TGA thermogram at a temperature between ambient temperature, such as 25 to 200°C. In one embodiment, crystalline polymorph B of psilocybin loses less than 2% by weight when tested for loss on drying, such as less than 1% by weight, such as less than 0.5% by weight. Loss on drying testing is carried out at 70°C.

In one embodiment, psilocybin crystalline polymorph B is a highly pure crystalline form of polymorph B, e.g., 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 B.

In another embodiment, crystalline polymorph B of psilocybin is chemically pure, for example, psilocybin has a chemical purity of greater than 97%, such as greater than 98%, or for example greater than 99% by HPLC. In one embodiment, crystalline polymorph B of psilocybin is free of individual impurities greater than 1%, more preferably less than 0.5%, including phosphoric acid as measured by ³¹ P NMR and psilocin as measured by HPLC. In one embodiment, crystalline polymorph B of psilocybin has a chemical purity of greater than 97 area%, more preferably greater than 98 area%, and most preferably greater than 99 area% by HPLC. In one embodiment, crystalline polymorph B of psilocybin is free of individual impurities greater than 1 area%, more preferably less than 0.5 area%, as measured by HPLC. In one embodiment, crystalline polymorph B of psilocybin does not contain psilocin at a level greater than 1% by area, more preferably less than 0.5% by area, as measured by HPLC. In one embodiment, psilocybin crystalline polymorph B is free of phosphoric acid at a level greater than 1 wt%, more preferably less than 0.5 wt%, as measured by ³¹ P NMR. In one embodiment, the psilocybin crystalline polymorph B has a chemical assay of at least 95 wt%, such as at least 96 wt%, or for example at least 98 wt%.

Psilocybin in accordance with this invention in the form of polymorph A or A' has the general properties illustrated in table. 5 below.

Table 5

Appearance: White or off-white solid Main endothermic event in DSC (onset temperature) (corresponding to melting): 210-215°С G playability: Psilocybin forms Hydrate A when humidity is high and when added to water, but water from hydration is quickly lost upon drying. Therefore, an anhydrous form is formed. Crystal Form: Anhydrous Polymorph A and/or A’ pKa (calculated): 1.74, 6.71, 9.75 Solubility approximately 15 mg/ml in water

Psilocybin corresponds to the spectrum presented in table. 6 below and shown in the spectrum in FIG. 10-13.

Table 6

Methodology Conclusions Proton (Ή) and carbon ( ¹³ C) NMR The location of the proton (Fig. 10) and carbon spectrum (Fig. I) corresponds to Psilocybin. PF-infrared spectroscopy (PF-IR) The location of the PF-IR spectrum (Fig. 12) corresponds to Psilocybin. Mass Spectroscopy (MS) Assignment of the mass spectrum (Fig. 13) is concordant with Psilocybin.

High purity is achieved by careful control of reaction conditions to ensure that potential organic impurities are significantly reduced.

Known and potential impurities in psilocybin are shown in Table. 7 below.

- 10 043831

Table 7

Impurity Relative retention time (RRT) Structure Origin Psilocin 1.65 \N- HE 03 N Starting material (stage 3). Also formed during the hydrolysis of Psilocybin. The only significant impurity observed in batches of psilocybin. Stage 4A ABOUT \ ΒηΟ-ψ Ν' ΒηΟ ''О 03 Η The starting product formed in the reaction in step 4. Converts to step 4 with stirring in THF. Converts to Psilocybin at stage 5. Stage 4 2.74 ο \ ^Βη Ν ⁺ 'ΒηΟ'' ^Χ Ο __' ο4 Η Intermediate connection N Densylated stage 4 0\ ΗθΑ No. ΒηΟ' Ό 03 Η MS was identified at stage 4. Converts to Psilocybin at stage 5. Stage 4 anhydride impurity OO \ / Γ, ΒηΟ.^Ο.β Ν ^{+ Βη} ΒηΟ Ό ^9 ο3 Η MS was identified at stage 4. Converts to stage 5 Pyrophosphoric acid impurity in stage 5. Stage 5 Pyrophosphoric acid impurity 0 Ο \Η ⁺ ΗΟ,^Ο^ Ν BUT Ό 9 03 Η MS identified. Formed from the anhydride of step 4. Removed in step 6 by recrystallization by a combination of hydrolysis to Psilocybin and increases solubility due to the extra phosphate group.

- 11 043831

Stage 5 (intermediates) 1.89 and 2.45 o \n ⁺ Ό-φ N- BnO' ^X O ' 03 no \ ^Vn no''o 03 n 2 intermediates are formed during hydrogenation. They are then converted into a product (chemistry-based structures). Tracked and controlled in the reaction at step 5.

Also, careful processing ensures that solvent levels remain below the levels shown in the table. 8.

Table 8

Solvent Controlled Chemical step in which a solvent is used Methanol 3000 ppm Stage 5 Ethanol 5000 ppm Stage 5 THF 720 ppm Stage 4 Toluene 890 ppm Formed as a by-product in stage 5

Through careful selection of the operating procedure, the psilocybin drug substance in accordance with this invention meets the acceptance criteria specified in table. 9 below.

Table 9

Sign of quality Eligibility Criteria Way testing 1. Appearance For information only. Visual 2. Identification by Ή NMR Compare with the reference. Fig. 10 Ή NMR 3. Identification by ¹³ C NMR Compare with the reference. Fig. AND ¹³ NMR 4. Identification by MS Compare with the reference. Fig. 12 MS 5. Identification by PF-IR Compare with the reference. Fig. 13 PF-IR 6. Loss on drying NBC 2% w/w European

- 12 043831

pharmacopoeia 2,2,32 7. Combustion residue NBC 0.5% w/w Pharmacopoeia USA <281> 8. Chemical purity NMC 97% area HPLC 9. Drug-related impurities No impurity NBC 1.0% area HPLC 10. Analysis (dry basis) 95-103% wt. HPLC 11. Residual solvent content Methanol NBC 3000 ppm. Ethanol NBC 5000 ppm. THF NBC 720 ppm. Toluene NBC 890 ppm. GHVR 12. Phosphoric acid content NBC 1% w/w ³¹ RNMR 13. Elemental analysis ICP-MS Cd NBC 1.5 ppm. Pb NBC 1.5 ppm. As NBC 4.5 ppm. Hg NBC 9.0 ppm. With NBC 15 ppm. V NBC 30 ppm. Ni NBC 60 ppm Li NBC 165 ppm Pd NBC 30 ppm. Pharmacopoeia USA <233> 15. Polymorphism Corresponds to the link. Fig. 7a PRD 16. Melting point Report result. Fig. 8a DSK

Abbreviations used in the table: NBC = no more than;

NMF = no less than.

The methodology used for verification is presented in the Detailed Description section. In fact, criteria 6-13 are significantly exceeded in practice, as noted in Table. 10 below.

Table 10

Sign of quality Eligibility Criteria Usually Test method 1. Loss on drying Typically less than 1% w/w. European Pharmacopoeia 2,2,32 2. Combustion residue Typically less than 0.2% w/w. Pharmacopoeia USA <281> 3. Chemical purity Usually NMC 99% HPLC

- 13 043831

4. Related drug impurities No impurity NBC 1.0% OVU 1.49: 0.06% OVU 1.69 (Psilocin): 0.39% OVU 1.70: 0.05% Other MP 0.05%: 0.22% HPLC 5. Analysis (dry basis) 95-103 98.65% HPLC 6. Residual solvent content Methanol NBC 3000 ppm. Ethanol NBC 5000 ppm. THF NBC 720 ppm. Toluene NBC 890 ppm. NBC 5 ppm NBC 10 ppm NBC 5 ppm NBC 5 ppm ΓΧΒΡ Content phosphoric acid NBC 1% w/w 0.2% No phosphoric acid (H ₃ PO ₄ ), which starts at approximately 0 ppm. ³¹ RNMR Elemental analysis by ICP-MS Cd NBC 1.5 ppm. Pb NBC 1.5 ppm. As NBC 4.5 ppm. Hg NBC 9.0 ppm. With NBC 15 ppm. V NBC 30 ppm. Ni NBC 60 ppm MP 0.5 ppm. MP 0.5 ppm. MP 1 ppm MP 1 ppm MP 5 ppm MP 20 ppm MP 10 ppm Pharmacopoeia USA <233> Li NBC 165 ppm Pd NBC 30 ppm. MP 20 ppm MP 5 ppm

Abbreviations used in the table:

NBC=no more than;

MCH=less than.

Thus, crystalline psilocybin in the form of polymorph A or polymorph A' has a spectrum that is confirmed by proton (1H) and carbon ( ¹³ C) NMR, PF-infrared spectroscopy (PF-IR) and mass spectroscopy (MS); fig. 10-13.

It also meets any criteria specified in the table. 9 or 10.

According to the second aspect of the present invention, there is provided a batch of crystalline psilocybin in the form of polymorph A or polymorph A' in accordance with the first aspect of the present invention. In one embodiment, there is a lot of crystalline psilocybin, polymorph A, or polymorph A' containing at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, there is a lot of crystalline psilocybin, polymorph A, or polymorph A', containing at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, there is a batch of high purity psilocybin containing at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, there is a batch of polymorph A of high purity psilocybin containing at least 10 g, more preferably at least 100 g, and most preferably at least 250 g. In one embodiment, there is a batch of polymorph A' high purity psilocybin containing at least 10 g, more preferably at least 100 g and most preferably at least 250 g.

Alternatively and independently, crystalline psilocybin can take the form of hydrate A or polymorph B.

- 14 043831

According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising crystalline psilocybin and one or more excipients.

In one embodiment, a pharmaceutical composition is provided comprising high purity psilocybin and one or more excipients. In another embodiment, a pharmaceutical composition is provided comprising crystalline polymorph A of psilocybin and one or more excipients. In another embodiment, a pharmaceutical composition is provided comprising a crystalline polymorph A' of psilocybin and one or more excipients. In another embodiment, a pharmaceutical composition is provided comprising high purity crystalline psilocybin, polymorph A or polymorph A', and one or more excipients. In another embodiment, a pharmaceutical composition is provided comprising a high purity crystalline polymorph A of psilocybin and one or more excipients. In another embodiment, a pharmaceutical composition is provided comprising a high purity crystalline polymorph A' of psilocybin and one or more excipients.

Alternatively and independently, the crystalline psilocybin in the formulation may take the form of hydrate A or polymorph B.

Preferred pharmaceutical excipients for oral administration include diluents such as microcrystalline cellulose, starch, mannitol, anhydrous calcium phosphate, or mixtures of silica, calcium carbonate, microcrystalline cellulose and talc; raising agents such as sodium starch glycolate or croscarmellose sodium; binding agents such as povidone, sopovidone or hydroxypropylcellulose; lubricants such as magnesium stearate or sodium stearyl fumarate; glidants such as colloidal silicon dioxide; and film coatings such as white Opadry II or brown PVA-based Opadry II.

Psilocybin is a difficult active ingredient to formulate for a variety of reasons. Firstly, it has poor flow characteristics, and secondly, it is used in relatively low doses, which together make it difficult to ensure uniformity of the contents when tableting.

A good mixture will have an Acceptance Criterion, IC value of less than 15 and more preferably less than 10.

It also has a percentage of stated value greater than 90%, more preferably greater than 94%.

Between them, these parameters show the appropriate dosing of psilocybin between pills.

For most pharmaceutical tablets, standard excipients, in particular excipients, can be used. However, during the process of formulating psilocybin tablets, the applicant discovered that a non-standard filler was preferred to obtain a satisfactory product.

In connection with this, a functional filler is chosen. The functional filler is a silicified filler, preferably silicified microcrystalline cellulose. Preferred forms contain highly compressible grades with particle sizes of about 45-150 microns.

In fact, a mixture of two functional fillers having different particle size ranges with mass fractions of the two favoring larger particle sizes can be used.

In one embodiment, the silicated microcrystalline filler may comprise a first filler having a particle size of about 45-80 microns in an amount of up to 30%, more preferably up to 20%, more preferably up to 15% or less, and a second filler having a particle size of about 90-150 microns in amounts up to 70%, more preferably up to 80% and more preferably up to 85% or more by weight.

The composition may further contain or consist of a disintegrant, preferably sodium starch glycolate, a glidant, preferably colloidal silica, and a lubricant, preferably sodium stearyl fumarate.

Other details of formulation development are given in Example 12.

It should be noted that the formulations may contain psilocybin in any form, not just the preferred polymorphic forms described.

In Students et al. (2011), J. Psychopharmacol., 25(11):1434-1452 classified oral doses of psilocybin as follows: very low doses 0.045 mg/kg, low doses 0.115-0.125 mg/kg, moderate doses 0.115-0.260 mg/kg and high doses 0.315 mg/kg.

Psilocybin is typically present in a formulated dose at levels ranging from 0.01 to 1 mg/kg. A typical human dose (for an adult weighing 60-80 kg) would be approximately 0.60 to 80 mg. In one embodiment, 2-50 mg of crystalline psilocybin most preferably polymorph A or polymorph A' is present in a formulated dose such as 2-40 mg, such as 2-10 mg, such as 5 mg, such as 5-30 mg, such as 5-15 mg, such as 10 mg, such as 20-30 mg or such as 25 mg. In one embodiment, 2-50 mg of crystalline psilocybin, particularly polymorph A, is present in a formulated dose, for example 2-40 mg, for example 2-10 mg, for example 5 mg, for example 5-30 mg, for example 5-15 mg, for example 10 mg, for example 20-30 mg or for example 25 mg. In one embodiment, 2-50 mg of crystalline psilocybin, particularly polymorph A', is present in a formulated dose, e.g. 2-40 mg, e.g. 2-10 mg, e.g. 5 mg, e.g. 5-30 mg, e.g. 5-15 mg, e.g. 10 mg, for example 20-30 mg

- 15 043831 or for example 25 mg.

Preferred dosages for adults are likely to range from 1 to 40 mg, preferably 2 to 30 mg, more preferably 15 to 30 mg, for example 5, 10 or 25 mg. Microdosing, usually tenths of these doses, is also possible with microdose formulations typically in the range of 0.05 to 2.5 mg.

The preferred pharmaceutical formulation is an oral form.

The oral dosage form may be a tablet or capsule.

The tablet requires the ability to accurately disperse the active substance. This is difficult due to the low doses and the hygroscopic and sticky nature of the active substance, which limits fluidity.

Psilocybin is present together with one or more excipients. Preferred excipients include microcrystalline cellulose and starch, more particularly silicated microcrystalline cellulose.

According to a fourth aspect of the present invention, crystalline psilocybin is provided in the form of polymorph A or polymorph A' according to the first aspect of the present invention for use in medicine. In one embodiment, a crystalline polymorph A of psilocybin is provided for medical use. In one embodiment, a crystalline polymorph A' of psilocybin is provided for medical use. In one embodiment, a high purity crystalline polymorph A of psilocybin is provided for medical use. In one embodiment, a high purity crystalline polymorph A' of psilocybin is provided for medical use.

Alternatively and independently, crystalline psilocybin can take the form of hydrate A or polymorph B.

According to a fifth aspect of the present invention, crystalline psilocybin is provided in the form of polymorph A or polymorph A' according to the first aspect of the present invention for use in the treatment of disorders of the central nervous system.

Alternatively and independently, crystalline psilocybin can take the form of hydrate A or polymorph B.

In one embodiment, crystalline psilocybin, polymorph A or polymorph A', is provided for use in the treatment of depression. In one embodiment, crystalline psilocybin, polymorph A or polymorph A', is provided for use in the treatment of drug-resistant depression. In one embodiment, a crystal polymorph A of psilocybin is provided for use in the treatment of drug-resistant depression. In one embodiment, a crystalline polymorph A' of psilocybin is provided for use in the treatment of drug-resistant depression. In one embodiment, a high purity crystalline polymorph A of psilocybin is provided for use in the treatment of drug-resistant depression. In one embodiment, a high purity crystalline polymorph A' of psilocybin is provided for use in the treatment of drug-resistant depression.

Other conditions that may be treated include anxiety disorders, including anxiety in advanced illness, such as cancer, and generalized anxiety disorder, depression, including clinical depression, histamine headaches, obsessive-compulsive disorder, personality disorders, including conduct disorder, medications disorders including: alcohol addiction, nicotine addiction, opioid addiction, cocaine addiction and other addictions, including gambling addiction, food addiction and nutrition disorder and body dysmorphic disorder. Another condition is pain management.

According to a sixth aspect of the present invention, there is provided a method for treating central nervous system disorders, comprising administering to a subject in need thereof an effective dose of crystalline psilocybin in the form of polymorph A or polymorph A' according to the first aspect of the present invention.

In one embodiment, a method of treating depression is provided, comprising administering to a subject in need thereof an effective dose of crystalline psilocybin in the form of polymorph A or polymorph A'. In one embodiment, a method of treating drug-resistant depression is provided, comprising administering to a subject in need thereof an effective dose of crystalline psilocybin in the form of polymorph A or polymorph A'. In one embodiment, a method of treating drug-resistant depression is provided, comprising administering to a subject in need thereof an effective dose of psilocybin polymorph A. In one embodiment, a method of treating drug-resistant depression is provided, comprising administering to a subject in need thereof an effective dose of psilocybin polymorph A'. In one embodiment, a method of treating drug-resistant depression is provided, comprising administering to a subject in need thereof an effective dose of high purity crystalline polymorph A of psilocybin. In one embodiment, a method of treating drug-resistant depression is provided, comprising administering to a subject in need thereof an effective dose of a high purity crystalline polymorph A' of psilocybin.

Alternatively and independently, crystalline psilocybin can take the form of hydrate A or polymorph B.

- 16 043831

To produce psilocybin in accordance with this invention, psilocybin is crystallized from water in a controlled manner.

According to a seventh aspect of the present invention, there is provided a method for large-scale production of psilocybin, characterized in that the method includes the step of aqueous crystallization of psilocybin with controlled drying to obtain crystalline polymorph A of psilocybin according to the first aspect of the present invention.

In one embodiment, a method for large-scale production of psilocybin is provided, characterized in that the method includes the step of aqueous crystallization of psilocybin with controlled drying to produce crystalline psilocybin polymorph A with a PRD diffractogram, which is shown in FIG. 7a and DSC and TGA thermograms, which are shown in Fig. 8a. In one embodiment, a method for large-scale production of psilocybin is provided, characterized in that the method includes the step of aqueous crystallization of psilocybin with controlled drying to produce high purity crystalline psilocybin polymorph A with a PXRD pattern as shown in FIG. 7a and DSC thermogram, which is shown in Fig. 8a.

Preferably, polymorph A is an isostructural variant with a PXRD pattern as shown in FIG. 7a, and the DSC thermogram, which is shown in FIG. 8a.

