DIOXYPHENYL)-2-(METHYLAMINO) PROPANE (MDMA)

FIELD OF INVENTION

[0001] The present invention relates to the field of organic synthesis.

[0002] In one form, the invention relates to an improved method of synthesising a pharmaceutically active organic compound.

[0003] Any discussion of material such as documents, devices, acts or knowledge in this specification is included to explain the context of the invention in terms of the inventor’s knowledge and experience and, accordingly, any such discussion should not be taken as an admission that any of the material forms part of the prior art base or the common general knowledge in the relevant art in Australia, or elsewhere, on or before the priority date of the disclosure and claims herein.

[0004] It is to be appreciated that any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the present invention. Further, the discussion throughout this specification comes about due to the realisation of the inventor and/or the identification of certain related art problems by the inventor.

BACKGROUND ART

[0005] 3,4-Methylenedioxymethamphetamine, otherwise known as l-(3’,4’- methylenedioxyphenyl)-2-(methylamino)propane (MDMA), is a psychoactive drug that acts primarily by increasing the activity of neurotransmitter substances such as serotonin, dopamine and noradrenaline in parts of the brain. Early synthesis of MDMA was reported in 1912 by Merck in German patent no. 274350 filed 24 December 1912. At that time, Merck was not interested in MDMA as a pharmaceutical in its own right, but as an intermediate for preparation of the styptic compound methylhydrasitine. Subsequently, scientists periodically explored its pharmacological effects but it was not until the 1960’s that substantial investigations were published.

[0006] Recreational use of MDMA developed substantially during the 1970’s and 1980’s. In 1985 the World Health Organization's Expert Committee on Drug Dependence recommended that MDMA be placed in Schedule I of the United Nations Convention on Psychotropic Substances (1971). By the end of the 1980s the use or possession of MDMA had been formally criminalised in many countries and most legitimate medical use had ceased by the 1990s. [0007] Nonetheless, in recent times MDMA has been investigated for clinical use, including clinical applications for enhancing psychotherapy. Administration of purified MDMA in a therapeutic setting has been found to increase the effectiveness of psychotherapy techniques, such as revisiting traumatic experiences with an appropriate level of emotional engagement.

[0008] In 2017 the United States Food and Drug Administration approved limited research on MDMA-assisted psychotherapy for post-traumatic stress disorder (PTSD), designating MDMA assisted psychotherapy as a Breakthrough Therapy after their assessment of preliminary results from Phase 2 clinical trials. Results of a Phase 3 trial in the United States have been reported in Mitchell et al., Nature Medicine, vol.27, June 2021, 1025-1033, https://doi.ort/10.1038/s41591- 021-01336-3. In recent times the Australian Therapeutic Goods Administration has issued a number of approvals for the use of MDMA as part of the psychotherapy for treatment of PTSD under its Special Access Scheme-B (SAS-B). The SAS-B allows certain health practitioners to access therapeutic goods that are not included in the Australian Register of Therapeutic Goods, for a single patient who is defined as seriously ill. MDMA is also being used under expanded access schemes in the United States, Switzerland and Israel.

[0009] Relevant studies are discussed for example in Bahji et al., Progress in Neuropsychopharmacology & Biological Psychiatry 96 (2020) 109735; Jerome et al., Psychopharmacology, https://doi .org/10.1007/s00213-020-05548 -2; and Mitchell et al., mentioned above.

[0010] Some of the studies have been sizeable. A recently completed Phase 3 study had 98 participants (half placebo) and a large effect size. This followed from a Phase 2 trial which had a similar effect size. The size of the first Phase 3 trial is likely to be statistically relevant because of the large effect size of the Phase 2 trial. All other factors being equal, the larger the effect size, the smaller the sample size require by the regulatory authorities. A second Phase 3 trial is anticipated.

[0011] However, legitimate studies are hampered due to a lack of licit, pharmacological quality MDMA. To ensure that patients receive safe, effective drugs, manufacture is closely reviewed by drug regulators for compliance with the requirements of current Good Manufacturing Process (cGMP). Although the first synthesis of MDMA was reported by Merck in 1912, it has taken over a century for the first fully validated cGMP to be described by Nair et al., in ACS Omega 2022, 7, 900-907, http://d0i.0rg/l /acsomega.1 c()5520 . [0012] Even when a legitimate source is available in one country, transport and supply to other countries is not practical or economical. National laws and the suite of United Nations Conventions against illicit trafficking of drugs and drug precursors also hampers access of licit transport across borders.