More preferably, psilocybin is recrystallized, usually in about 10-20 volumes of water, heated with stirring to a temperature of at least 70°C, further purified on a filter with a suitable cutoff (usually below 5 microns), sown at a temperature of about 70°C and cooled under control to about 5°C for more than 2 hours.

More preferably, the method includes controlled cooling that lowers the temperature by about 5-15°C/hour, more preferably about 10°C/hour.

Preferably, the filter post-treatment step is carried out through a suitable sized filter, such as a 1.2 µm in-line filter.

Preferably, mixing is carried out by stirring at about 400-500 rpm, usually about 450 rpm.

Preferably the primer is psilocybin hydrate A. In one embodiment, 0.1% by weight or less is added to the process.

Preferably, crystalline psilocybin is isolated by vacuum filtration.

In one embodiment, the isolated crystals are dried in vacuum at a temperature of at least 30°C, such as 30-50°C or such as 40-50°C. In one embodiment, the isolated crystals are dried under vacuum for at least 10 hours, such as 12-18 hours or for example about 30 hours. In one embodiment, the isolated crystals are dried under vacuum at a temperature of at least 30° C., such as 30-50° C, or such as 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 the isolated crystals have lost less than 2% by weight in loss on drying test, for example less than 0.5% by mass.

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

The resulting crystals are typically relatively large (50-200 microns) and uniform when viewed under a 10x microscope, as shown in FIG. 16a.

This is in contrast to crystals produced without controlled cooling, which are much smaller in size (typically 5-50 microns) when viewed under a 10x microscope, as shown in FIG. 16b.

According to the eighth aspect of the present invention, there is provided psilocybin according to the first aspect of the present invention obtained by the crystallization method in accordance with the present invention.

According to the ninth aspect of the present invention, there is provided a pharmaceutical composition containing psilocybin, according to the first aspect of the present invention, obtained by the crystallization method in accordance with the present invention.

Psilocybin produced before crystallization can be produced by any method: synthetic or biological, such as fermentation, or obtained by extraction from mushrooms.

Preferred production methods use psilocin or 4-hydroxyindole as the starting material.

According to a tenth aspect of the present invention, there is provided a method for the large-scale production of psilocybin from psilocin, comprising the following steps:

i) step 4 - reaction of psilocin with tetrabenzyl pyrophosphate to produce benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1 H-indol-4-yl phosphate; and ii) step 5 - reacting benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate with hydrogen to produce psilocybin.

According to an eleventh aspect of the present invention, there is provided a method for the large-scale production of psilocybin from 4-hydroxyindole, comprising the following steps:

i) stage 1 - reaction of 4-hydroxyindole with acetic anhydride to obtain acetate

- 17 043831

N-indol-4-yl;

ii) step 2 - reaction of 1H-indol-4-yl acetate with oxalyl chloride and dimethylamine to obtain 3-[(dimethylcarbamoyl)carbonyl]-1H-indol-4-yl acetate;

iii) stage 3 - reaction of 3-[(dimethylcarbamoyl)carbonyl]-1H-indol-4-yl acetate with lithium aluminum hydride to produce psilocin;

iv) step 4 - reaction of psilocin with tetrabenzyl pyrophosphate to produce benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1 H-indol-4-yl phosphate; And

v) step 5 - reaction of benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate with hydrogen to produce psilocybin.

According to a twelfth aspect of the present invention, there is provided a method for large-scale production of psilocybin according to the tenth or eleventh aspect of the present invention, further comprising vi) aqueous crystallization step 6 with controlled drying to produce crystalline psilocybin polymorph A according to the first aspect of the present invention.

In one embodiment, a method for large-scale production of psilocybin according to the tenth or eleventh aspect of the present invention is provided, further comprising vi) aqueous crystallization step 6 with controlled drying to produce crystalline psilocybin polymorph A with a PRD diffractogram which is substantially shown in FIG. 7a, and a DSC thermogram, which is essentially shown in FIG. 8a.

In one embodiment, a method for large-scale production of psilocybin according to the tenth or eleventh aspect of the present invention is provided, further comprising vi) aqueous crystallization step 6 with controlled drying to produce high purity crystalline psilocybin polymorph A with a PRD diffractogram substantially as shown in FIG. 7a, and a DSC thermogram, which is essentially shown in FIG. 8a.

Preferably, the crystalline psilocybin is polymorph A.

In developing a method for the large-scale production of psilocin or psilocybin, the applicant has overcome one or more significant problems in each of steps 1-5, and while these problems are considered in the context of each stage of the large-scale production of psilocin or psilocybin, or rather methods for overcoming each problem are considered separate and independent inventions, since they are used in the production of other active substances, whether they are intermediates for psilocin, psilocybin, or other derivatives, salts, esters and the like, which provide a prodrug.

Preferably, reaction step 4 (i) involves the use of sodium hexamethyldisalazide (NaHMDS).

It has advantages over the use of butyllithium in that

i) easier to handle; and ii) it does not introduce lithium into the reaction, which could raise issues downstream in the production process.

Preferably, the solvent used in the reaction is THF.

The advantage is that the resulting product is obtained with significantly greater purity.

Preferably, in (i) the reaction is started at a temperature below -50°C.

The benefit is the reduction of impurity levels (m/z 295.2 by LCMS), which significantly impacts downstream purity.

More preferably, THF is used as a solvent in step 4 (ii).

The advantage is to ensure that there is no thickening and to facilitate simple mixing to obtain the product.

Preferably, step 4 (ii) involves a stirring process to produce benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate.

The mixing process has the advantage that the process is simplified and the yield is improved.

To ensure that the reaction in step 4 (ii) is complete, the levels of intermediate 4A are monitored and, when complete, the benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate is filtered and oven dried.

The advantage is that impurities are minimized and a cleaner product is obtained.

Preferably, the reaction in Step 5 is monitored for levels of intermediates by HPLC using relative retention time (RRT) and completion is determined by the presence of intermediates of less than 0.2%.

Crude psilocybin (Step 5 product, (12)) has Stage 5 based impurities whose HPLC relative retention time (RRT) is about 1.89 and 2.45, respectively, and psilocin (RTR 1.66). These impurities are shown in table. 7. Usually unrefined psilocybin (product

- 18 043831 stage 5 (12)) has 0.24% of the impurity area with an OHC of 1.89, 0.03% of the impurity area with an OHC of 2.45 and 1.86% of the area of psilocin. In addition, the impurity pyrophosphoric acid (OVU 0.31) is present in crude psilocybin, for example, at a level of about 2-6 area% by HPLC.

At this level, subsequent crystallization processes can be carried out to obtain substantially pure psilocybin, for example, psilocybin having a purity of at least 95 area % HPLC, such as at least 98 area %, or such as at least 99 area % purity. In one embodiment, the pyrophosphoric acid impurity (OVV 0.31) is present in substantially pure psilocybin at a level of less than 0.3 area % by HPLC, for example less than 0.2 area % or for example less than 0.1 area %.

Additionally, during this step, water is added to the reaction mixture to keep the psilocybin in solution.

Preferably, the catalyst is recovered by filtration.

Preferably, in step 1 the reaction is carried out in DCM and pyridine.

The advantage is that there are no flammable solvents.

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

The advantage is that intermediate 2A can be isolated, allowing purification without excess oxalyl chloride.

More preferably, the DCM phase is then washed with sodium bicarbonate at a pH of about 8.

The advantage is cleaner processing.

Preferably, 1H-indol-4-yl acetate is precipitated in heptane.

This helps precipitation and overcomes some of the partial solubility issues.

Preferably, magnesium sulfate is used as a drying agent.

The preferred solvents used are tert-butyl methyl ether (MTBE) and tetrahydrofuran (THF).

Preferably, the reaction with oxalyl chloride is carried out at about 30-40°C.

The benefit is to provide faster response rates resulting in improved levels of completion.

Preferably, intermediate 2A is isolated by filtration.

The advantage is that the intermediate is purified without excess oxalyl chloride.

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

Preferably, intermediate 2A is washed with MTBE.

Preferably, heptane is added to precipitate additional intermediate 2A.

Preferably, in step 2, step (ii), dimethylamine is used in excess.

The benefit is a significantly improved impurity profile and yield obtained.

Preferably, the pH is maintained at or above pH 7.

Preferably the reaction is carried out in MTBE.

Preferably, this step further includes a purification step that removes the dimethylamine salts.

The benefit is improved cleanliness.

Preferably, this step includes suspension and filtration steps.

The benefit is improved processability and cleanliness.

More preferably, it involves suspending with water and/or IPA, filtering and drying the isolated 3-[(dimethylcarbamoyl)carbonyl]-1H-indol-4-yl acetate.

The benefit is improved purity and yield and reduced hydrolysis.

Preferably, in step 3, the reaction is carried out in the solvent THF.

The advantage is to obtain a suspension/emulsion without thickening.

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

Preferably, the reaction is quenched with acetone, then citric acid, ensuring that the mixture remains strongly alkaline (pH 11 or higher).

The advantage is to obtain a higher yield.

Preferably, the psilocin is filtered and washed in THF and suspended in ProAc:MTBE, filtered, washed in MTBE and dried.

The advantage is to obtain a product of high purity, eg at least 95% HPLC pure, eg at least 98% HPLC pure, or eg at least 99% HPLC pure.

The preferred production method includes each of steps 1-6, but it should be understood that each of the characteristics may remain alone or may be used in combination with any other characteristic from the same or a different reaction step.

- 19 043831

Psilocybin of this form, polymorph A or polymorph A', and psilocybin of such high purity have not previously been obtained, and applicants are aware that their preparation of polymorph A and polymorph A', in particular (which are shown in Figs. 7a, 7b and 8a, 8b) , is new. Indeed, the production of large quantities of polymorph A is new. The result of the crystallization technique in accordance with this invention and part of the manufacturing process is to obtain such high chemical purity of crystalline psilocybin.

Moreover, given the unstable nature of the compound, they obtained a crystalline form that was found to be stable under harsh conditions (described below) for at least 12 months.

Polymorph A and A' (Fig. 7a and 7b) are different from polymorph B (Fig. 7c), hydrate A (Fig. 7d) and ethanol solvate (Fig. 7e) and mixture (Fig. 7f (top)), which is obvious from their SRD diffractograms and DSC thermograms, as described below.

The relationship between the various polymorphs is shown in FIG. 9.

Indeed, the size and shape of the crystals are determined by the crystallization technique, and these, in turn, can affect the stability and compoundability of the product.

In a particularly preferred embodiment, psilocybin is produced in a 6-step process as shown below.

According to another aspect of the present invention, there is provided a method for producing crystalline psilocybin according to the first aspect of the present invention, characterized in that the method comprises aqueous crystallization of psilocybin with controlled drying to produce crystalline psilocybin polymorph A or polymorph A' according to the first aspect of the present invention. In one embodiment, there is provided a method for producing crystalline psilocybin according to the first aspect of the present invention, characterized in that the method comprises aqueous crystallization, with controlled drying, to produce crystalline psilocybin polymorph A or polymorph A' with a PRD diffractogram that is substantially shown in FIG. 7a or 7b, and a DSC thermogram, which is essentially shown in FIG. 8a or 8b. In one embodiment, a method for producing psilocybin according to the first aspect of the present invention is provided, characterized in that the method comprises aqueous crystallization with controlled drying to produce high purity crystalline psilocybin polymorph A or polymorph A' with a PRD diffractogram as shown in FIG. 7a or 7b and the DSC thermogram, which is shown in FIG. 8a or 8b.

Preferably, polymorph A and polymorph A' are isostructural variants with PXRD patterns substantially shown in FIG. 7a and 7b, and DSC thermograms, which are essentially shown in FIGS. 8a and 8b.

More preferably, psilocybin is recrystallized in about 10-20 volumes of water, heated with stirring to a temperature of at least 70°C, further purified on a filter with a suitable cut (usually below 5 microns), sown at a temperature of about 70°C and cooled under control to about 5°C for more than 2 hours.

More preferably, the method includes controlled cooling that reduces the temperature by about 5-15°C per hour, more preferably about 10°C/hour.

Preferably, the filter post-treatment step is carried out through a suitable filter size, such as a 1.2 or 0.45 micron in-line filter.

Preferably, mixing is carried out by stirring at about 400-500 rpm, usually about 450 rpm.

Preferably the primer is psilocybin hydrate A. In one embodiment, 0.1% by weight or less of the seed is added to the process.

Preferably, crystalline psilocybin is isolated by vacuum filtration.

In one embodiment, the isolated crystals are dried in vacuum at a temperature of at least 30°C, such as 30-50°C or such as 40-50°C. In one embodiment, the isolated crystals are dried under vacuum for at least 10 hours, such as 12-18 hours or for example about 30 hours. In one embodiment, the isolated crystals are dried under vacuum at a temperature of at least 30° C., such as 30-50° C or such as 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 the isolated crystals have lost less than 2% in the loss test when drying, for example, less than 0.5% by weight.

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

The resulting crystals are typically relatively large (50-200 microns) and uniform when viewed under a 10x microscope, as shown in FIG. 16a.

This is in contrast to crystals produced without controlled cooling, which are much smaller in size (typically 5-50 microns) when viewed under a 10x microscope, as shown in FIG. 16b.

- 20 043831

Stage 1. Synthesis of 1H-indol-4-yl acetate (3).

The main reaction is the reaction of 4-hydroxyindole (1) with acetic anhydride (2) to produce 1H-indol-4-yl acetate (3) (Fig. 2).

Most preferably, step 1 proceeds as follows.

4-Hydroxyindole (1), DCM (12) and pyridine (13) are added to the vessel and cooled to about 0-5°C. Acetic anhydride (2) is added dropwise, the mixture is heated to about 20-25°C and stirred until completely dissolved by HPLC. The reagents are washed with aqueous citric acid (14) and aqueous NaHCO ₃ (15), dried over MgSO ₄ (16), filtered and evaporated to approximately half the volume. Heptane (17) is added and distillation is carried out to remove the bulk of the DCM. The mixture is cooled to about 5-25°C, filtered, washed with heptane and dried in a vacuum oven overnight to isolate 1H-indol-4-yl acetate (3) as a solid suitable for use in the next step.

Step 2. Synthesis of 3-[(dimethylcarbamoyl)carbonyl]-1H-indol-4-yl acetate (6).

The main reaction is the reaction of 3-indol-4-yl acetate (3) with oxalyl chloride (4) and dimethylamine (5) to produce 3-[(dimethylcarbamoyl)carbonyl]-1H-indol-4-yl acetate (6) (Fig. 3).

Most preferably, step 2 proceeds as follows.

1H-indol-4-yl acetate (3) is dissolved in a mixture of THF (19) and MTBE (18) at room temperature. Oxalyl chloride (4) is added dropwise, allowing an exothermic reaction at about 35-40°C. The temperature range is maintained throughout the addition. The reaction mixture is then stirred at about 40°C until complete by HPLC. The reaction mixture is cooled to room temperature and heptane (17) is added, which causes additional solid to precipitate. The suspension is stirred, then allowed to settle, then the bulk of the solvent is removed (18/19) by decanting. The solid is washed twice in the vessel with heptane (17). MTBE (18) is added to produce a yellow suspension and the mixture is cooled to about -20°C. Add dimethylamine solution (5), maintaining the temperature between -20 and -10°C. The reaction mixture is then warmed to room temperature and stirred until complete, adding additional dimethylamine if necessary. The reaction mixture is filtered, washed with heptane (17) and dried in a vacuum oven. Crude 3-[(dimethylcarbamoyl)carbonyl]1H-indol-4-yl acetate (6) is then purified by suspending in water (20), then IPA (21) and then dried in a vacuum oven to give (6) as a solid for application at the next stage.

Step 3. Synthesis of 3-(2-(dimethylamino)ethyl)-1H-indol-4-ol (psilocin) (8).

The main reaction is the reaction of 3-[(dimethylcarbamoyl)carbonyl]-III-indol-4-yl acetate (6) with lithium aluminum hydride (7) to produce psilocin (8) (Fig. 4).

Most preferably, step 3 proceeds as follows.

3-[(dimethylcarbamoyl)carbonyl]-1H-indol-4-yl acetate (6) is stirred in THF (19) and cooled to about 0°C. THF solution of LiAlH ₄ (7) is added dropwise, maintaining the temperature at about 0-20°C. The reaction mixture is then refluxed until complete by HPLC. The reaction mixture is cooled to 0°C and the excess LiAlH ₄ is quenched by adding acetone (22), then an aqueous solution of citric acid (14). The batch is then filtered to remove lithium and aluminum salts. The filtrate is dried over MgSO ₄ (16), filtered and concentrated and loaded onto a layer of silica (23). The layer is eluted with THF (19) and the fractions containing the product are evaporated. The resulting solid is suspended in iPrOAc:MTBE (24/18), filtered and washed with MTBE. The solid is dried in an oven to obtain high purity psilocin (8) as a whitish substance.

Step 4. Synthesis of benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10).

The main reaction is the reaction of psilocin (8) with tetrabenzyl pyrophosphate (9) to produce benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10) (Fig. 5).

Most preferably, step 4 proceeds as follows.

Load psilocin (8) into the vessel, then add THF (19). The reaction mixture is cooled to a temperature of -50 to -70°C and NaHMDS (25) is added dropwise at a temperature of about -45 to -70°C. The temperature is adjusted to -45 to -60°C and tetrabenzyl pyrophosphate in THF is added. The batch is heated to 0°C, then solid by-products are removed by filtration and the filtrate is concentrated in vacuo. The concentrated mixture is then heated to about 40°C and stirred until the intermediate is converted to the product of step 4(10), monitored by monitoring and using HPLC. The batch is cooled to about 0-5°C and the resulting solid is removed by filtration and dried in vacuo to give benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10) as a solid.

Step 5. Synthesis of the intermediate 3-[2-(dimethylazaniumyl)ethyl]-1H-indol-4-yl hydrogen phosphate (crude psilocybin) (12).

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

Most preferably, step 5 proceeds as follows.

Pd/C (26), methanol (24) and 3-[2-(benzyldimethylazaniumyl)ethyl]-1H- 21 043831 indol-4-yl phosphate (10) are loaded into the vessel and the resulting mixture is purged with hydrogen (11) until completion. HPLC. Purified water (20) is added during this process to keep the product in solution. The mixture is heated to approx.

35-45°C and then filtered through a layer of celite (27), washing with methanol (24) and purified water (20).

The filtrate is evaporated in vacuo by azeotroping with ethanol (28), yielding the intermediate psilocybin (12).

Stage 6. Synthesis of 3-[2-(dimethylazaniumyl)ethyl]-1H-indol-4-yl hydrogen phosphate (psilocybin).

The primary purification/polymorph determination step is an aqueous crystallization step followed by a controlled cooling and drying step to obtain high purity crystalline psilocybin, polymorph A, or polymorph A'.

Most preferably, step 6 proceeds as follows.

Intermediate psilocybin (12) (step 5) is loaded into a vessel with purified water (20) and the mixture is heated until the psilocybin (12) dissolves. The resulting stock solution is then purified by filtration into a preheated vessel. The temperature is adjusted to preferably about 68°C-70°C and the psilocybin hydrate seed (ie hydrate A) is added to the reaction mixture. The batch is then cooled under control to about 0-10°C and stirred and the solids are collected by filtration and washed with purified water. The isolated solids are then dried under vacuum to obtain high purity crystalline psilocybin, polymorph A or A', as an off-white solid.

Brief description of drawings

Embodiments of the present invention are described below with reference to the accompanying drawings.

In fig. 1 shows the reaction scheme described in JNP.

In fig. 2 is a reaction diagram from Step 1 of one aspect of the present invention.

In fig. 3 is a reaction diagram from Step 2 of one aspect of the present invention.

In fig. 4 is a reaction diagram from Step 3 of one aspect of the present invention.

In fig. 5 is a reaction diagram from Step 4 of one aspect of the present invention.

In fig. 6 is a reaction diagram from Step 5 of one aspect of the present invention.

In fig. Figure 7a shows a PXRD diffraction pattern of polymorph A (GM764B).

In fig. Figure 7b shows the SRD diffractogram of polymorph A' (JCCA2160F).

In fig. Figure 7c shows the SRD diffractogram of polymorph B; (JCCA2160-F-TM2).

In fig. Figure 7d shows the SRD diffractogram of hydrate A (JCCA2157E).

In fig. Figure 7f shows a PRD diffraction pattern of ethanol solvate (JCCA2158D).

In fig. 7f shows the PXRD pattern of the product obtained during process development (CB 646-E) (top), compared with the XRD patterns of polymorph A' (JCCA2160F) (middle) and polymorph B (JCCA2160-TM2) (bottom).

In fig. Figure 8a shows a DSC and TGA thermogram of polymorph A (GM764B).

In fig. Figure 8b shows a DSC and TGA thermogram of polymorph A' (JCCA2160F).

In fig. Figure 8c shows a DSC thermogram of polymorph B (GM748A).

In fig. Figure 8d shows the DSC and TGA thermogram of hydrate A (JCCA2157E).

In fig. Figure 8f shows the DSC and TGA thermogram of the ethanol solvate (JCCA2158D).

In fig. 9 Shape phase diagram showing shape relationships in water-based systems.

In fig. Figure 10 shows the 1H NMR spectrum of psilocybin (read together with the corresponding example 7).

In fig. Figure 11 shows ^{the 13} C NMR spectrum of psilocybin (read together with the corresponding example 7).

In fig. Figure 12 shows the PF-IR spectrum of psilocybin.

In fig. Figure 13 shows the mass spectrum of psilocybin.

In fig. Figure 14 shows the numbered structural form of psilocybin.

In fig. Figure 15 shows the solubility versus temperature curve for psilocybin in water.

In fig. 16a is a micrograph showing crystals obtained under controlled cooling.

In fig. 16b is a micrograph showing crystals obtained by uncontrolled cooling drying.

In fig. Figure 17 is a shape phase diagram showing the relationships between shapes in different solvent systems.

In fig. Figure 18 shows a PRD diffraction pattern - template C for solids isolated at 25 and 50°C.

In fig. Figure 19 shows PRD diffraction patterns - templates D, E and F for solids isolated at 25 and 50°C.

In fig. Figure 20 shows a comparison of PRD diffractograms obtained for solids isolated by equilibrating amorphous psilocybin in solvents A-H.

In fig. Figure 21 shows a comparison of PRD diffraction patterns obtained for solids isolated by equilibrating amorphous psilocybin in solvents I-P.

In fig. Figure 22 shows a comparison of PRD diffractograms obtained for solids isolated by equilibrating amorphous psilocybin in solvents R-Y.

Detailed description

In contrast to the prior art, this invention contemplates the production of psilocybin on a commercial scale in quantities or batches of at least 100 g and more preferably at least 250 g levels at 1 log or 2 log higher than the levels described in JNP, where described large-scale method of obtaining quantities in grams at the 10 g scale level.

To demonstrate the many significant stages of development from JNP, the description below provides details of experiments and studies conducted at each of the process stages, which illustrate the choices made to overcome the multiple technical challenges encountered in producing psilocybin (7) for GMP on a large scale (including various intermediates (2-6)), starting with 4-hydroxyindole (1).

A reference to a particular numerical value includes at least that particular value unless the context clearly requires otherwise. When a range of values is expressed, another embodiment includes from one specific value and/or to another specific value. In addition, reference to values specified in intervals includes each and every value within that interval. All intervals are inclusive and combinable.

When values are expressed as approximations using the preceding approximation, it should be clear that a particular value constitutes another option.

As used here, singular forms include the plural.

The term about when used in reference to numerical ranges, limits, or specific values is used to indicate that the values given may vary by up to 10% of the stated value. Since many of the numerical values used herein are determined experimentally, those skilled in the art will appreciate that such determinations can, and often will, vary between different experiments. The values used herein should not be construed as unduly limiting due to this inherent variation. Thus, the term about is used to include changes of ±10% or less, changes of ±5% or less, changes of ±1% or less, changes of ±0.5% or less, or changes of ±0.1% or less of the specified value.

As used herein, the term treatment and similar terms refer to reducing the severity and/or frequency of symptoms, eliminating symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of symptoms and/or the underlying cause thereof, delaying, preventing and/or slowing the progression of diseases and /or disorders and improvement or elimination of damage caused, directly or indirectly, by diseases and/or disorders.

The following abbreviations are used here:

DSC - differential scanning calorimetry;

RT - room temperature;

MTBE - methyl tert-butyl ether;

TGA - thermogravimetric analysis;

THF - tetrahydrofuran;

rel. - in a relationship;

PXD - powder x-ray diffraction.

Example 1. Step 6. Crystallization method and resulting polymorphs.

Experiment with obtaining form A'.

1.0 g of crude psilocybin is charged into a 25 ml flask. Add water (12.8 ml/16 vol. depending on the activity of the starting material). The mixture is stirred and heated to 80°C. A dark brown solution with visible undissolved solids is obtained. The mixture is further purified by filtration through a heated 0.45 μm filter into a hot 25 ml flask. Undissolved solids are removed to obtain a dark brown solution. The solution is re-equilibrated at 75°C and then slowly cooled (10°C/h) to ambient temperature. The resulting pale brown solution is equilibrated at ambient temperature for 16 hours. The suspension is cooled to 5° C. until the solid is isolated by vacuum filtration. The filter cake is washed with water (0.8 ml/1 vol.) and dried in vacuum at 50°C for 16 hours. Yield 75%, chemical purity 99%, NMR analysis >98%.

The method described above is repeated with 14 volumes (11.2 ml) of water. Yield 69%, chemical purity 99%, NMR analysis >98%.

In both cases, dissolution of crude psilocybin is achieved at about 75°C. With gradual cooling, precipitation is observed at about 60°C.

In both cases, polymorph A' of psilocybin is obtained, confirmed by PRD (diffraction pattern corresponds to Fig. 7b) and DSC (thermogram corresponds to Fig. 8b).

Experiment with obtaining form A.

g Crude psilocybin obtained from step 5 (about 93% purity by HPLC with about

- 23 043831

4% pyrophosphate impurity) is subjected to aqueous recrystallization as follows.

The protocol used a sufficient amount of water (12 volumes), fast stirring speed (450 rpm) and a controlled cooling profile (10°C/h).

Psilocybin (94.0 g) (CB650E) is loaded into a 2 L flask. Add water (902 ml, 12 volumes depending on the activity of the starting material). The mixture is stirred and heated to about 78°C. A dark brown solution with visible undissolved solids is obtained. The mixture is further purified by filtration through a 1.2 μm built-in filter into a hot 5 L flask equipped with an overhead stirrer (450 rpm). Undissolved solids are removed to obtain a clear, dark brown solution. The solution is re-equilibrated at about 75°C and then slowly cooled (10°C/h) to ambient temperature. The solution is inoculated with psilocybin hydrate A (OM758A-PRD diffractogram corresponds to Fig. 7d) followed by maturation in water at 68-70°C. The resulting pale brown suspension was equilibrated at ambient temperature for 16 hours. The suspension was cooled to 5°C for 1 hour until a solid was isolated by vacuum filtration. The filter cake is washed with water (282 ml, 3 volumes) and dried in vacuum at about 50°C for 30 hours.

The method is successfully completed with a yield of 75%. The chemical purity of the solid is confirmed to be 99.3%. Analysis of the PRD solid after drying for 30 h showed polymorph A (Fig. 7a). A characteristic disturbance is observed at about ~17° 2θ, for example 17.5° 2θ, and was noticeable in bulk material.

Characterization of the solid state of polymorph A and polymorph A'.

The DSC and TGA thermograms (Fig. 8a) obtained for polymorph A were comparable to the DSC and TGA thermograms obtained for polymorph A' (Fig. 8b). TGA thermograms (Fig. 8a) obtained for polymorph A and polymorph A' showed no mass loss before decomposition. This suggests that the difference between the PRDs obtained for polymorph A (Fig. 7a; perturbation present at about ~17° 2θ) and for polymorph A', which is obtained on a small scale (Fig. 7b; perturbation absent) was not due to - for excess hydration.

Microscopy of the solid (Fig. 16a) showed rod-shaped crystals with good uniformity with a size range of 50 to 200 microns.

The PXD diffractogram obtained for polymorph A' does not show disturbance at about ~17° 2θ to the same extent as polymorph A. The perturbation in the PXD diffractogram at about ~17° 2θ is more pronounced for psilocybin produced on a large scale (vs. with that obtained on a small scale) and was unexpected. The Applicant has demonstrated that hydrate A is the only polymorph that exists in the temperature range with no diffraction peak in the 17° 2θ region (see Fig. 7d). This strongly suggests that hydrate A breaks down upon dehydration to produce polymorph A or A' that varies in scale, and that polymorph A is the true form with polymorph A' produced on a small scale being atypical.

To test the reliability of this theory and demonstrate the return to polymorph A, a small portion of the volume is re-dried after another soak in water (to recreate hydrate A). A small sample (250 mg - psilocybin polymorph A) was equilibrated in water (10 vol) for 1 hour. The suspension was filtered and wet solid analysis confirmed that hydrate A had been obtained (Fig. 7d), without disturbance at 17° 2θ . The product is dried under vacuum for 16 hours and the solid is re-evaluated by PRD. The product polymorph A' confirms the PRD (Fig. 7b) with a decrease in the observed PRD perturbation. Additional drying of the initial free-flowing solid and keeping it at ambient temperature does not change the PRD diffraction pattern of the solid. Two variants of the solid are obtained; the PRD diffractograms for polymorph A and polymorph A' are virtually identical, except for the -17.5° 2θ peak. The thermal properties are also identical. The difference between the PRD diffraction patterns for polymorph A and polymorph A' is negligible, and both polymorphs kinetically convert to the hydrated state very quickly.

Additional experiments were conducted to verify that the differences in the PRD diffraction patterns for polymorph A and polymorph A' were due to the large-scale crystallization process producing solids with large particle sizes, which then did not dry as efficiently and cause changes, or whether whether the reason was the difference in the crystallization form and the size of the crystalline solid. Psilocybin polymorph A (the polymorphic form confirmed prior to the experiment) was crushed in a mortar and pestle and the PRD was assessed. No changes in the polymorph are noted. Another portion (51 mg) is loaded with water (<1 ml) and assessed in the wet state to confirm hydrate formation. Both batches are vacuum dried at 50°C for about 18 hours and the PRD is re-evaluated. The crushed sample remains polymorph A. The hydrated sample after dehydration is determined to be polymorph A' (i.e., no reflection at ~17.5° 2θ). This suggests that crystallization size/shape alone is not the sole cause for the original reflectance peaks.

TGA evaluation showed that the input batch exhibited negligible mass loss (0.139%

- 24 043831 mass) at about 70°C. The particle size decreases and then the dried solid shows a greater mass loss of 0.343 wt.%, at about 75°C, while the hydrated and dried solid shows a smaller mass loss of 0.069 wt.%, at about 80°C. The particle size is reduced and the dried solid is then held at 80°C for 10 min (beyond the TGA mass loss point), but the PDP evaluation shows no change in the starting material, which means that low levels of hydration and partial swelling of the crystalline matrix are not the reason for the changes.

It is possible to obtain polymorph A' through hydration of polymorph A and subsequent drying of the isolated solid on a small scale.

Polymorph A and polymorph A' of psilocybin, about 60 mg each, are loaded with water, 0.2 ml, to obtain hydrate A from both batches. Half of each hydrate A is dried under vacuum at 25° C. for about 171/4 hours, and the remaining each hydrate A is dried at ambient temperature under N ₂ flow for about 171/4 hours. The solid is isolated after drying and the PRD is assessed. Evaluation of the PDP of solids isolated from the starting polymorph A confirms that hydrate A was successfully prepared and that the solids were dried to polymorph A' by both drying methods. Evaluation of the PDP of solids isolated from the original polymorph A' confirms that hydrate A was successfully obtained and that the dried solids remained polymorph A' under both drying methods.

On the small scale examined, polymorph A and polymorph A' dry to produce polymorph A' through conversion to hydrate A.

For polymorph A of psilocybin (100 mg), the particle size is reduced by grinding in a mortar and pestle. The ground batch is subjected to two different drying regimes to evaluate whether the reduction in particle size affects sample dehydration. The first sample is kept at 80°C for 10 minutes and the second sample is kept at 110°C for 10 minutes. Both solids evaluate to PRD, which shows that polymorph A is retained. It was considered whether the crushed batch had been exposed to a previous isothermal stress at 110°C for long enough to affect the shape, and therefore a portion of the crushed batch was vacuum dried at 110°C for about 24 hours. minor changes in shape with reflections of polymorph A still present at around 17, but with slightly reduced intensity.

It was concluded that polymorph A is not easily converted to polymorph A' through particle size reduction and/or high temperature drying.

Methodology.

Stability assessments of psilocybin free of pyrophosphate impurity indicate that at temperatures above 80°C, the level of impurity of the Stage 3 intermediate (psilocin) formed by hydrolysis of psilocybin is a problem. For example, when an 83 mg/mL aqueous solution of psilocybin is heated to 90°C and analyzed by HPLC after 1, 2, and 4 hours, the impurity level from step 3 is determined to be 0.28, 1.82, and 7.79 area%, respectively. Conversely, when 50 mg of psilocybin is dissolved in water (1.2-1.8 ml; volume sufficient to maintain the solution) and heated to 70, 75 and 80°C for 4 hours, the level of impurity from step 3 is determined by HPLC , as 0.53, 0.74 and 2.30% of the area, respectively. Recrystallization heats the crude psilocybin to 75°C-80°C to achieve dissolution and further purification by filtration. Immediate cooling of the solution limits the extent of psilocybin hydrolysis by reducing the retention time of the product to excess temperature.

Further test recrystallization of psilocybin is carried out by introducing the following changes:

changing the volumes of water used;

mixing change;

presence of a controlled cooling profile;

the presence of a fast (uncontrolled) cooling profile.

The use of smaller volumes of water (up to 12 vol.) does not impede the recrystallization process, and the dissolution of psilocybin is achieved at a temperature that allows the post-purification stage by filtration. Different cooling rates have been shown to produce different crystal size distributions; slow controlled cooling at about 10°C/h gives a relatively large and more uniform average particle size (Fig. 16a), while the rapid cooling profile produces smaller crystals (Fig. 16b). A controlled cooling profile is preferred and this is reflected in the improved cleanliness for controlled cooling.

Using this method produces psilocybin with a chemical purity of 99.3% with a 75% yield. The thermal characteristics of the solid are as desired. Differences in the PXRD pattern of the dry solid suggest that the drying profile may be important in determining how hydrate A breaks down to form the preferred solid form. Polymorph A has been demonstrated to be stable under accelerated stability testing conditions over 12 months.

- 25 043831

Experiment.

Stage 5 is loaded into a vessel under N2 followed by the addition of water (approximately 12-15 vol depending on the active substance from stage 5). The mixture is heated to about 80°C to dissolve and further purified by filtration through a 1.2 µm inline filter into a clean new flask heated to 80°C. The stirring speed is set to high (450 rpm) and the solution is equilibrated at 70-75°C. The solution is cooled to ambient temperature at a rate of approximately 10°C/hour, inoculated with psilocybin hydrate A (0.001 x step 5 loading) at 68-70°C. The suspension is kept at ambient temperature overnight, then cooled to approximately 5°C and incubated for 1 hour. The suspension is filtered, washing with water (2-3 volumes depending on the active load of stage 5). Pure psilocybin is dried in a vacuum at 50°C. A crystalline psilocybin product is produced (polymorph A or polymorph A' depending on scale), for example, using 94 g of input psilocybin produces polymorph A and using 1 g of input psilocybin produces polymorph A'. Typically, lot sizes greater than 5 g produce polymorph A, and lot sizes less than 5 g produce polymorph A'.

The differences from JNP and the benefits can be summarized as follows.

i) An additional crystallization step gives a specific crystalline form - polymorph A (or A').

ii) Heating to about 80°C for a short period of time has the advantage that solubility is maximized (and hydrolysis is avoided), resulting in good yield.

iii) At about 70-80°C, post-treatment by filtration can be used to remove insoluble impurities. This is best achieved by using a built-in filter - usually around 1.2 microns. This ensures good chemical purity.

iv) The use of high stirring speed (typically about 450 rpm) ensures rapid dissolution, minimizing the time the solution is held at 80°C, thereby avoiding increased levels of intermediate impurity from stage 3 formed by hydrolysis of psilocybin.

v) Providing controlled cooling, typically cooling at about 10°C/h, produces a more uniform crystal size and maintains its shape as crystalline hydrate A.

vi) Inoculating the solution at about 70°C with psilocybin hydrate A promotes crystallization as hydrate A.

vii) The crystals are washed in water and dried at about 50°C in order to maximize purity and production of polymorph A or A', depending on the scale.

Examples 2-6. Stages 1-5. Receiving psilocybin.

The following examples show significant developments in the method described in JNP and shown in FIG. 1, as described here above.

Example 2.

Stage 1 (Fig. 2).