Chemistry

[0013] MDMA is in the class of chemicals known as substituted amphetamine or substituted methylenedioxyphenethylamines. As a free base, MDMA is a colourless oil that is insoluble in water. The most common salt of MDMA is the crystalline, water soluble hydrochloride salt. MDMA is usually obtained as a racemic mixture of (R)-MDMA and (S)-MDMA enantiomers having the following structures:

(R)-MDMA (S)-MDMA

[0014] The original synthesis described in the Merck patent mentioned above involves an initial step of brominating safrole to form l-(3,4-methylenedioxyphenyl)-2-bromopropane according to Reaction Scheme 1(a). safrole

Reaction Scheme 1(a)

[0015] The l-(3,4-methylenedioxyphenyl)-2-bromopropane is reacted (1) with Nal and hexamine, then (2) reacted with ammonia to form 3,4-methylenedioxyamphetamine (MDA). The MDA is (3) reacted methylamine to form MDMA, then (4) transformed to 3,4-methylenedioxy- N-ethylamphetamine (MDEA) by reaction with ethylamine according to Reaction Scheme 1(b): 1 N I h i

Reaction Scheme 1(b)

Starting Materials

[0016] Piperonal and safrole are well known starting products for MDMA production.

[0017] The starting products are usually converted to an intermediate, (3,4- methylenedioxyphenyl)-2-propanone (MDP2P), depicted in Reaction Scheme 2:

Reaction Scheme 2

[0018] One conversion method includes isomerisation of safrole to isosafrole in the presence of a strong base, followed by oxidisation of the isosafrole to MDP2P. Another method uses the Wacker process to oxidize safrole directly to the MDP2P intermediate using a palladium catalyst according to Reaction Scheme 3:

Reaction Scheme 3 [0019] However, this reaction sequence has a number of drawbacks. It is difficult to source safrole for use in pharmaceutical synthesis, partly due to its listing as a Table I precursor under the United Nations Convention Against Illicit Traffic in Narcotic Drugs and Psychotropic Substances. Furthermore, benzoquinone is hazardous to handle in large amounts and the palladium (II) catalyst is expensive.

[0020] Another problem is that the reaction produces competing aldehyde and ketone products. Specifically, the reaction generates a large proportion of an aldehyde by-product instead of the desired ketone. Separation of the aldehyde and ketone via sodium bisulphite extraction is possible, but the yield of ketone is poor.

Use of MDP2P Precursor

[0021] The majority of illicit MDMA synthesis is based on the use of MDP2P as a precursor. The first step utilises a Leuckart reductive amination of MDP2P in the presence of N-methyl formamide and formic acid to produce N-formyl-MDMA according to the Reaction Scheme 4:

Reaction Scheme 4

[0022] The Leuckart reaction sequence is challenging to perform and requires carefully controlled heating.

[0023] The next step is acid mediated hydrolysis of N-formyl-MDMA, followed by pH adjustment to produce MDMA oil according to Reaction Scheme 5a:

Reaction Scheme 5a

[0024] However, this reaction sequence provides a low yield of low purity MDMA. Licit commercial use of this reaction scheme is not practical due to the low yield and the cost of the extensive purification process to remove multiple impurities and by-products to achieve a pharmaceutically acceptable product. cGMP Synthesis

[0025] The first fully validated cGMP described by Nair et al., in ACS Omega 2022, 7, 900- 907, http://doi.org/10.1021/aesomega. lcO552O is a four-stage process capable of reproducibly synthesizing up to 5kg (approximately 30,000 patient doses) of MDMA-HC1 with an overall yield of 41.8-54.6% and a minimum purity of 99.4% w/w by HPLC assay.

[0026] Reaction scheme 5(b) illustrates the cGMP. The first step comprises performing Grignard reaction between 5-bromo-l,3-benzodioxole and 1,2- propylene oxide in tetrahydrofuran (THF) followed by distillation to produce l-(3,4-methylenedioxy-phenyl)-2-propanol. The following three synthetic steps rely on well-known synthetic transformations. The l-(3,4- methylenedioxy-phenyl)-2-propanol was oxidised to methyl piperonyl ketone with a biphasic (DCM/water) TEMPO/KBr/bleach reagent system, followed by aqueous workup, filtration and removal of solvent. The crude methyl piperonyl ketone was subjected to reductive amination using aqueous methylamine and NaOH/Na BH4. A complex acid/base treatment led to isolation of MDMA.HC1.

Reaction Scheme 5(b)

[0027] The synthetic steps for the above described cGMP are well-known, but the workup is relatively complex.

[0028] Accordingly, there is an ongoing need for a commercially practical and economically viable method of obtaining pharmaceutically acceptable MDMA. SUMMARY OF INVENTION

[0029] In one aspect the present invention seeks to provide an improved method of synthesising MDMA that is of sufficient purity that it can be used for pharmaceutical applications.