Stage 1 conditions in JNP include the use of 1.1 eq. Ac ₂ O and 1.2 eq. Pyridine in DCM solvent. The reaction is considered complete (99% product, 0% IM) after stirring overnight. The reaction mixture is washed with water and concentrated in vacuo to give a brown oil. In the literature, the oil is placed in EtOAc and concentrated by evaporation, causing solids to precipitate at low volume.

Study.

However, applicants have not observed precipitation of solids from EtOAc. Precipitation of solids is induced by trituration with heptane, but this does not provide an adaptable method. The solids are collected to yield the product from Step 1 in high purity (75% yield, >95% NMR purity).

Although the reaction works well, the isolation method requires further development in order to obtain an easy-to-handle solid. It is hoped that separation of solids by filtration will also provide purification.

The reaction is first tried in EtOAc to see if precipitation of solids can be stimulated by allowing separation directly from the reaction mixture. However, the reaction profile in EtOAc was found to be less favorable than in DCM and therefore the reaction was abolished.

The applicant washes out the pyridine from the DCM reaction mixture as it is believed that this may interfere with the recrystallization of the product. The reaction is repeated (0.4% IM complete, 98.7% product by HPLC), and the reaction mixture is washed with 20% citric acid to pH 2/3, removing pyridine, and then adding saturated NaHCO ₃ (aq) to to avoid low pH during the evaporation stages. The organic phases are dried and solvent exchange to heptane is carried out to obtain the precipitation of Step 1. The solids are collected by filtration to obtain pure Step 1 after vacuum drying (87% yield, >95% NMR purity).

Stability tests are carried out to ensure that the reaction mixture is stable overnight when stirred with 20% citric acid as well as saturated NaHCO ₃ . The product is stable when dried in an oven at 40 and 60°C.

Scaling.

The reaction from step 1 was successfully scaled up by treating >100 g of 4-hydroxyindole. The reaction proceeds as expected and gives the product from step 1 (93% yield, ~98% NMR purity).

Synthesis of GMP starting material.

The large scale reaction from step 1 is carried out to obtain the GMP starting material (treating >500 g of 4-hydroxyindole). The reaction proceeds as expected, consuming IM according to HPLC (99.2% product, <0.1% IM). The reaction mixture is worked up using the established procedure to obtain the product from step 1 after drying (94% yield, 99.1% HPLC, 99% NMR analysis).

During development, it was also noted that the Stage 1 procedure was effective in removing minor impurities present in some batches of 4-hydroxyindole. Low levels of impurities present in 4-hydroxyindole are completely removed after reaction from Step 1, yielding a pure product with high yield (89%) and purity (99% HPLC, 99% NMR).

Experiment.

4-hydroxyindole (1 eq. limiting reagent) is charged into the vessel under N2, then DCM (dichloromethane; 6 volumes based on the loading of 4-hydroxyindole). The reaction mixture is cooled to 0-5°C and pyridine (1.2 eq.) is added dropwise at 0-5°C. Acetic anhydride (1.1 eq.) is added dropwise at 0-5°C and the reaction mixture is heated to 20-25°C for 1-1.5 s and stirred at 20-25°C for another 3 hours Take a sample of the reaction mixture and analyze the completion. The reaction mixture is then washed three times with 20% aqueous citric acid (3x3 volume based on 4-hydroxyindole loading) and once with sat. NaHCO ₃ (3 vol. based on 4-hydroxyindole loading). The DCM solution is dried over MgSO ₄ and filtered, and the DCM layer is concentrated to half volume by distillation. Heptane (6 vol based on loading of 4-hydroxyindole) is added and additional DCM is removed by distillation until complete precipitation of step 1. The reaction mixture is cooled to 15-25°C and the solids are collected by filtration, washed with heptane (1 vol based on loading 4-hydroxyindole), dried in vacuum overnight at 60°C.

The differences from JNP and the benefits can be summarized as follows.

i) The Applicant washes out pyridine using citric acid at a pH of about 2-3. This promotes improved release and crystallization. In practice, the DCM phase is separated and the aqueous citric acid phase is discarded.

ii) Additional washing in sodium bicarbonate allows further improvement.

iii) Solvent replacement with heptane improves solids precipitation, maximizing yield and giving a reproducible step 1 of high purity.

Example 3.

Stage 2 (Fig. 3).

Step i - formation of acid chloride.

Formation of reactive intermediate 2A by reacting the product from step 1 with oxalyl chloride (1.5 eq) is first tested in a mixture of MTBE and THF (6/1 vol) to determine whether it is a viable alternative to the volatile and flammable Et ₂ O used in literature. The reaction is completed after ~18 h with a similar solubility profile as for Et ₂ O (step 1 in solution, precipitation step 2A).

Since the acid chloride intermediate is susceptible to hydrolysis leading to variable assay results, a more reliable sample is obtained and an assay is performed in which the reaction is quenched in TFO/NMe ₂ (to obtain step 2) and then analyzed by HPLC.

The ratio of MTBE to THF is optimized to obtain the highest purity and yield of the intermediate, where the preferred MTBE:THF ratio of 6:1 is selected for scale-up. Other MTBE:THF ratios may be used.

The scale-up reaction is carried out using the preferred solvent mixture (1 vol. THF, 6 vol. MTBE), but the addition of oxalyl chloride is carried out at 30-35°C. The resulting solution is then heated at 40°C for 2.5 hours to obtain completion with ~1% of the remaining product from step 1. Conducting hot addition to maintain the solution allows for a high reaction rate and gives an improved level of completion with much shorter reaction time (2, 5 hours compared to night). The product still precipitated at temperature after ~15 min, and no detrimental effect on the reaction profile was observed by HPLC.

Since the stability of intermediate 2A is not known, telescoping the material to step 2 is attempted rather than isolating the intermediate and risking degradation (hydrolysis). The reaction profile is complex, with many components present at low levels. Add MTBE and collect the precipitate. However, it was also identified as a complex mixture by HPLC/NMR.

Due to the poor reaction profile, it was deemed necessary to isolate intermediate 2A to purify excess oxalyl chloride. The reaction is repeated, the yellow precipitate is collected by filtration and washed with MTBE to remove excess oxalyl chloride (80% yield). NMR analysis confirms the sufficient purity of the product (~95% by NMR). However, despite storage under nitrogen, some degradation was noted within a few days, causing partial hydrolysis, including deprotection of the acetate group.

To try to reduce the likelihood of hydrolysis of the intermediate acid chloride during isolation, further research is being carried out on the telescoping method. It has been found that by allowing the reaction mixture to precipitate easily, MTBE solutions can be easily decanted and then the residual solids washed with another portion of MTBE in the same manner. This allows intermediate 2A to be purified of excess oxalyl chloride while minimizing exposure to moisture.

It is felt that some reaction yield may be lost due to partial solubility of the intermediate in the THF/MTBE mixture. This is confirmed by the addition of heptane to the decanted liquids, which causes additional solids to precipitate. To limit this solubility, the addition of heptane (8 vol.) is carried out before decanting. Instead of washing the MTBE solid, heptane is also used for washes (3x6 vol), which maximizes yield while maintaining high purity of the intermediate. This technique has been successfully scaled up and is the preferred method.

Step ii - reaction with dimethylamine.

Literature (Synthesis, 1999, 6, 935-938, DE Nichols) suggests that HNMe ₂ gas is effective for this transformation. However, to simplify the large-scale process, it is replaced by either solid HNMe ₂ -HCl with additional excess base, or a solution of HNMe ₂ in THF. JNP uses HNMe ₂ in the presence of excess base (pyridine).

The initially isolated intermediate 2A was used to optimize the reaction with dimethylamine through a series of trial reactions (see Table 11).

Table 11

Attempts to optimize the reaction at stage 2

# Source of dimethyls on Base Solvent Completion by HPLC Isolated solids 1 2M ΗΝΜε ₂ /ΤΓΦ 1.2 eq. Pyridine 1.3 eq. THF/MTBE 80% product 64% yield, -70% pure by NMR 2 HNMe ₂ .HCl 1.5 eq. K ₂ CO ₃ THF/N ₂ O 70% product 40% yield -98% by NMR. 3 2M ΗΝΜε ₂ /ΤΓΦ 1.33 eq. Pyridine 3.6 eq. Et ₂ O 81% product 63% yield, -90% by NMR. 4 2M ΗΝΜε ₂ /ΤΓΦ 2.9 eq. But Et ₂ O 93% product 72% yield, -98% by NMR.

The conditions proposed in the literature with pyridine (#1) were tried together with the same reaction in Et ₂ O (#3), a two-phase reaction using Me ₂ NH.HCl and aqueous K ₂ CO ₃ (#2) and a reaction with an excess of 2 M Me ₂ NH in THF (#4). The main component by HPLC was the desired product in all cases, where conditions using aqueous base and excess Me2NH were generally purer than conditions with pyridine. Although a significant amount of hydrolysis product was visible in all cases, it is believed that this is the result of unreacted intermediate 2A that was quenched during sample acquisition for HPLC analysis. The reaction mixtures are treated with the addition of water and then the organic solvent is removed in vacuo to precipitate solids.

The reaction with excess amine showed a significantly improved profile, which results in higher yield (72 to 63%) and purity (98 to 90%). This approach limits the water content of the reaction and therefore minimizes the likelihood of hydrolysis occurring. Purification is also expected to be easier due to the absence of pyridine in the recovered solids. For these reasons, conditions with excess HNMe ₂ as the base were chosen for scaling.

Reaction in Et ₂ O gives a clean (#4) profile. However, to facilitate large-scale processing, substitution with a less volatile solvent such as MTBE has been shown to be advantageous. This will make it easier for the acid chloride to telescope into this reaction. For these reasons, the reaction was chosen to be carried out in MTBE using an excess of 2 M Me ₂ NH in THF.

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It is believed that the addition of water will aid processing by solubilizing the salts present

HNMe2-HCl, which give a very thick mixture and slow down the filtration. We tried this approach. However, when water was added to the reaction mixtures, MTBE and THF received poor recovery, and analysis of the liquids showed additional impurities and significant deprotection of the acetate (phenolic product). Therefore, further development of purification is required.

Development of purification.

It would be desirable to develop a purification strategy that would remove the hydrolysis product and other resulting impurities. It would also be desirable to include water in the crystallization to reduce the salt content of the isolated product (presumably HNMe2-HCl). For this purpose, a series of 15 solvents and solvent mixtures were screened (scale 100 mg, 10 vol. solvent, cyclic heating to 60°C).

Table 12

Stage 2 cleanup attempts

Solvent Observations Recovery HPLC Purity (Hydrolysis Impurities of Intermediate 2A) MTBE Suspension 47 mg* 96.4% (2.8%) DXM Suspension 60 mg 99.5% (0.5%) Toluene Suspension 30 mg* 95.8% (3.4%) EtOAc Suspension 66 mg* 97.8% (1.6%) iPrOAc Suspension 40 mg* 97.3% (2.0%) IPS Suspension 65 mg 99.4% (0.4%) EtOH/ _H2O , 1:1 Suspension 62 mg 99.4% (0.5%) MeCN/ _H2O , 1:1 Partial solution at CT Solution at 60°C 30 mg 99.0% (0.7%) Acetone/H ₂ O, 1:1 Suspension at CT 51 mg 99.4% (0.5%) Solution at 60°C THF/N ₂ O, 1:1 Partial solution at CT Solution at 60°C No sediment n/a Heptane Suspension 34 mg* 95.0% (3.6%) MIBK Suspension 65 mg 97.9% (1.4%) IEC Suspension 60 mg 99.6% (0.3%) Cyclohexane Suspension 41 mg* 94.4% (4%) Xylene Suspension 56 mg* 96.1% (3%)

*Recoveries are not representative due to thick suspensions and solids adhering to the glass tube.

Of the solvents tested, acetone/water recrystallizes with negligible observed solubility at room temperature. Since this is an aqueous system, it has the advantage of helping to blow Me ₂ NH-HCl out of the solids.

Recrystallization from acetone/water was scaled up. A solution is prepared at (5 vol acetone, 1 vol water) before adding additional water (4 vol) and the mixture is cooled to RT to give crystallization (62% recovery, >99% HPLC purity). This method is then scaled up further by adding more water to aid recovery (total 5 vol acetone, 10 vol water, 78% recovery).

The process is scaled up further (30 g) and the crude solid is obtained by recrystallization. Although the purity of the product is high, there is a sharp decrease in yield (56% yield, 99% by NMR analysis, 99.4% by HPLC).

To improve recovery, the added amount of water is increased from 10 to 15 vol. This maintains product purity at over 99% and gives high recovery on a small scale (90% recovery, 56-70% previously obtained). However, scaling up this replaced technique

- 29 043831 again gives low recovery (58% yield). Consequently, due to problems encountered when scaling up recrystallization, an alternative purification method was found based on the initial screening of the suspension performed (Table 12 above).

Reconstruction of the purification strategy is carried out with the product isolated from the large-scale reaction, step 2. The reaction proceeds as expected and gives the crude product after oven drying (70% by NMR analysis, 79% yield of active substance). To remove the significant salt component (presumably HNMe ₂ -HCl), a portion is suspended in water at RT. After drying it gives a 75% recovery (95% by NMR analysis), showing that it is an effective means of reducing salt content. HPLC purity remains unchanged at 93%. Then a way was found to increase the chemical purity of solids.

After initial screening, both EtOH:H2O, IPA and acetone: _H2O appear to produce a high purity product with good recovery and these solvent systems are therefore selected for further investigation. Input purity is 92.7% with major impurities at levels of 1.4, 1.0 and 0.8%.

Table 13

Further development of purification (suspension at 40°C for 1 hour, filtration at RT, 1 volume rinse)

Solvent system Recovery HPLC Purity (Levels of Major Impurities) Product t Etc. 1 Etc. 2 Etc. 3 Acetone/water 6/16 vol. 67% 98.8% 0.7% 0.2% 0.1% 1:1EUN:N ₂ O 10 vol. 79% 98.0% 0.9% 0.5% 0.2% IPS 10 vol. 90% 97.5% 0.8% 0.8% 0.3% IPS 5 vol. 92% 97.2% 1.0% 0.7% 0.3% 4.5:2.5 IPS:N ₂ O* 7 vol. 79% 98.7% 0.6% 0.4% 0.1% 6:1 IPS:N ₂ O 7 vol. 82%** 98.0% 0.9% 0.6% 0.2%

* The reaction initially takes place in 1:1 IPA:H ₂ O in 5 vol. However, it becomes too thick to stir, so add another 2 vol. IPS.

**The mixture was thick and the solids present were very fine, making filtration difficult as some of the solids clogged the filter.

The results of these attempts suggest that good recovery is possible for these systems, particularly those based on IPS. EtOH:H2O gives a much better impurity profile than IPA alone; however, recovery was not very good (79 to 90%). The IPA impurity profile improved significantly in the presence of water (98.7 to ~97.5%), however this resulted in low recovery (79 to 90%). This suggests a certain level of solubility of the compound in water. A final attempt in IPA and EtOH:water is carried out at reduced volumes of water to see if a balance of the resulting high purity and ~90% recovery can be found. Although this system improves yield, filtration was slow and therefore other solvent mixtures were also evaluated.

Table 14

Study of other solvent mixtures (500 mg scale, suspension at 40°C for 1 hour, filtration at RT, 1 volume wash)

Solvent system Recovery HPLC Purity (Levels of Major Impurities) Product t Etc. 1 Etc. 2 Etc. 3 IPS 10 vol. 460 mg (92%) 97.6% 1.1% 0.7% 0.3% 1:3 E1OH:H2O 5 vol. 451 mg (90%) 96.2% 1.4% 0.8% 0.5% 1:2EUN:H2O5 vol. 440 mg (88%) 97.5% 1.3% 0.7% 0.4% 1:1 EtOH:H2O 5 vol. 375 mg (75%) 97.9% 1.0% 0.7% 0.3% 2:1 EtOH:H2O 5 vol. 354 mg (71%) 97.6% 1.0% 0.6% 0.4% 3:1 E1OH:H2O 5 vol. 369 mg (74%) 97.9% 0.9% 0.5% 0.3%

At 5 rpm The EtOH/water suspension was too thick and did not process well. Since the purity of the solids was comparable across all attempts (minor variations likely due to the number of filtrations and washes), the 100% IPA conditions were scaled up as some offer high recovery and the resulting suspension is easily processed.

Initial scaling of the preferred suspension gives (92% recovery) with HPLC purity of 96.4% (impurity levels 1.2, 0.7, 0.4%). Analysis of the liquids showed that they were enriched with all major impurities - 72% (8.6, 3.8, 3.4%) by HPLC. This method is recognized as a suitable purification method offering high recovery and the product is used for testing in the next stage to ensure that impurities are tracked and removed further (>99% in stage 3, no individual impurities >0.5%) .

The suspension is considered scalable when the remaining crude product from step 2 (70% analysis) is further suspended in water to remove inorganics, and then suspended in IPA to give the product of improved purity (97% by NMR analysis, 76% yield for step 2, 96. 8% by HPLC, impurities 1.1, 0.8, 0.4%).

Synthesis of GMP starting material.

The large scale reaction from step 2 is carried out to supply GMP. The reaction proceeds as expected to yield a crude product, which is suspended in water and filtered to obtain Step 2 which is 93% pure by HPLC. It is further suspended in 8 vol. IPA and filter to obtain the product of stage 2 (93.7% by HPLC, 92% analysis, 66% yield of active substance). Since the purity obtained was less than that observed during development, testing is carried out to confirm that the resulting high purity Stage 3 is suitable for further processing (GMP starting material).

A second batch was prepared under identical conditions to yield a crude product that, after suspension in water, was 90% pure by HPLC. This product was then suspended in water and purified with IPA suspension to give 384 g of Step 2 product (93.0% HPLC, 91% NMR, 60% active yield).

The third batch is received to replenish the GMP synthesis. The crude product is then purified with water followed by IPA slurry to deliver Step 2 (79% yield) with increased purity over previous batches (97.3% HPLC, 96% NMR).

Experiment.

Stage 1 (1 eq. limiting reagent) is loaded into a vessel under N2, then THF (1 vol. relative to stage 1 loading) and MTBE (6 vol. relative to stage 1 loading). Oxalyl chloride (1.5 eq.) is then added dropwise to the vessel, allowing the ectotherm to initially increase the temperature to 35-40°C, and then cooled as necessary to maintain 35-40°C. Immediately after addition, the reaction mixture is heated to 40°C and suspended for 2-6 hours. Sample the reaction mixture and analyze completion, then cool to RT and add heptane (8 vol. relative to the loading of stage 1), causing the precipitation of additional solids substances. The reaction mixture is stirred for 10 minutes and then the solids precipitate. Most of the solvent is decanted from the solid, which is then washed twice with heptane (2x6 vol. relative to the loading of stage 1), decanting the same after each wash. Samples of the solids are then taken and analyzed. MTBE is loaded into the vessel (4 vol. rel. loading stage 1) to obtain a yellow suspension, which is cooled to -20°C using a dry ice/acetone bath. A 2 M solution of Me ₂ NH in THF (2 eq.) is added dropwise into the vessel over ~15 min, maintaining the temperature from -20 to -10°C. The reaction mixture is slowly warmed to RT and stirred overnight. At this point, add more Me ₂ NH, if necessary. Samples of the reaction mixture are taken and completion is analyzed. The reaction mixture is filtered, washed with heptane (2x2 vol. relative to the loading of stage 1), and the isolated solids are dried at 60°C in vacuum. The crude stage 2 is suspended in water (8 vol. relative to the loading of stage 1) for 2-18 hours and then filtered, washing with water (2 vol. relative to the loading of stage 1). The solids are dried at 60°C in vacuo to obtain crude step 2 with <2% w/w water (determined by Karl Fischer (KF) titration). Crude step 2 is suspended in IPA (10 vol) for 2-18 hours and then filtered, washed with IPA (1 vol relative to the weight of crude step 2) and dried in a vacuum oven at 60°C.

The differences from JNP and the benefits can be summarized as follows.

Step i.

i) Firstly, the use of a THF/MTBE solvent system instead of diethyl ether was less volatile and flammable.

ii) Secondly, the addition of oxalyl chloride is carried out at elevated temperature, heated to 40°C, achieving improved solubility and preventing the product of stage 1 from being trapped in the precipitate. It also provides high response speeds, improved completion levels and shorter reaction times.

iii) Third, intermediate 2A is isolated to remove excess oxalyl chloride. iv) Fourth, heptane is added to promote precipitation of intermediate 2A. Stage 2.

v) By using the amine in excess, significantly improved purity and yield are achieved due to the minimal presence of water and therefore reduced hydrolysis.

vi) Finally, the use of water and IPA suspensions gives good purity of the stage 2 product.

Example 4.

Stage 3.

The initial test reaction of Step 3 is carried out using the purified product from Step 2

- 31 043831 (>99% by HPLC) and given reaction conditions. The input from Step 2 was found to be substantially insoluble in THF, so instead of adding the NiAlH ₄ solution of Step 2, a reverse addition was made. 4 eq. NiAlH ₄ is used as a 1 M solution in THF with the addition carried out at 20-25°C for ~2 hours. At this point, a 10% product is observed with the presence of several types of intermediate compounds. The reaction mixture is heated at reflux for ~7 hours to obtain 93% conversion of the product (by HPLC). The reaction mixture is processed to obtain the crude product of step 3 (~90% by HPLC, ~90% by NMR, ~87% corrected yield).

A trial reaction is carried out in which the applied NiAlH ₄ charge is successfully reduced (3 to 4 eq.). It is hoped that this will benefit processing by reducing the amount of Li and Al salts formed. After prolonged reflux (10–18 h), the reaction intermediates were largely consumed (2–3% residue), with ~95% product by HPLC.

Development of processing.

Although the first pilot reaction worked successfully using Rochelle salt, the volumes used were too high (~100 vol) and the technique did not form a valuable process for scale-up. A variety of alternative processing techniques have been explored in an attempt to reduce the volumes required and aid in the removal of Li/Al salts.

Tried low volume quenching with EtOAc and then Rochelle salt. The gray solid was present as a thick paste that settled to the bottom of the flask. Although filtration fails, the liquids can be decanted and the solids resuspended in THF/EtOAc to extract the product. Then water treatment is carried out and the product is isolated by concentration. This gives a product of good purity (90-95% by NMR) in good yield (94% uncorrected for purity). However, the method is not easily scalable.

The reaction is quenched by adding EtOAc and then sat. Na ₂ SO ₄ in the presence of anhydrous Na ₂ SO ₄ acting as a coupling agent. The reaction produces granular solids that can be easily filtered. Then aqueous treatment is carried out, and the product is isolated by concentration. Good yields are obtained (~94% not corrected for purity), but the product contains high levels of major impurity by NMR (10% to the 2-4% typically obtained).

The reaction is quenched with 20% AcOH at 0°C, resulting in the formation of a gel that cannot be filtered. They refuse to react.

The reaction is quenched with EtOAc and then 20% citric acid to give solids which can be separated by filtration. The liquids are concentrated to obtain the product. Although this procedure gave a slightly lower yield (~77% not corrected for purity), the product had very high purity (>95%).

The other reaction is quenched by adding EtOAc and then water (3 ml per g LiAlH ₄ in THF). A gel formed that could not be easily filtered and the reaction was abandoned.

Finally, the reaction is quenched using the Fischer method. Add water (1 ml per g LiAlH4), then 15% NaOH (1 ml per g LiAlH ₄ ) and finally water (3 ml per g LiAlH ₄ ). This produces a solid that can be filtered from the reaction mixture. The liquids are then separated and concentrated in vacuo (87% yield, 90-95% by NMR).

These experiments are summarized in Table. 15 below.

Table 15

Summary of Alternative Processing Conditions

No. Processing technique Exit Purity 3.1 Quenching EtOAc Rochelle salt (reduced volume) 2.8 g (94% unadjusted) 90-95% by NMR 3.2 Quenching of EtOAc in the presence of Na ₂ SO ₄ 2.8 g (94% unadjusted) -80-85% according to NMR, 10% etc. 3.3 Quenching 20% AcOH Emulsion (reaction discarded) 3.4 Quenching EtOAc 20% citric acid i) 529 mg (71% unadjusted) ii) 2.3 g (77% unadjusted) >95% purity by NMR 3.5 Extinguishing with water Emulsion (reaction discarded) 3.6 Quenching with water/NaOH i) 615 mg (83% unadjusted) ii) 2.6 g (87% unadjusted) 90-95% by NMR

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Both quenching with citric acid and NaOH produces solids that can be easily filtered from the reaction mixture and require minimal volumes of solvent. Although NaOH conditions gave a higher yield (~10%), the product obtained by this method was less pure and would likely require further purification before use in the next step. The lower yield with citric acid was probably due to some precipitation of the citrate of the product. This had a cleansing effect, yielding a pure product immediately after concentration. These conditions were selected for scale-up and it is hoped that further optimization of the citric acid loading will allow the isolation of pure product in high yield from this process.

The reaction is repeated with a slightly reduced citric acid load to attempt and maximize recovery. This reaction gave product in 57% yield with a further 20% yield obtained by resuspending the filter cake in THF (both samples 97.7% by HPLC).

The reaction is scaled up. However, during the EtOAc quench, where pre-thickening of the reaction mixture was observed, the reaction mixture clumped together in the flask as a thick mass with limited mixing. Although the addition of citric acid further gave a conventional suspension/gel, this is not a valuable method. This reaction was run with a filter cake resuspended in THF to maximize recovery, yielding 76% yield, 95.0% by HPLC.

The reaction is repeated to develop better quenching and to avoid the formation of gum produced with EtOAc. Part is quenched by adding acetone, which gives an easily stirred suspension/emulsion without signs of thickening. Then the mixture is treated with citric acid to obtain a filterable mixture. This quench was successfully carried out on the remainder of the reaction mixture and processed to obtain the crude product in good yield (71% analysis, 82% corrected yield, 98.0% HPLC).

After quenching, the reaction mixture typically has a pH of 8/9. As part of the process optimization process, different pH values are examined. The reaction is split into a workup where half receives a slightly reduced citric acid load (resulting in a pH of 11/12 after quenching) and the other half is adjusted to pH 7 by adding additional citric acid. The pH 11 reaction mixture was treated to give 85% NMR product (73% yield) and the pH 7 reaction mixture gave 60% NMR product (62% yield). It is clear from these results that obtaining the correct pH after quenching is critical to obtain >70% yield. With a very slight reduction in citric acid loading (approximately 2 vol. 20% citric acid) the yield increases by approximately 8%. Given this information, the pH of future reactions is monitored during quenching to ensure that the mixture remains strongly alkaline.

Development of purification.

Purification screening is carried out using 100 mg portions of the crude psilocin product, which is suspended in 10 vol. solvent with cyclic heating to 60°C. The suspensions are cooled to RT over the weekend, and then the resulting solids are collected by filtration. Acid and base resistance are also tested in terms of acid/base treatment. The screening results are presented in table. 16 below.

Table 16

Psilocin purity screening (input purity and 3 impurity levels: 90.2, 3.8, 0.9, 0)

Solvent Observations Recovery (approx.) HPLC Purity (and Major Impurity Levels) MeOH Solution at CT on on EtOH Solution at 60°C Sediment at CT 35 mg 97.2%, 0.4%, 1.1%, 0.8% IPS Suspension at 60°C 51 mg 97.6%, 0.5%, 0.5%, 1.1% MeCN Solution at 60°C Sediment at CT 46 mg 96.8%, 0.6%, 0.4%, 1.6%

- 33 043831

EtOAc Suspension at 60°C 58 mg 97.1%, 0.9%, 0.2%, 1.3% iPrOAc Suspension at 60°C 58 mg 98.2%, 0.8%, 0.2%, 0.4% Toluene Suspension at 60°C 70 mg 93.3%, 3.6%, 0.2%, 0.8% Heptane Suspension at 60°C 77 mg 91.3%, 3.8%, 0.2%, 0.7% Acetone Solution at 60°C Sediment at CT 30 mg 97.7%, 0.4%, 0.3%, 0.9% mek Solution at 60°C Sediment at CT 24 mg 97.3%, 0.5%, 0.5%, 1.2% MIBK Suspension at 60°C 49 mg 97.4%, 0.6%, 0.2%, 1.3% THF Solution at CT on on MTBE Suspension at 60°C 67 mg 95.5%, 2.0%, 0.1%, 1.5% DXM Solution at CT on on 1MNS1 Solution at CT on 83.7%, new approx. 8% 1MKON Suspension at CT (black) on 89.1%, 5.4%, 0.9%, 2.2%

The first of the three impurities isolated corresponds to the most stable reaction intermediate, which is observed at ~70%, where the addition of LiAlH4 is completed (requiring reflux to convert the product). The third impurity was not present at the inlet and was likely formed during suspension. Of the solvents left in suspension, ⁱ PrOAc gives the greatest purity. Several recrystallizations have been found with MeCN having the ability to remove impurities during crystallization and having a recovery that is potentially improved during development. Some decomposition is observed in both the acid and base with the KOH sample quickly turning black.

The purification of the crude product from step 3 is enhanced using two of the most promising solvents (MeCN and iPrOAc). Solvent volumes are reduced to a minimum in order to improve recovery. The results of these tests are presented in table. 17 below.

Table 17

Further development of purification with MeCN/iPrOAc

Solvent Observations Recovery (approx.) HPLC Purity (and Major Impurity Levels) MeCN (5 vol.) Recrystallization at 5 vol. 512 mg (51%) 97.6%, 0.7%, 0.8%, 0% 'PgOAc (3 vol.) Suspension in 3 vol. 706 mg (71%) 95.8%, 1.3%, 0.6%, 0%

Recrystallization is obtained from MeCN at 5 vol. and a hot suspension is obtained in iPrOAc at 3 vol. (both at 75 °C). Recovery from MeCN was again poor despite the reduced volumes, but the product was of high purity (>>95% by NMR). Recovery from iPrOAc was better with a significant increase in product purity by NMR analysis (~95%).

Although the purity of the product from the iPrOAc suspension was high by HPLC and NMR, a low analytical result was observed (85% by NMR analysis). To improve the result of the analysis of the material, as well as to remove color (all products obtained at this point were bright purple, green or brown), purification through a layer of silicon dioxide is examined.

Crude psilocin (71% assay, 98.0% HPLC) was passed through 4 eq. silica, eluting with THF. An 80% recovery of a whitish solid was obtained with slightly improved HPLC purity (98.4%) and assay result (~82% assay). This has proven to be an effective means of enhancing product analysis and is therefore considered as part of the reaction mixture workup.

A series of suspensions in ⁱ PrOAc/antisolvent (Table 18) are then carried out using silica-treated feedstock (100 mg per suspension) to attempt to improve recovery while maintaining chemical purity (entry purity 98.4%).

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Table 18

Results of iPrOAc/antisolvent additions

Solvent system Recovery HPLC Purity 'ProgOAc 5 vol. 78% 99.7% 1:1 TgOAsteptan 5 vol. 70% 99.6% 1:1 TgOAc:MTBE 5 vol. 83% 99.7% 1:1 TrOAc:Toluene 5 vol. 84% 99.4% BIOAc 5 vol. 80% 99.6%

Since all purities were comparable, two solvent systems are selected for scaling based on the highest recovery results obtained. The two most preferred suspensions (MTBE and toluene as antisolvent) are scaled (1.0 g per suspension) to better assess recovery.

Table 19

Scaling Preferred Cleaning Methods

Solvent system Recovery HPLC Purity 1:1 FgOAc:MTBE 5 rev. 79.9% 99.1% 1:1 TrOAc: Toluene 5 rev. 79.4% 99.6%

Both of these options produce a product with HPLC purity >99% with ~80% recovery and, when combined with a silica layer, are an effective means of purifying the psilocin product. Additional color is removed from the liquids during suspension to produce psilocin as a white solid. All impurities are effectively removed to levels below 0.5%. The iPrOAc:MTBE suspension is chosen for scale-up because it uses non-toxic ICH class 3 solvents.

Scaling.

The resulting step 3 conditions were scaled up and the reaction progressed to yield 94.4% product complete with 2.9% reaction intermediate present by HPLC after refluxing overnight (typical method). After a layer of silicon dioxide, psilocin is obtained with 83% purity by NMR analysis, 66% yield of the active substance, 97.0% by HPLC. This product was then purified by suspension in iPrOAc/MTBE to give 100% product by NMR analysis with 62% yield and 99.7% by HPLC.

Because the crude reaction yield was lower than expected (66 to ~75%), the filter cake and silica layer were re-examined to sample and recover additional product. However, to no avail.

A lower than expected yield may have been due to degradation of the product during processing, although previous extreme testing indicated that the product was stable under the conditions used. To further investigate this phenomenon, repeat the reaction. The crude product is isolated to the silica layer and additional samples are taken for extreme testing to confirm that the product has not degraded during processing.

The reaction proceeds as expected to completion (93.7% product, 2.9% intermediates) and a concentrated yield of crude product is obtained (77% NMR analysis, 66% yield of active compound). The filter cake is resuspended in THF/MeOH but does not release any significant amount of psilocin. To try and replace any product that coordinates with aluminum salts, additional citric acid is added to bring the pH to 4 (from pH 8) and the cake is resuspended in THF, but again no significant amount of psilocin is released. The mass balance is not obtained from the reaction with 66% yield of the active substance, exactly the same as previously obtained. This batch was purified on a silica pad and slurried in iPrOAc/MTBE to give a 62% yield of high purity product (99.8% by HPLC).

Despite the use of relatively high volumes of solvent and the need for a layer of silica to remove aluminum and lithium compounds, the process is still most suitable for the scale required.

The step 3 reaction is further scaled up to the method. The reaction proceeds as expected with completion after 18 hours (~91% product, ~3% reaction intermediates remaining). Treatment on a layer of silica and suspension gives a 57% yield of high purity psilocin (>99% HPLC, 99% NMR, 0.35% w/w, Karl Fischer water).

Experiment.

Step 2 (1 eq. limiting reagent) is loaded into the vessel, then add (5 vol. rel. load

- 35 043831 ki stage 2). The mixture is cooled to 0°C and a 1 M THF solution of LiAlH ₄ (3 equiv.) is added dropwise over 30-45 minutes, maintaining the temperature at 0-20°C. After addition, the reaction mixture is stirred for 30 minutes at 10-20°C and then heated to reflux and stirred for ~16 hours. Take a sample of the reaction mixture and analyze the completion, cool to 0°C and quench by adding acetone dropwise (9.3 eq.) at 0-30°C, then 20% aq. citric acid solution (1.9 vol. relative to stage 2 loading) at 0-30°C. Monitor the pH of the addition to ensure it remains at pH>11 and stop the addition early if necessary. The resulting suspension is stirred for 1 hour and filtered, washing with THF (2 vol. relative to stage 2 loading) to remove Li and Al salts. The filter cake is suspended in THF (12.5 vol. relative to stage 2 loading) for ~1 hour and filtered, washing with THF (5 vol. relative to stage 2 loading) to recover the product from the Li and Al salts. The combined organic phases are dried over MgSO ₄ and filtered. The filtrate is evaporated in vacuo to approximately 10 vol. (relative to stage 2 loading) and this solution is applied to a layer of silicon dioxide (3 equivalents relative to stage 2 loading). The silica layer is eluted with THF, the product fractions are combined and evaporated to dryness in vacuo. Crude step 3 (psilocin) is suspended in 1:1 iPrOAc:MTBE (5 vol/w in step 18) for 2-18 hours, filtered, washed with MTBE (2.5 vol/w in step 18) and dried in a vacuum at 40°C to release psilocin.

The differences from JNP and the benefits can be summarized as follows.

i) First, using THF as a solvent, the reaction is quenched using acetone. This gives a suspension/emulsion without thickening.

ii) Secondly, the applicant quenches with citric acid, maintaining an alkaline pH, usually around 11. Control of the pH ensures high yield.

iii) Thirdly, after purification on a pad of silica to remove residual Li/Al salts, eluting with THF, an iPrOAc:MTBE suspension is obtained, giving a highly purified product, which is then dried.

Example 5.

Stage 4.

First apply the conditions described in the literature to treat 2.58 g of sample to obtain ~88% conversion to intermediate 4A by HPLC analysis. The product is purified by addition of aminopropyl silica and filtration through celite. The resulting green oil (5.5 g) was suspended in DCM to give benzyl conversion and precipitate zwitterionic step 4 (4.1 g, 70% yield, ~95% by NMR).

Step i.

Initial development in this step is focused on finding an alternative ^{to nBuLi} that would be easier to handle and ideally would not introduce additional lithium into the synthesis. Conduct an initial screening of alternative conditions, including the following bases: LitBuO, KtBuO, NaH, NaHMDS and NaNH ₂ . All reactions produce a product where NaHMDS works the same as ⁿ BuLi. All reaction mixtures become very thick with visible gelation, so an overhead stirrer is recommended for effective mixing.

Initial screening suggests that NaHMDS may be a suitable alternative ^{to n} BuLi (81% conversion to intermediate 4A). These conditions are scaled up to 1.5 g along with the reference reactions with ⁿ BuLi. Top mixing is used in both cases.