[0030] In another aspect the present invention provides an improved method of synthesising MDMA that is achievable using readily available reactants or which is at least commercially practical.

[0031] The present invention also seeks to overcome or alleviate at least one of the above noted drawbacks of related art syntheses or to at least provide a useful alternative to related art syntheses.

[0032] In its broadest form, a first aspect of embodiments of the present invention provides a method of synthesising MDMA, which includes both (R)-MDMA and (S)-MDMA, in high yield in three steps from piperonal starting material. Preferably the three steps comprise a condensation reaction, followed by oxidative hydrolysis to form MDP2P as an intermediate, then reductive amination.

[0033] In a second aspect of embodiments described herein there is provided a method of synthesising MDMA comprising the steps of;

(a) condensation of piperonal and nitroethane in the presence of a catalysing base to form l-(3’,4’-methylenedioxyphenyl)-2-nitroprop-l-ene (MDP-2- nitropropene),

(b) oxidative hydrolysis of MDP-2-nitropropene to form 1- (3’,4’methylenedioxyphenyl)-propan-2-one (MDP2P), and

(c) reductive amination of MDP2P to l-(3’,4’-methylenedioxyphenyl)-2- (methylamino)propane (MDMA).

[0034] Optionally, the MDMA formed according to the above methods is converted to a pharmaceutically acceptable salt, preferably a hydrochloride salt - l-(3’,4’- methylenedioxyphenyl)-2-(methylamino)propane hydrochloride monohydrate (MDMA.HCl).

[0035] Preferably, the yield of MDP-2-nitropropene from piperonal according to Step (a) is greater than 90%, more preferably greater than 95%. [0036] Preferably, the yield of MDP2P from MDP-2-nitropropene according to Step (b) is greater than 75%, more preferably greater than 85%.

[0037] Preferably, the yield of MDMA from MDP2P according to Step (c) is at least 90%, more preferably 95%.

[0038] It has advantageously been found that the Step (a) condensation of piperonal and nitroethane in the presence of a catalysing base can be carried out in the absence of solvent to form MDP-2-nitropropene in high yield. This is contrary to previous teaching that the reaction must be carried out in the presence of an acid, such as an acid solvent. Preferably Step (a) comprises reacting piperonal and base in a mole ratio of 1:0.01- 2 of piperonal to catalysing base. This is preferably carried out in the presence of an excess of nitroethane, in a mole ratio of about 1:8 of piperonal to nitroethane, more preferably about 2:6 of piperonal to nitroethane.

[0039] Preferably, the catalysing base of Step (a) is an amine. It is preferred that the amine is a cycloamine such as cyclohexylamine or alkylamine such as ethylene diamine.

[0040] The Step (a) condensation is typically carried out at 115-125 °C, preferably about 120 °C. In a particularly preferred embodiment Step (a) is microwave assisted.

[0041] Preferably, Step (b) comprises oxidative hydrolysis of MDP-2-nitropropene in the presence of an excess of an oxidative catalyst. Contrary to prior art teaching that recommends a 10-to- 12-fold excess of catalyst, the present invention may be carried out using, for example, an iron catalyst in no more than a 5-fold excess.

[0042] Advantageously, the reactions of steps (b) and (c) may proceed at ambient temperature without requiring special heating or cooling conditions.

[0043] The intermediates formed during the synthesis Steps (a), (b), and (c) are all readily isolable solid materials and may be crystallised to reduce the content of impurities, without the need for recrystallisation or chromatography. However, preferably the MDP2P product of Step (b) and the MDMA product of Step (c) are recrystallised to further reduce unwanted by-products.

[0044] In a third aspect of embodiments described herein there is provided a method of producing MDMA according to the Reaction Scheme 6:

Reaction Scheme 6

[0045] Step (c), the reductive amination of MDP2P to MDMA with sodium cyanoborohydride in the presence of methylamine hydrochloride, should be conducted at ambient temperature. It has been found that increasing the temperature of reaction is likely to cause the further, undesirable reduction of the ketone (MDP2P) to the corresponding alcohol l-(3’,4’-MDP)-2-propanol.

[0046] Preferably Step (c) comprises reacting MDP2P with a slight molar excess of sodium cyanoborohydride, in the presence of a large excess of methylamine hydrocholoride. Preferably the methylamine hydrochloride is present in a mole ratio of 10:12 of MDP2P to methylamine hydrochloride.

[0047] In a fourth aspect of embodiments described herein there is provided MDMA comprising (R)-MDMA and (S)-MDMA, produced according to any of the aforementioned methods.