Table 20

Comparison ^{of n} BuLi and NaHMDS

Time HPLC - ^nBuLi HPLC - NaHMDS 1 hour, -30°С 6.6% Art. 3, 78% Prom 4A, 1% St. 4 <1% St 3, 78% Prom 4A, 4% St 4 2 hours, 0°C 6.5% St 3, 77% Prom 4A, 1% St 4 <1% St 3, 76% Prom 4A, 4% St 4 Unrefined product 4.89 g <1% St 3, 2% Prom 4A, 60% St 4 4.38 g <1% St 3, 5% Prom 4A, 68% St 4

Abbreviations used in the table:

St 3=stage 3;

Prom 4A=intermediate 4A;

St 4=stage 4.

The reaction profile obtained in both cases was very similar, and the reaction with NaHMDS had step 3 consumption. Both reaction mixtures were filtered on celite to remove the white precipitate, and concentrated. By NMR, an excess of benzyl protons is present in both cases (especially in the example with ⁿ BuLi), and the yield obtained is >100%. NaHMDS conditions were found to be successful, producing a favorable response profile, and were selected for further scale-up. However, development of processing and purification is required.

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Step ii.

HPLC data shows that the product isolated in the above attempts using NaHMDS and ⁿ BuLi rearranged to give zwitterionic step 4 at concentration. They tried to purify this product from benzylphosphoric acid by-products and other impurities by suspending it in a variety of solvents.

Table 21

Test purification of stage 4 crude product

Solvent Recovered mass HPLC Purity DXM White solid 84% St 4 EtOH Gum - EtOAc Gum - IPS White solid 88% St 4 Toluene Gum - MTBE White solid 62% St 4 MIBK Gum - MeCN Gum - Acetone White solid 86% St 4

Abbreviations used in the table: St 4=stage 4.

* Filtration is poor.

A white solid is produced from several solvents, however the solids produced from DCM and MTBE turned to a pale purple gum when stored over the weekend. The material produced from IPA and acetone remained as free-flowing white solids during storage, suggesting that the stability of these solids was likely greater and that they may allow for easier processing.

Suspensions in IPA and acetone were scaled up to 1 g. However, gum formation was noted immediately after the addition of the solvent. The gum is slowly dispersed with vigorous stirring and signs of crystallization are eventually seen to form a white slurry after stirring overnight. However, this method is not suitable for scaling. Solids are isolated in good yield, with IPA giving the highest purity.

THF is also being explored as it has the advantage of also being a reaction solvent. However, when testing it, the initial formation of gum is again observed (~80% yield, ~92% by HPLC). To try to avoid gum formation and obtain a more controlled crystallization, the crude step 4 is first solubilized in a low volume of DMSO (2 vol). THF (10 vol) is then added and the solution is stirred over the weekend. This gives a slow precipitation of the product, which is collected by filtration and washed with THF to give step 4 (86% yield) with 96% HPLC purity (>95% NMR).

Since crystallization in THF was successful, and, as noted earlier, complete conversion to zwitterionic step 4 occurs while concentrating the reaction liquids (THF/EtOAc) at 40°C, it is hoped that solvent changes can be avoided and the product crystallized directly by stirring reaction mixture at 40°C.

Two 4 g reactions were carried out in NaHMDS where both reactions reached completion with ~80% conversion to Intermediate 4A. One reaction mixture is diluted with EtOAc and the other with THF, and both are filtered to remove phosphate by-products. To further reduce the levels of phosphate impurities, salt is deposited and the organic phases are dried and concentrated to 10 vol. These solutions are stirred overnight at 40°C to obtain the conversion and precipitation of step 4 (~1% step 3, ~0.2% intermediate 4A, ~82% step 4). The solids were collected by filtration to provide 8.03 g (88% yield) from EtOAc/THF and 5.65 g (62% yield) from THF. The brown/gray solids produced from EtOAc/THF were less pure (~90% by HPLC, 78% analysis) compared to the white solids obtained from THF (97% by HPLC, 88% analysis). Analysis of the aqueous layer from the reaction with THF indicated the presence of product, and additional losses were noted upon final filtration of THF.

Due to the high purity obtained from THF, this solvent was further investigated to optimize the recovery. Salt precipitation is not carried out due to loss of product into the aqueous layer, and the reaction mixture is further concentrated after the reaction to minimize losses during the final filtration step. This new technique is blown on a 75 g scale with portions of the reaction mixture concentrated to 8 and 6 vol. Upon filtration, no difference in yield was noted between the two portions, with a total yield of 140.4 g (90% NMR, 74% active, 90% HPLC).

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Impurity tracking.

Three major impurities were detected in the isolated product, and two of these species were identified based on MS data.

A debenzylated impurity (typically ~2-5% by HPLC) has been shown to produce psilocybin during subsequent hydrogenation and can therefore be tolerated at high levels. The main impurity observed in the isolated step 4 (usually ~5-8% by HPLC) is an anhydride impurity. It is monitored during subsequent hydrogenation and is shown to be readily removed by recrystallization from water as a highly soluble pyrophosphate impurity that arises during debenzylation. The other major observed impurity (m/z 295.2 observed by LCMS) can be controlled to less than 2% by limiting the reaction temperature (below -50°C) and is not observed in psilocybin after hydrogenation.

The impurity profile of the 140 g batch obtained above showed 90.0% stage 4, 6.4% anhydride impurity, 0.2% N-debenzylated impurity and 1.2% m/z 295.2 impurity.

GMP synthesis.

The first large scale batch of Step 3 (input 544 g) was prepared using the established procedure to yield 213.5 g (53% yield, 99% HPLC). The second batch (input 628.2 g) was also successfully processed to yield 295.2 g (66% yield, 99% HPLC).

Some variability in yield at this stage was noted for 3 large-scale batches (57, 53 and 66%). This is likely a consequence of minor differences in processing and quenching techniques.

Experiment.

Stage 3 is loaded into the vessel, then THF is added (15 vol. relative to stage 3 loading) and cooled to <-50°C using a dry ice/acetone bath. Load a 1M solution of NaHMDS in THF (1.13 eq.), keeping the temperature <-45°C, target <-50°C. The reaction mixture is stirred for 30 minutes at -60 to -50°C. Tetrabenzyl pyrophosphate (2.26 eq.) is loaded into the reaction mixture in one portion, then more THF (20 vol.) is added, maintaining the reaction temperature <-30°C. The reaction mixture is heated to 0°C for 1.5-2 hours and a sample is taken to complete. The reaction mixture is filtered to remove phosphate salts, washing with THF (8 vol). The filtrate is concentrated to 6-8 vol. residue and stir overnight at 40° C. to convert Intermediate 4A to the product of step 4. Take a sample of the reaction mixture to determine completion and then filter and wash the solid with THF (2 vol). The product from step 4 is dried in a vacuum oven at 40°C.

The differences from JNP and the benefits can be summarized as follows.

Step i.

i) First, sodium hexamethyldisalazide is administered to maintain deprotonation. It provides an effective alternative to butyllithium that is easier to process and does not introduce additional lithium into the reaction.

ii) Secondly, diluting the reaction mixture with THF allows for greater purity of intermediate 4A.

iii) Thirdly, by controlling the reaction temperature below -50°C, the unwanted mz 295.2 observed by LCMS is controlled to less than 2%.

Step ii.

iv) Fourth, by monitoring the impurity levels of Step 4A, particularly the N-debenzylated Step 4 (Table 7) and the anhydride Step 4 (Table 7), a pure product can be reproducibly obtained.

v) The conversion of the intermediate from Step 4A to Step 4 can be carried out in a reaction solvent, avoiding the need to waste time changing the solvent.

vi) Finally, the resulting solid is washed with THF and dried in an oven to obtain step 4.

Example 6.

Stage 5.

Catalyst poisoning is noted during the development of this stage and it is necessary to include a carbon treatment step in the process, if necessary, to prevent incomplete hydrogenation. However, charcoal treatment is not usually required.

After purging with hydrogen for 3 h, typical reactions showed high yields at completion (>90% product, 3-5% IM remaining). A small amount of water is added to aid solubility, and after purging with hydrogen for a further 1 hour, stage 4 consumption is achieved.

A successful reaction is treated by filtration, then evaporation to remove the methanol, leaving the product as a thick suspension in water. Ethanol is added and the solid is filtered to give psilocybin in 69% yield. 1H NMR confirms the identity of the product but shows the minor presence of a related impurity. LCMS analysis shows a purity of 95.2% with the main impurity (4.1%) identified as pyrophosphoric acid impurity (Table 7), derived from

- 38 043831 anhydride impurity from stage stage 4. It is further shown that this impurity is effectively purged during the final recrystallization of the product (stage 6).

An additional reaction is then carried out using the product from step 4 from the completed THF treatment, which is 88.0% pure by HPLC and contains 7.3% N-debenzylated step 4 (converted to product) with no anhydride impurity. Completion is again noted and the reaction mixture is processed as before to obtain psilocybin in 46% yield. It is believed that the low yield is a result of product precipitation during the catalyst filtration step. 1H NMR confirms the identity of the product and HPLC shows 98.9% purity.

Further development of the reaction conditions is carried out to optimize the volume of water used, and minimize product losses during the filtration step. After the reaction, the solution is prepared by adding 10 vol. water when heated to 40°C. This allows the catalyst to be removed by filtration without loss of product on the filter.

Some step 3 is generated by hydrolysis during reaction and processing at a level of approximately 1-2.5%, which appears to be typical for the process. Stage 3 recovery was demonstrated during the final recrystallization of the product.

Scaling.

A large-scale Stage 4 (non-GMP) batch is processed as one batch (148 g of active ingredient input). Stage 4 consumption is achieved with 88% of the product and 0.9% of Stage 3 obtained by hydrolysis. The anhydride impurity (6.4%) is completely converted into the corresponding pyrophosphoric acid impurity (5.2%).

The large scale hydrogenation product was filtered and concentrated to yield 109 g of crude product after removal from ethanol to reduce water content (~71% NMR, 86% yield).

Experiment.

10% Pd/C (~50% water-wetted, type 87L, 0.1x stage 4 charge) is loaded into the vessel under N2, then methanol (20 vol. rel. stage 4) and stage 4. N2 is replaced by H ₂ , and the reaction the mixture is stirred under H ₂ (atmospheric pressure) for 1-2 hours. Take a sample of the reaction mixture to confirm completion and then add water (10 vol. rel. step 4), maintaining the temperature <25°C. The mixture is stirred for another 1-2 hours under H2 (atmospheric pressure). Take a sample of the reaction mixture and check for completion.

If the reaction is not complete, reload H ₂ and continue the reaction for another 1-12 hours until completion. The reaction mixture is then placed under N2 and heated to 40°C and maintained for 15-45 minutes. The reaction mixture is filtered through celite to remove the catalyst, washing with methanol (13.3 vol. relative to stage 4 loading) and water (6.7 vol. relative to stage 4 loading). The filtrate is concentrated in vacuo, azeotroping with ethanol to remove water, until a solid is obtained. The differences from JNP and the benefits can be summarized as follows:

First, the reaction is monitored for levels of intermediates by HPLC using relative retention time (RRT), and completion is monitored when intermediates are present at levels less than 0.2%. The pyrophosphoric acid impurity from step 5 is also carefully monitored to ensure that it can be controlled during the final recrystallization.

The final method of step 6 is as described in example 1.

Example 7.

Testing methods and protocols.

For testing purity, etc. The following methodology/protocols are used.

1. NMR.

The 1H and ^13C NMR spectrum of psilocybin in D2O was obtained using a 400 MHz spectrometer. Chemical shifts are reported in ppm relative to D2O in 1H NMR (δ=4.75 ppm) and relative to MeOH (δ=49.5 ppm), which is added as a reference in the ¹³ C NMR spectrum. In the literature, values for psilocybin are shown in JNP. NMR analysis of psilocybin produces data that is consistent with the structure and consistent with those presented in the literature, with minor variations in chemical shifts for protons near ionizable groups expected since the zwitterionic nature of the compound makes the product very sensitive to minor changes in pH .

The 1H NMR and ^13C NMR data are presented below and the spectra are shown in FIG. 10 and 11.

^1H NMR data (400 MHz, D2O): 2.79 (s, 3H), 3.18 (t, J=7.4 Hz, 2H), 3.31 (t, J=7.4 Hz, 2H ), 6.97 (d, J=8.0 Hz, 1H), 7.08 (s, 1H), 7.10 (t, J=8.0 Hz, 1H), 7.19 (d, 8 ,2 Hz, 1H).

¹³ C NMR data (400 MHz, D ₂ O (+ traces of MeOH): 22.3 (1 x CH), 43.4 (2 x CH ₃ ), 59.6 (1 x x CH ₂ ), 108.4 ( 1 x CH), 108.6 (1 x C), 109.5 (1 x CH), 119.1 (d, ³ Jp-h=6.7 Hz, 1 x C), 123.3 (1 x CH2), 124.8 (1 x CH), 139.3 (1 x C), 146.3 (d, ² Jp-h=6.7 Hz, 1 x C)

2. PF-IR.

Data are collected on a Perkin Elmer Spectrum Two™ spectrometer with the UATR Two application. Analysis

- 39 043831 psilocybin (lot: AR755) PF-IR spectroscopy gives a spectrum (Fig. 12) that corresponds to that proposed in the literature. The broad peak at 3244 cm ^-1 is typical of an amine salt. The remaining peaks are in the characteristic region and therefore cannot be assigned individually.

3. Mass spectrometry.

The mass spectrum of psilocybin (AR755) was obtained on a Bruker Esquire 3000 plus Ion Trap mass spectrometer and was consistent with the structure. The mass spectrum (Fig. 13) showed a main peak at m/z=284.8 and 568.1, which corresponds to (M+H)+ and (2M+H) ⁺ of psilocybin. This implies that the molecular ion m/z 284 corresponds to the molecular formula of psilocybin (C ₁₂ H ₁₇ N ₂ O ₄ P) (Fig. 14).

4. Incineration residue.

The combustion residue method repeats the method from the pharmacopoeia with one adjustment. Inconsistent results were obtained when the crucible was heated to 500°C, and this is believed to be due to the volatility of the phosphate residues that are formed. The temperature is then raised to 800°C for psilocybin and consistent and accurate results are obtained.

5. HPLC - analysis and determination of purity.

The HPLC method that is used for the analysis, chemical purification and quantitative analysis of psilocybin impurities is a gradient HPLC-UV method, the conditions of which are shown in table. 22. External standards are used for quantitative analysis. Approximately 1 mg/ml psilocybin is dissolved in purified water:MeOH (95:5). Treat with ultrasound to ensure complete dissolution.

HPLC purity is calculated based on area % and correlated to a known retention time standard.

HPLC analysis is calculated on an anhydrous basis, on a wt% basis, to a standard of known purity and composition.

Table 22

Typical HPLC Conditions for Identification, Purity and Analysis

Parameter Conditions System Agilent 1100 Series Liquid Chromatograph or equivalent Column XBridge C18, 4.6^150 mm; 3.5 µm (Ex; waters PN: 186003034) Flow rate 1.0 ml/min' ¹ Injection volume 5 µl Definition UV @ 267 nm Temperature speakers 30°C Mobile phase A - purified water: methanol:TF A (95:5:0.1) B - methanol:purified water:TFA (95:5:0.1) Gradient Time (min) %A %B 0 100 o 2 100 0 15 θ 100 20 0 100 22 100 o

6. Residual solvent content according to HRGC.

The HRGC method for quantitative analysis of residual solvents is a headspace method and is described in Table. 23 below.

Table 23

Typical GC method for determining residual solvent

Parameter Conditions System Agilent 6890/7890 HRGC or similar Column DB-624 60 m x 0.32 mm, 1.80 µm film thickness (or

- 40 043831

equivalent) Oven program 40°С (holding time 15 min) then linear increase (ΖΟθΟΜΗΗ' ¹ ) to 200°С (holding time 5 min) Vapor phase parameters furnace temperature loop temperature transition line temperature 125°C 140°C 150°C Flow division 10:1 Injection temperature 200°C Sensor temperature 250°С, FID Inlet pressure 15 psi, constant pressure Carrier gas Nitrogen Column flow 2.0 ml.min' ¹ @ 40°C Internal standard 1,2-difluorobenzene

Determine levels of the following solvents and reagents: methanol, ethanol, THF, and toluene.

7. Melting point according to DSC.

DSC data are collected on a PerkinElmer Pyris 6000 DSC (or similar). The instrument is tested for energy and temperature calibration using certified indium. The sample is weighed (usually 0.5-3.0 mg) into an aluminum sample pan with pinholes. The cuvette is crimped with an aluminum cuvette lid. Then the cuvette is heated at a rate of 20°C/min from 30 to 300°C while purging with dry nitrogen at a rate of 20 ml/min. During the melting procedure, each batch of polymorph A or A' of psilocybin exhibits two endothermic events of the latter, the first of which is attributed to the solid-to-solid transition of polymorph A or A' to polymorph B and the second of which is attributed to the melting of polymorph B.

8. Polymorphism in PRD.

The solid form of psilocybin is determined by the PRD. PXD diffraction patterns are collected on a diffractometer (such as a PANalytical X'Pert PRO or equivalent) using Cu Ka irradiation (45 kV, 40 mA), θ goniometer, focusing mirror, divergence slit (1/2), Soller slits for both incident and divergent beams (4 mm) under ambient conditions. The data acquisition interval is 3-35° 2Θ with a continuous scan rate of 0.2° s ^-1 . The resulting diffraction pattern is compared with the reference diffraction pattern of polymorph A or A' to ensure their similarity (Fig. 7a or 7b, respectively).

9. Thermogravimetric analysis (TGA).

TGA data is collected on a PerkinElmer Pyris 1 TGA (or similar). The instrument is calibrated using certified weights and certified Alumel and Perkalloy for temperature. A predetermined amount of sample (usually about 5 mg) is loaded into an aluminum crucible and heated at a rate of 20°C/min from ambient temperature to 400°C. Nitrogen purge of the sample is continued at a rate of 20 ml/min.

10. Loss on drying.

Determine the loss on drying of the sample twice using a 1 g portion, accurately weighed, dried at 70°C in vacuum to constant weight.

Calculation.

% Loss on drying = (M _I сх ~ ^W KOH) x 100 where W _ISH = initial mass of the cup and sample before drying (g);

W _KOH = final weight of cup and dried sample (g);

W _OBR = sample mass (g).

Example 8.

Forced decomposition studies.

The drug substance psilocybin is stressed under different conditions in solution and in the solid state to obtain samples to evaluate the sensitivity of the analytical method.

Forced degradation studies are conducted on psilocybin based on ICH Q1A(R2) requirements. Testing under adverse conditions provides information about potential degradation pathways and inherent stability of psilocybin. The optimized analytical method used demonstrates specificity for psilocybin; It has been shown to be suitable and changes in product identity, purity and potency can be determined using this method. The method used was also shown to be free from interference from possible impurities and degradation products according to ICH Q2(R1) (Validation of Analytical Procedures) with reference to specificity.