[0048] In a fifth aspect of embodiments described herein there is provided a pharmaceutical composition comprising a therapeutically effective amount of MDMA, (R)-MDMA or (S)- MDMA produced according to any of the aforementioned methods, and at least one pharmaceutically acceptable excipient.

[0049] In a sixth aspect of embodiments described herein there is provided a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of MDMA, (R)-MDMA or (S)-MDMA produced according to any of the aforementioned methods, and at least one pharmaceutically acceptable excipient.

[0050] In a seventh aspect of embodiments described herein there is provided the use of MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof produced according to any of the aforementioned methods in the preparation of a medicament for MDMA- assisted psychotherapy. The medicament may be used in combination with one or more other kinds of therapeutic agents and /or therapeutic methods for the treatment, amelioration and/or prevention of psychological disorders.

[0051] Typically, the medicament would be in the form of capsules containing a powder mix or tablets, typically pressed tablets. The powder mix for a capsule would typically include MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof combined with any excipients, such as binders, disintegrants, fillers, glidant and preservatives. The press mix for a tablet would typically include MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof combined with excipients, such as binders, disintegrants, fillers, glidant, lubricant and preservatives.

[0052] In an eighth aspect of embodiments described herein there is provided a method of treating, ameliorating and/or preventing a psychological disorder comprising administering to a subject in need thereof, a therapeutically effective amount of MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof, produced according to any of the aforementioned methods. The method may be used in combination with one or more other kinds of therapeutic agents and /or therapeutic methods for the treatment, amelioration and/or prevention of psychological disorders. A typical treatment regimen would include a patient receiving an initial dose of 1 to 250 mg, more typically 60 to 150 mg, preferably 80 to 120 mg, the exact amount depending on body mass. If the patient and clinician believe it would be helpful, after a couple of hours the patient may receive a subsequent dose of between 20 to 80 mg, 40mg to 60mg.

[0053] Other aspects and preferred forms are disclosed in the specification and/or defined in the appended claims, forming a part of the description of the invention.

[0054] In essence, embodiments of the present invention stem from the realization that it is possible to isolate high yields of MDMA from piperonal in three synthetically straightforward steps by careful control of the acid and base components of the synthesis. Specifically, it has been found that it is advantageous to use a first step involving condensation of piperonal and nitroethane in the presence of a base, but the absence of an acid. It has also been realised that careful control of the temperature of reaction advantageously avoids undesirable side reactions and much of the synthesis can be carried out at ambient temperature.

[0055] Advantages provided by the synthetic method of the present invention comprise the following: readily available and cost-effective precursors and reaction materials; • provides desired compounds in useful yields;

• improved production of MDP-2-nitropropene by removal of acid catalyst;

• has a small number of synthetic steps;

• piperonal (in contrast to safrole) is commercially available because it is used in other industries, notably the flavour and fragrance industries;

• minimal side-reactions with concomitant reduction in by-products;

• scalable; and

• manageable chemical risks associated with reaction materials and methods.

[0056] Further scope of applicability of embodiments of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure herein will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Further disclosure, objects, advantages and aspects of preferred and other embodiments of the present application may be better understood by those skilled in the relevant art by reference to the following description of embodiments taken in conjunction with the accompanying drawings, which are given by way of illustration only, and thus are not limitative of the disclosure herein, and in which:

Figure 1 illustrates spectral analyses for the condensation of piperonal and nitroethane in the presence of a cyclohexylamine base to form MDP-2-nitropropene as described in Example 1A including: Fig.lA - ’ H NMR spectrum; Fig. IB - ¹³ C NMR spectrum

Figure 2 illustrates analyses for the condensation of piperonal and nitroethane in the presence of ethylenediamine base to form MDP-2-nitropropene as described in Example IB including: Fig.2A - ¹ H NMR spectrum; Fig.2B - ¹³ C NMR spectrum; Fig.2C - HPLC; Fig.2D - HPLC-MS; Fig.2E - HPLC-nMS ² Collision energy -10 Volts; Fig.2F - HPLC-nMS ² Collision energy -20 Volts; Fig.2G - HPLC-nMS ² Collision energy -35 Volts; Figure 3 illustrates analyses for the oxidative hydrolysis of MDP-2-nitropropene with iron to form MDP2 -propanone as described in Example 2 including: Fig.3A - ¹ H NMR spectrum; Fig.3B - ¹³ C NMR spectrum; Fig.3C - HPLC-UV; Fig.3D - HPLC-MS; Fig.3E - HPLC- nMS ² Collision energy -10 Volts; Fig.3F - HPLC-nMS ² Collision energy -20 Volts; Fig.3G - HPLC-nMS ² Collision energy -35 Volts;