Therefore, the HPLC method is considered suitable for determining the purity of psilocybin and related impurities.

The control psilocybin sample was stable in solution during the study period (the study period was 7 days for non-light-stable samples). Psilocybin decomposes slowly when heated in solution, producing psilocin as the main impurity. Psilocybin was also stable under acidic conditions at room temperature. However, at 60°C there was a slow and constant decomposition, producing psilocin as the main impurity. Psilocybin was slightly unstable at room temperature in the presence of base, with slow degradation into several impurities over the study period. Only minor levels of impurities were formed under peroxide conditions with a ~0.5% drop in overall purity. In the solid state, slow chemical decomposition was noted (3 days at 150°C), mainly producing psilocin (stage 3) as an impurity. Psilocybin was stable under photostability conditions in both solid and solution.

Stability studies.

Stability studies were performed on two batches of psilocybin, as shown in Table 1. 24.

Table 24

Study number/start of study No. batches of drugs special substances a Package Place production Application of the batch Conditions storage Desired time points/study status ONYXST GM764B Double swollen Link. 2-8°C 1, 3, 6 months AB0138 food grade polyethylene new packages. Outer polyethylene container Scientifi with Std. 25°C/60% OB 40°С/75% OB continuous 1, 3, 6 months continuous 1, 3, 6 months continuous ONYXST 170231 Double swollen Kleene 2-8°C 1, 3, 6, 9, 12, 18, AB0139 food grade polyethylene new packages. Outer polyethylene container Scienti fiс logical 25°C/60% OB 40°С/75% OB 24, 36 months continuous 1, 3, 6, 9, 12, 18, 24, 36 months continuous 1, 3, 6 months continuous

Samples are double-packed in food-grade plastic bags and sealed in an outer plastic container and stored at 2-8°C, 25°C/60% RH and 45°C/75% RH, a desiccant bag is placed between the plastic bags to prevent moisture absorption. Appearance, water content, purity are tested and analyzed.

The protocols of the two studies are shown in Table. 25 and 26.

Stability data after one month and after three months for batch GM764B are detailed in the table. 27 and 28 below. Stability data after one, three, six, nine and twelve months for GMP lot 170231 are presented in Table. 29, 30, 31, 32 and 33 respectively (below).

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Stability test protocol

Table 25

Product: Psilocybin Swelling test number: ONYXSTAB0138 Batch number GM764B Expected start date: March 10, 2017 Test Method: n/a Date of manufacture: 06 February 2017 Additional Information: 1200 mg of product is required in each container. Packaging components: Double lined polyethylene bags contained in a 300 ml HDPE container (food grade). Added desiccant between two plastic bags. Test parameters Regular tests Appearance Analysis (anhydrous base) Ή-NMR Water content at loss during drying Chemical purity/impurities by HPLC Months 1 3 6 Spares Total 2°С-8°С X X X 2 5 25°C/60% ov X X X 2 5 40°С/75% OB X X X 0 3 date April 10th June 10th 10 September 13 expiration deadline 2017 2017 2017

Stability test protocol

Table 26

Product: Psilocybin Onyx Test Number: ONYXSTAB0139 Batch number: 170231 Expected start date: March 31, 2017 Test method: SS/PSILOCYBIN/ Date of production: February 27, 2017 Additional information: 2200 mg of product is required in each container. Packaging components: Double lined polyethylene bags contained in a 300 ml HDPE container (food grade). Added desiccant between two plastic bags. Test parameters Regular Appearance tests Analysis (anhydrous base) Ή-NMR Water content at loss during drying Chemical purity/impurities by HPLC Moment time 1 month 3 months 6 months 9 months 12 months 18 months 24 months 36 months Spares Total 2-8°C X X X X X X X X 2 10 25°C/60% RH X X X X X X X X 2 10 40°C/75% RH X X X 1 4 expiration date 31 April 30 June 2017 September 30th 31th of December March 31 2018 September 30th March 31 2019 March 31 2020 24 deadline 2017 2017 2017 2018

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Stability data after one month for batch GM764B

Table 27

Test Permissible limit T=0 T=1 month T=1 month T=1 month Condition ON ON 2°С-8°С 25°C/60% RH 40°C/75% RH Appearance For information only. Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Ή-NMR Analysis For information only. 97% w/w 99% w/w 98% w/w 96% w/w Water content at loss during drying For information only. 0.86% w/w 0.35% w/w 0.20% w/w 0.14% w/w Chemical purity by HPLC For information only. 99.24% 99.24% 99.22% 99.23% Impurities according to HPLC: Only for 0.05% 0.05% 0.05% 0.05% (All warheads are indicated information. 0.05% 0.09% 0.10% 0.10% 0.05%) OVU 0.86 OVU 1.46 OVU 1.59 (Psilocin) Total impurities 0.37% 0.76% 0.35% 0.76% 0.34% 0.78% 0.34% 0.77%

Stability data after three months for batch GM764B

Table 28

Test Permissible limit T=0 T=3 months T=3 months T=3 months Condition ON ON 2°С-8°С 25°C-60% RH 40°C-75% RH Appearance For information only. Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Ή-NMR Analysis For information only. 97% w/w 97% w/w 99% w/w 97% w/w Water content at loss during drying For information only. 0.86% w/w 0.26% w/w 0.08% w/w 0.14% w/w Chemical purity by HPLC For information only. 99.24% 99.31% 99.27% 99.26% Impurities according to HPLC: (all BC 0.05% are indicated) For information only. OVU 0.86 OVU 1.46 OVU 1.59 (Psilocin) Total impurities 0.05% 0.05% 0.37% 0.76% MP 0.05% 0.10% 0.37% 0.69% MP 0.05% 0.09% 0.36% 0.73% MP 0.05% 0.10% 0.37% 0.74%

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Table 29

Stability data after one month for batch 170231

Test Permissible limit T=0 T=1 month T=1 month T=1 month Condition ON ON 2°С-8°С 25°C/60% RH 40°C/75% RH Appearance Only for Whitish solid Whitish solid Whitish solid Whitish solid information. substance. Not contains visible contaminants substance. Not contains visible contaminants substance. Not contains visible contaminants substance. Contains no visible contaminants Chemical purity by HPLC For information only. 99.28% 99.20% 99.16% 99.17% Impurities according to HPLC: Only for 0.06% 0.05% 0.05% 0.06% (All warheads are indicated information. 0.39% 0.36% 0.37% 0.36% 0.05%) 0.05% MP 0.05% MP 0.05% MP 0.05% OVU 1.49 MP 0.05% MP 0.05% MP 0.05% MP 0.05% OVU 1.62 (Psilocin) MP 0.05% MP 0.05% MP 0.05% MP 0.05% OVU 1.70 0.22% 0.39% 0.42% 0.41% Impurities at ORV 1.89 Impurities at OVU 2.45 Impurities MP 0.05% Total impurities 0.72% 0.80% 0.84% 0.83% HPLC analysis (dry basis) For information only 98.65% w/w 98.76% w/w 97.98% w/w 98.52% w/w Water content at loss during drying For information only. 0.32% w/w 0.27% w/w 0.17% w/w 0.19% w/w

Stability data after three months for batch 170231

Table 30

Test Permissible limit T=0 T=3 months T=3 months T=3 months Condition ON ON 2°С-8°С 25°C/60% RH 40°C/75% RH Appearance Only for Whitish solid Whitish solid Whitish solid Whitish solid information. substance. Not contains visible contaminants substance. Not contains visible contaminants substance. Not contains visible contaminants substance. Not contains visible contaminants Chemical purity by HPLC For information only. 99.28% 99.30% 99.31% 99.17% Impurities according to HPLC: Only for MP 0.05% 0.05% MP 0.05% MP 0.05% (All warheads are indicated information. 0.06% 0.05% 0.05% 0.06% 0.05%) 0.39% 0.37% 0.36% 0.39% OVU 0.69 0.05% MP 0.05% MP 0.05% MP 0.05% OVU 1.49 MP 0.05% MP 0.05% MP 0.05% MP 0.05% OVU 1.62 (Psilocin) MP 0.05% MP 0.05% MP 0.05% MP 0.05% OVU 1.70 0.22% 0.22% 0.27% 0.34% Impurities at ORV 1.89 Impurities at OVU 2.45 Impurities MP 0.05% Total impurities 0.72% 0.70% 0.69% 0.79% HPLC analysis (dry basis) For information only 98.65% w/w 98.45% w/w 99.46% w/w 98.64% w/w Water content at loss during drying For information only. 0.32% w/w 0.17% w/w 0.01% w/w 0.19% w/w

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Stability data after six months for batch 170231

Table 31

Test Permissible limit T=0 T=6 months T=6 months T=6 months Condition ON ON 2°С-8°С 25°C-60% RH 40°C-75% RH Appearance For information only. Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Chemical purity by HPLC For information only. 99.28% 99.20% 99.19% 99.12% Impurities according to HPLC: (all BC 0.05% are indicated) OVU 0.69 OVU 1.49 OVU 1.62 (Psilocin) OVU 1.70 Impurities at ORV 1.89 Impurities at ORV 2.45 Impurities MP 0.05% Total impurities For information only. MP 0.05% 0.06% 0.39% 0.05% MP 0.05% MP 0.05% 0.22% 0.72% 0.06% 0.07% MP 0.05% 0.35% MP 0.05% MP 0.05% MP 0.05% 0.32% 0.80% 0.06% 0.07% MP 0.05% 0.34% MP 0.05% MP 0.05% MP 0.05% 0.34% 0.81% 0.06% 0.08% MP 0.05% 0.38% MP 0.05% MP 0.05% MP 0.05% 0.36% 0.88% HPLC analysis (dry basis) For information only 98.65% w/w 97.97% w/w 98.04% w/w 100.10% w/w Water content at loss during drying For information only. 0.32% w/w 0.06% w/w 0.32% w/w 2.26% w/w

Stability data after nine months for batch 170231

Table 32

Test Permissible limit T=0 T=9 months T=9 months Condition ON ON 2°С-8°С 25°C-60% RH Appearance For information only. Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Chemical purity according to HPLC For information only. 99.28% 99.16% 99.16% Impurities according to HPLC: (all BC are indicated 0.05%) TOC 0.69 OVU 1.49 OVU 1.62 (Psilocin) OVU 1.70 Impurities at ORV 1.89 Impurities at ORV 2.45 Impurities MP 0.05% Total impurities For information only. MP 0.05% 0.06% 0.39% 0.05% MP 0.05% MP 0.05% 0.22% 0.72% MP 0.05% MP 0.05% 0.07% 0.06% 0.37% MP 0.05% MP 0.05% MP 0.05% 0.34% 0.84% MP 0.05% MP 0.05% 0.05% 0.06% 0.37% MP 0.05% MP 0.05% MP 0.05% 0.35% 0.84% HPLC analysis (dry basis) For information only 98.65% w/w 97.53% w/w 98.12% w/w Water content at loss during drying For information only. 0.32% w/w 0.21% w/w 0.10% w/w

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Table 33

Stability data after twelve months for batch 170231

Test Permissible limit T=0 T=12 months T=12 months Condition ON ON 2°С-8°С 25°C/60% RH Appearance For information only. Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Whitish solid. Contains no visible contaminants Chemical purity according to HPLC For information only. 99.28% 99.25% 99.25% Impurities according to HPLC: (all BC are indicated 0.05%) TOC 0.69 OVU 1.49 OVU 1.62 (Psilocin) OVU 1.70 Impurities at ORV 1.89 Impurities at ORV 2.45 Impurities MP 0.05% Total impurities For information only. MP 0.05% 0.06% 0.39% 0.05% MP 0.05% MP 0.05% 0.22% 0.72% MP 0.05% MP 0.05% 0.37% MP 0.05% MP 0.05% MP 0.05% 0.38% 0.75% MP 0.05% MP 0.05% 0.37% MP 0.05% ND MP 0.05% 0.38% 0.75% HPLC analysis (dry basis) For information only 98.65% w/w 99.63% w/w 98.97% w/w Water content at loss during drying For information only. 0.32% w/w 0.49% w/w 0.61% w/w

During the first 12 months of the ICH stability study, psilocybin was proven to be chemically stable at low temperature (2-8°C), ambient (25°C/60% RH) and forced (40°C/75% RH) conditions. ) conditions. There was no change in appearance and the HPLC analysis also remained constant. Water content varies across all samples due to initial exposure and subsequent aging of the desiccant in the study.

Example 9. Experiment with the production of hydrate A.

Psilocybin (200 mg) is loaded into a crystallization tube, followed by deionized water (4 ml). The mixture is equilibrated at 25 °C for 2 hours, then the solid is isolated by vacuum filtration. The product is divided into two equal portions. One portion is not subjected to subsequent drying to obtain hydrate A, lot GK2, by PXRD and DSC (diffraction pattern and thermogram correspond to Fig. 7d and 8d, respectively).

Example 10. Experiment with obtaining polymorph B.

Psilocybin polymorph A (250 mg) is placed in a round bottom flask, heated to 173°C using an oil bath and held at temperature for 5 minutes. The solid is cooled to ambient temperature and isolated to yield Lot GK3 with 93% recovery. PXRD and DSC analysis showed that lot GK3 is polymorph B (diffraction pattern and thermogram correspond to Fig. 7c and 8c, respectively).

Example 11. Studies of solid matter.

They do a lot of research on polymorphism. The set of solid forms found is shown in Fig. 17. Most of the forms found were obtained by changing the solvent; in some cases stoichiometric solvates were isolated, in other cases non-stoichiometric solvates were isolated.

Suspensions of polymorph A.

Solvent-mediated equilibrations of psilocybin template A were performed as a primary pathway in solid form modification and to visually assess product solubility in a series of 24 solvents at 25-50°C.

Psilocybin Template A (40 mg) is dosed into test tubes at room temperature and the solvents listed in Table 1 are added. 34 in aliquots of 0.4 ml (10 vol.) to a total volume of 1.2 ml (30 vol.) and note the observations. The mixtures are constantly stirred. Cyclic heating is carried out as follows: 50°C for 18 hours, cooling to 20°C for 2 hours, maturing for 4 hours, heating to 50°C for 4 hours, cooling to 20°C for 2 hours, ripening for 18 hours. Repeat the cycle of 50-20°C for 24 hours and apply the following:

release after heating to 50°C, where sufficient solid = A series;

release after cooling to 20°C, where sufficient solid = B series.

All isolated solids are dried in vacuum at 50°C for 24 h and analyzed by PRD. Observations are presented in table. 34.

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The API was largely insoluble in solvents, and solvent mixtures tested at 30 vol. at 50°C, gave thick suspensions. Water does not solubilize psilocybin at 50°C.

Table 34

Tabulated observations of the maturation of suspensions during cyclic heating and the use of template A mixture as an input

Vvo d Solvent Nab l. 20°C, 0.4 ml Nab l. 20°C, 0.8 ml Observation 20°C, 1.2 ml Observation 50°С PRD A series PRD B series 1 Cyclohexane Susp. Susp. Susp. Susp. A A 2 Chlorobenzene Susp. Susp. Susp. Susp. A A 3 2-Chlorobutane Susp. Susp. Susp. Susp. A A 4 Benzotrifluoride Susp. Susp. Susp. Susp. A A 5 Anisole Susp. Susp. Susp. Susp. A A 6 Nitromethane Susp. Susp. Susp. Susp. WITH WITH 7 HPME Susp. Susp. Susp. Susp. A A 8 Heptane Susp. Susp. Susp. Susp. A A 9 MTBE Susp. Susp. Susp. Susp. WITH A 10 MIBK Susp. Susp. Susp. Susp. A A AND IEC Susp. Susp. Susp. Susp. A A 12 iPrOAc Susp. Susp. Susp. Susp. WITH WITH 13 EtOAc Susp. Susp. Susp. Susp. A A 14 Toluene Susp. Susp. Susp. Susp. A A 15 THF Susp. Susp. Susp. Susp. A A 16 SNS1 ₃ Susp. Susp. Susp. Susp. A A 17 MeOH Susp. Susp. Susp. Susp. D D 18 EtOH Susp. Susp. Susp. Susp. E E 19 ANN Susp. Susp. Susp. Susp. F F 20 MeCN Susp. Susp. Susp. Susp. WITH A 21 Water Susp. Susp. Susp. Solution on A 22 4:1 EtOH/water Susp. Susp. Susp. Susp. A A 23 4:1 THF/water Susp. Susp. Susp. Susp. A Hydrate A 24 4:1 IPA/water Susp. Susp. Susp. Susp. A WITH

Results.

In fig. 18 and 19 25C means the release of solid at 25°C and 50C means the release of solid at 50°C. For example, GM832-2O_5O_A9 means GM832 input 20 (MeCN) isolated at 50°C.

Suspensions at 50°C.

Entries 1, 2, 3, 4, 5, 7, 8, 10, 11, 13, 14, 15, 16, 22, 23, 24: PXRD pattern mainly corresponds to polymorph A, but with an additional peak of varying intensity at 18° 2θ.

Entries 6, 9, 12, 20: The PXRD pattern obtained for the isolated solids is generally consistent with (see Fig. 18) with additional peaks at 10°2Θ and 13.2°2θ observed for some samples. This PRD pattern is designated pattern C. There is no chemotypic correlation between the solvents (CH3NO2, MTBE, iPrOAc and CH3CN).

Input 17: the resulting PRD diffraction pattern has many diffraction peaks (Fig. 19). The PXRD diffraction pattern was designated pattern D.

Input 18: The resulting PRD diffraction pattern has many diffraction peaks (Fig. 19). The PXRD diffraction pattern was designated pattern E.

Input 19: the resulting PXD diffraction pattern has many diffraction peaks (Fig. 19). The PXRD diffraction pattern was designated pattern F.

Suspensions at 25°C.

Inputs 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 20, 21, 22: all PRD diffraction patterns are similar to the one

- 48 043831 which was obtained for polymorph A.

Entries 6, 12, 24: PXRD patterns obtained for the isolated solids generally correspond (see Fig. 18) to pattern C.

Input 23: PRD analysis showed the formation of hydrate A.

Input 17: the resulting PRD diffraction pattern has many diffraction peaks (Fig. 19). The PXRD diffraction pattern was designated pattern D.

Input 18: The resulting PRD diffraction pattern has many diffraction peaks (Fig. 19). The PXRD diffraction pattern was designated pattern E.

Input 19: the resulting PXD diffraction pattern has many diffraction peaks (Fig. 19). The PXRD diffraction pattern was designated pattern F.

Analysis of results.

PRD diffraction patterns for solids isolated at 25°C are essentially the same as PRD diffraction patterns obtained for solids isolated at 50°C.

Templates D, E and F are derived from alcohols (MeOH, EtOH and IPA). Solvated states have been proposed taking into account the ethanol solvate isolated earlier during development. The PRD diffraction patterns for the ethanol solvate are not identical, however, given that precise changes in solvent level can result in different states of lattice order, comparison of these PRD diffraction patterns strongly suggests that these larger phase changes are caused by solvent entrainment.

The PXRD patterns D, E and F (Figure 19) are all different from the PXRD pattern of the hydrate (Figure 7d).

A direct comparison of the PRD diffraction patterns obtained from a layer of solids isolated from MeOH, EtOH, and IPA (patterns D, E, and F; Fig. 19) isolated at two temperatures showed matching diffraction patterns; the two diffraction patterns of MeOH are similar, and EtOH and IPA are directly comparable.

Perform DSC analysis of the isolated solids and, if sufficient sample is available, TGA. The solids that produce patterns D, E and F all show endotherms at around 170-180°C but otherwise produce different thermal profiles. TGA analysis of the solid isolated from the MeOH suspension showed one sustained weight loss from about 25-190°C (3.1%). The stoichiometric methanol solvate requires 10.3% by weight solvent. TGA analysis of the solid isolated from EtOH showed two distinct mass loss steps. The first, occurring up to 100°C (0.3% by weight), is considered to be due to water, and the second large loss (11.5% by weight) is due to the solvent. Stoichiometric ethanol solvate requires 13.9% by weight solvent. TGA analysis of the solid isolated from the IPA suspension showed two distinct mass loss steps. The first weight loss up to 100°C (0.4% by weight) is considered to be due to water, and the second large loss of weight (13.9% by weight) is considered to be due to residual solvent. Stoichiometric IPA solvate requires 17.5% by weight solvent.

Suspensions of amorphous psilocybin.

To obtain an amorphous product, a small sample of psilocybin (0.5 g) is dissolved in water (0.5 l, 1000 vol.), further purified on a filter and lyophilized. Psilocybin is reconstituted as a whitish fibrous product (lot MC1368A; 412 mg, 82%, PRD amorphous).

To visually assess the solubility of the amorphous API and to modify the form, a series of maturations in suspension are carried out as follows.

Psilocybin (15 mg) is loaded into test tubes. The solvent is then added at ambient temperature (20°C, 0.3 ml, 20 vol.) and the suspensions are stirred. They make observations. After 1 hour of stirring, the samples are heated to 45°C for 18 hours and observations are made. The samples are heated to 50°C for 8 hours and observations are made. Samples are stirred for 72 h at 25 °C and subjected to a final heating cycle until release. Observations are shown in table. 35.

Table 35

Observations of amorphous psilocybin during the maturation of suspensions under heating cycling and shape behavior

Enter Solvent Observation at 20°С Observation at 45°С Observation at 50°C PRD data A Cyclohexane Sushi. Sushi. Sushi. Semi-crystalline IN Chlorobenzene Sushi. Sushi. Sushi. Semi-crystalline WITH Chlorobutane Sushi. Sushi. Sushi. Template B

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D Benzotrifluoride Sushi. Sushi. Sushi. Semi-crystalline E Anisole Sushi. Sushi. Sushi. Semi-crystalline F Nitromethane Sushi. Sushi. Sushi. Template B G HPME Sushi. Sushi. Sushi. Semi-crystalline N Heptane Sushi. Sushi. Sushi. Semi-crystalline I MTBE Sushi. Sushi. Sushi. Semi-crystalline J MIBK Sushi. Sushi. Sushi. Semi-crystalline TO IEC Sushi. Sushi. Sushi. Semi-crystalline L iPrOAc Sushi. Sushi. Sushi. Semi-crystalline M EtOAc Sushi. Sushi. Sushi. Semi-crystalline N Toluene Sushi. Sushi. Sushi. Similar to Solvat A ABOUT THF Sushi. Sushi. Sushi. Semi-crystalline R Chloroform Sushi. Sushi. Sushi. Similar to Template E R MeOH Sushi. Sushi. Sushi. Semi-crystalline S EtOH Sushi. Sushi. Sushi. Template D T IPS Sushi. Sushi. Sushi. Template B And Acetonitrile Sushi. Sushi. Sushi. Amorphous V Water Sushi. Sushi. Sushi. Similar to Template A W 4:1 EtOH/water Sushi. Sushi. Sushi. Similar to Pattern D X 4:1 THF/water Sushi. Sushi. Sushi. Similar to Template A Y 4:1 IPA/water Sushi. Sushi. Sushi. Similar to Template A

Results.

Most solvents yielded a solid that is considered semicrystalline (mostly with appreciable reflection at about 18° 2θ).

A truly amorphous substance was obtained by equilibration in MeCN.

Polymorph B was obtained from chlorobutane, nitromethane and IPA (Figs. 20 and 22).

Template D, which was isolated from MeOH in the polymorph A suspension experiments discussed above, was obtained by equilibration in EtOH, and MeOH in this study gave a semicrystalline solid.

Solids similar to template A were recovered from water, THF:water and IPA:water (4:1).

A solid similar to template D was recovered from EtOH:water (4:1), confirming the discovery of the isolation of template D from pure EtOH.

A solid similar to template E was recovered from chloroform.

From nine solvents tested, the true polymorph A or A' was isolated after prolonged equilibration and thermal maturation of amorphous psilocybin.

Example 12. Composition development.

The initial series of experiments are carried out using the compositions indicated in table. 36 below. The goal was to identify a suitable single excipient or combination of excipients for a large-scale formulation.

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Table 36

Product name Batch No. (% w/w) APL-117- 6085-01 APL-117- 6085-02 APL-117- 6085-03 Psilocybin 1.0 1.0 1.0 Microcrystalline cellulose Ph 102 91.5 49.5 81.5 Pregelatinized starch (Starch 1500) - 45.0 - Compact Cel MAB - - 10 Hydroxypropyl cellulose (Klucel EXF) 3.0 3.0 3.0 Sodium starch glycolate 3.0 3.0 3.0 Colloidal silicon dioxide 0.5 0.5 0.5 Magnesium Stearate (Vegetable Derived) 1.0 1.0 1.0 Sodium stearyl fumarate - - - TOTAL 100.0 100.0 100.0

The result in the form of key physicochemical properties: product flowability, mixture uniformity and content uniformity are presented in table. 37 below.

Table 37

Batch number Durable (mg) Product flowability (coefficient Carrisa) Mixture homogeneity Content Uniformity APL-117- 6085-01 1.0 19.1 Top=127.9 Average = 106.4 Bottom = 104.5 Average=112.9 %OS=U.9 % declared=92, 4 SR=7.9 APL-117- 6085-02 1.0 19.1 Top=115.9 Middle= 106.6 Bottom = 106.1 % of declared=9 5.2 Average = 109.6% RSD = 4.9 SR=5.9 APL-117- 6085-03 1.0 22.4 Top=105.0 Average = 101.4 Bottom=98.7 Average=101.7 %OS=3.8 % of declared=9 6.3 SR=4.6

Although the batch (APL-117-6085-03) showed good mixture homogeneity in the different samples analyzed (top, middle and bottom) and very good content homogeneity, its flow properties (based on the Carr index) tend to the upper limit and were predicted to that the composition will not accommodate higher drug loads.

For this reason, several alternative formulations have been tested. The goal was to consider other combinations of fillers to improve powder flowability as well as achieve good mixture uniformity and content uniformity.

Formulations containing less Compacted MAB and higher amounts of glidant compared to Lot 3 (APL-117-6085-03) were also tested.

These additional compositions are presented in table. 38 below.

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Table 38

Product name Batch No. (% w/w) APL- 117- 6085-05 APL- 117- 6085-06 APL- 1176085-07 Psilocybin 1.0 1.0 5.0 Microcrystalline cellulose Ph 102 - 89.0 85.0 Pregelatinized starch (Starch 1500) 45.0 - - Compact Cel MAB - 5.0 5.0 Microcrystalline cellulose CEOLUS UF 702 49.5 - - Sodium starch glycolate h,o 3.0 3.0 Colloidal silicon dioxide 0.5 1.0 1.0 Sodium stearyl fumarate 1.0 1.0 1.0 TOTAL 100.0 100.0 100.0

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

Table 39

No. Durable Product flowability Mixture homogeneity Uniformity parties t (mg) (coefficient Carrisa) content APL- 117- 6085-05 1.0 20.9 TOP=130.0 MIDDLE=105.4 BOTTOM=107.2 Average=114.2%SD=12.6% % From declared=88 ,3 SR=16.5 APL- 117- 6085-06 1.0 20.0 TOP=107.0 Mid=96.2 Bottom=95.5 Mean=99.6% RSD=6.5 % From declared=96 ,2 SR=10.5 APL- 117- 6085-07 5.0 24.3 TOP=91.5 Mid=94.2 Bottom=94.8 Mean=93.5% RSD=7.0 % From declared=96 .0 SR=11.9

APL-117-6085-05 did not achieve good mixture homogeneity and does not meet the content uniformity criterion.

APL-117-6085-06 and APL-117-6085-07 showed improved powder flow, but mixture uniformity for both formulations was worse than APL-117-6085-03.

Consequently, the applicant has considered modified excipients and more specifically silicated fillers with different particle sizes. These compositions are shown in table. 40 below.

-

Claims (58)

Table 40 Product name Batch No. (% w/w) APL-117-6085-11 APL-117-6085-12 Psilocybin 5.0 1.0 Prosolv 50 10.5 15.5 Prosolv 90 80.0 79.0 Sodium starch glycolate h,o h,o Colloidal silicon dioxide 0.5 0.5 Sodium stearyl fumarate 1.0 1.0 TOTAL 100.0 100.0 Prosolv is a silicated microcrystalline cellulose, and two options were selected to determine whether particle size affected the result. Compared to standard microcrystalline cellulose (typical size range, depending on screening, 80-350 microns), Prosolv has a finer particle size distribution and this results in increased surface area. It has been suggested that increased surface area may provide superior flowability and increased compressibility together with improved content uniformity and formulation stability. The ratio of Prosolv 50 to Prosolv 90 was such as to obtain a particle size distribution of both smaller and larger particles. The results are shown in table. 41 below. Table 41 No. parties Durable (mg) Product flowability (Carris coefficient) Mixture homogeneity Content Uniformity ΑΡΕ- Ι 17- 6085-11 5.0 24.3 TOP= 103.4 Average = 100.4 Bottom = 100.2 Average = 101.5% RSD = 2.0 % From declared=94 ,1 SR=6.0 ΑΡΕ- Ι 17- 6085-12 1.0 21.1 TOP=101.9 Mid=98.4 Bottom=99.9 Mean= 100.1% RSD=3.8% % From stated = 10 0.5 SR=5.8 It can be seen that the key parameters of content uniformity (more than 90% and actually more than 94%) and CP (less than 10 and actually less than 7) are excellent, as is mixture uniformity (more than 95% with margin for error). CLAIM 1. Psilobicin polymorph A, characterized by the following peaks in the powder X-ray diffraction pattern (XRD): 11.5±0.1, 12.0±0.1, 14.5±0.1, 17.5±0.1 and 19.7 ±0.1° 2θ, where psilocybin has a chemical purity of more than 97%. 2. Polymorph A according to claim 1, having a chemical purity of more than 98%. 3. Polymorph A according to claim 1 or 2, having a chemical purity of more than 99%. 4. Polymorph A according to any one of claims 1-3, not containing individual impurities in an amount of more than 1% according to HPLC analysis. 5. Polymorph A according to any one of claims 1-4, additionally characterized by a water content of <0.5% w/w. 6. Polymorph A according to any one of claims 1 to 5, further characterized by an endothermic event in the DSC thermogram having an onset temperature of 205 to 220°C. 7. Polymorph A according to any one of claims 1 to 5, further characterized by one or more of the following: a) loss on drying not more than 2% w/w; b) combustion residue not more than 0.5% w/w; c) analysis (on a dry basis) 95-103% by weight, measured by HPLC; - 53 043831 d) residual solvent content not more than 3000 ppm methanol, 5000 ppm ethanol, 720 ppm THF and 890 ppm toluene, measured by HRGC; And e) elemental analysis by inductively coupled plasma mass spectrometry (ICP-MS) i) not more than 1.5 ppm Cd, ii) not more than 1.5 ppm Pb, iii) not more than 4.5 ppm As, iv) not more than 9.0 ppm Hg, v) not more than 15 ppm Co, vi) not more than 30 ppm V, vii) not more than 60 ppm Ni, viii) not more than 165 ppm Li, and ix) not more than 30 ppm Pd. 8. Polymorph A according to claim 1, 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. 9. Polymorph A according to claim 1, characterized by a PRD pattern the same as shown in FIG. 7a. 10. Polymorph A according to claim 1, additionally characterized by a water content of <0.4 wt.%. 11. Polymorph A according to claim 1, additionally characterized by a water content of <0.3 wt.%. 12. Polymorph A according to claim 1, additionally characterized by a water content of <0.2 wt.%. 13. Polymorph A according to claim 1, additionally characterized by a water content of <0.1 wt.%. 14. Polymorph A according to claim 1, additionally characterized by a loss of <0.5 wt.% on the TGA thermogram at temperatures from 25 to 200°C. 15. Polymorph A according to claim 1, additionally characterized by an endothermic event on the DSC thermogram with an onset temperature of 145 to 155°C. 16. Polymorph A according to claim 1, containing less than 1% psilocin according to HPLC analysis. 17. Polymorph A according to claim 1, containing less than 1% phosphoric acid according to ³¹ P NMR data. 18. Polymorph A according to claim 1, containing crystals ranging in size from 50 to 200 microns. 19. A pharmaceutical composition containing a therapeutically effective amount of polymorph A according to claim 1 and at least one pharmaceutically acceptable excipient. 20. The pharmaceutical composition according to claim 19, wherein the at least one pharmaceutically acceptable excipient is a diluent. 21. The pharmaceutical composition according to claim 19, characterized in that the diluent is microcrystalline cellulose, starch, mannitol, anhydrous calcium phosphate, silicon dioxide, calcium carbonate, talc or combinations thereof. 22. The pharmaceutical composition according to claim 20, in which the diluent contains microcrystalline cellulose. 23. The pharmaceutical composition according to claim 22, wherein the microcrystalline cellulose includes silicated microcrystalline cellulose. 24. The pharmaceutical composition according to claim 23, wherein the microcrystalline cellulose comprises a mixture of two silicated microcrystalline cellulose variants. 25. The pharmaceutical composition of claim 23, wherein the first embodiment of silicated microcrystalline cellulose has a particle size of about 45 to 80 microns, and the second embodiment has a particle size of about 90 to 150 microns. 26. The pharmaceutical composition of claim 25, wherein about 30% or less microcrystalline cellulose is a first embodiment having a particle size of about 45 to 80 microns, and about 70% or more microcrystalline cellulose is a second embodiment having a particle size of about 90 to 150 microns. 27. The pharmaceutical composition of claim 26, wherein about 20% or less microcrystalline cellulose is a first embodiment having a particle size of about 45 to 80 microns, and about 80% or more microcrystalline cellulose is a second embodiment having a particle size of about from 90 to 150 microns. 28. The pharmaceutical composition of claim 26, wherein about 15% or less microcrystalline cellulose comprises the first embodiment having a particle size of from about 45 to 80 microns, and about 85% or more microcrystalline cellulose comprises the second embodiment having a particle size of about 90 up to 150 microns. 29. The pharmaceutical composition according to claim 19, wherein at least one excipient contains a disintegrant. 30. Pharmaceutical composition according to claim 29, characterized in that the disintegrant contains sodium starch glycolate or sodium croscarmellose. 31. The pharmaceutical composition according to claim 19, wherein at least one excipient contains a binder. 32. The pharmaceutical composition according to claim 31, in which the binder contains povidone, co- - 54 043831 povidone or hydroxypropylcellulose. 33. The pharmaceutical composition according to claim 19, wherein at least one excipient contains a lubricant. 34. The pharmaceutical composition according to claim 33, wherein the lubricant is magnesium stearate or sodium stearyl fumarate. 35. The pharmaceutical composition according to claim 19, wherein at least one excipient is a glidant. 36. The pharmaceutical composition according to claim 19, in which the glidant is colloidal silicon dioxide. 37. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is a capsule composition. 38. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is a tablet composition. 39. The pharmaceutical composition according to claim 19, containing from about 1 mg to about 40 mg of polymorph A. 40. Pharmaceutical composition according to claim 19, containing about 1 mg of polymorph A. 41. Pharmaceutical composition according to claim 19, containing about 25 mg of polymorph A. 42. Pharmaceutical composition according to claim 19, characterized in that polymorph A has a chemical purity of more than 99%. 43. The pharmaceutical composition according to claim 19, in which polymorph A has a water content of <0.5% w/w. 44. The pharmaceutical composition according to claim 19, containing less than 1% psilocin according to HPLC analysis. 45. Pharmaceutical composition according to claim 19, containing less than 1% phosphoric acid according to ³¹ P NMR data. 46. The pharmaceutical composition according to claim 40, in which polymorph A has a chemical purity of more than 99%. 47. The pharmaceutical composition according to claim 40, wherein polymorph A has a water content of <0.5% w/w. 48. The pharmaceutical composition according to claim 40, containing less than 1% psilocin according to HPLC analysis. 49. Pharmaceutical composition according to claim 40, containing less than 1% phosphoric acid according to ³¹ P NMR data. 50. The pharmaceutical composition according to claim 41, in which polymorph A has a chemical purity of more than 99%. 51. The pharmaceutical composition according to claim 41, in which polymorph A has a water content of <0.5% w/w. 52. The pharmaceutical composition according to claim 41, containing less than 1% psilocin according to HPLC analysis. 53. Pharmaceutical composition according to claim 41, containing less than 1% phosphoric acid according to ³¹ P NMR data. 54. Use of polymorph A according to any one of claims 1 to 18 or a pharmaceutical composition according to any of claims 19 to 53 for the treatment of a central nervous system disorder selected from generalized anxiety disorder, personality disorder, drug disorder, gaming disorder, food addiction, body dysmorphic disorder , pain and depression. 55. Use according to claim 54, wherein the generalized anxiety disorder is clinical depression (CD), histamine headache or obsessive-compulsive disorder (OCD). 56. Use according to claim 54, wherein the personality disorder is a conduct disorder. 57. Use according to claim 54, wherein the drug disorder is alcohol dependence, nicotine dependence, opioid dependence or cocaine dependence. 58. Use according to claim 54, wherein the depression is drug-resistant depression. -