Figure 4 illustrates analyses for the reductive amination of MDP2P with sodium cyanoborohydride in the presence of methylamine hydrochloride as described in Example 3 including: Fig.4A - ’ H NMR spectrum; Fig.4B - ¹³ C NMR spectrum; Fig.4C - HPLC-UV; Fig.4D - HPLC-MS; Fig.4E - HPLC-nMS ² Collision energy 10 Volts; Fig.4F - HPLC-nMS ² Collision energy 20 Volts; Fig.4G - HPLC-nMS ² Collision energy 35 Volts; Fig.4H - HPLC-nMS ³ Collision energy -10 Volts; Fig.41 - HPLC-nMS ³ Collision energy -20 Volts; Fig.4J - HPLC-nMS ³ Collision energy -35 Volts;

Figure 5 illustrates analyses for the formation of the hydrochloride salt of MDMA from MDMA free base as described in Example 4 including: Fig.5A - ¹ H NMR spectrum; Fig.5B - ¹³ C NMR spectrum; Fig.5C - HPLC-nMS ² , collision energy -10 Volts; Fig.5D - HPLC- nMS ² , collision energy -20 Volts; Fig.5E - HPLC-nMS ² , collision energy -35 Volts;

DETAILED DESCRIPTION

[0058] It is to be understood that the specific devices and processes illustrated in the attached Figures and described in the following specification are simply exemplary embodiments of the inventive concepts defined in the appended claims. Hence, specific physical characteristics relating to the embodiments disclosed herein are not to be considered as limiting, unless the claims expressly state otherwise.

[0059] The preparation of MDMA according to the present invention comprises subjecting piperonal to three steps, preferably a condensation step, followed by oxidative hydrolysis of the condensate, then reductive amination.

[0060] Preferably, the preparation of MDMA involves synthesis of MDP2P as intermediate.

Steps (a) & (b) - Preparation of MDP2P Intermediate

[0061] Piperonal is a preferred starting material because it is readily available in high purity and comparatively inexpensive. [0062] In a preferred embodiment, the MDP2P is prepared by the following two steps;

(a) condensation of piperonal and nitroethane in the presence of a catalysing base to form MDP-2-nitropropene, and

(b) oxidative hydrolysis of MDP-2-nitropropene with iron to form MDP2P.

[0063] This two-step reaction has the advantage of having a relatively simple reaction workup.

Step (a) - Condensation of piperonal

[0064] Preferably, the condensation step is a modified Henry reaction. The prior art as described in Yang et al. (2012) Chin.J.Chem. 30, 2827-2833 (DOI: 10.1002/cjoc. 20120194) teaches the use of ethylene diamine as a highly effective catalysing base for one-pot synthesis of aryl nitroalkenes via the Henry reaction.

[0065] Similarly, PiHKAL A Chemical Love Story by Shulgin and Shulgin, (1991) Transform Press describes the reaction of piperonal in glacial acetic acid with nitroethane in the presence of cyclohexylamine .

[0066] In respect to the present invention, as described at Example 1A and Example IB, it has been found that the preferred catalysing base is an amine. It has further been found that in a particularly preferred embodiment the catalysing base is cyclohexylamine (as per the following Reaction Scheme 7 described at Example 1A)

Reaction Scheme 7 or more preferably, ethylene diamine (as per the following reaction Scheme 8 described at Example IB):

Reaction Scheme 8 Step (b) - Oxidative Hydrolysis of MDP-2-nitropropene with Iron.

[0067] The second step comprises oxidative hydrolysis of MDP-2-nitropropene in acetic acid with iron catalyst to form MDP2P according to Reaction Scheme 9 as described at Example 2:

Reaction Scheme 9

[0068] A number of prior art publications describe the general synthesis including Gallager et al. (2012) Forensic Sci. Int. Vol.223, Issues 1-3, 30 Nov. 2012, pp.306-313 (D01:http://dx.doi.org/10.1016/j.forsciint.2012.10.006)

[0069] MDP2P can be generated easily and in high purity from MDP-2-nitropropene with few side-reactions. The second step is highly tolerant of lower purity feedstock.

Step (c) - Reductive Amination of MDP2P to MDMA

[0070] The third step, step (c), comprises the reductive amination of MDP2P in methanol with sodium cyanoborohydride in the presence of methylamine hydrochloride according to Reaction Scheme 10:

Reaction Scheme 10

[0071] Step (c) needs to be conducted at ambient temperatures because elevated temperatures of reaction have been observed to cause the further, undesirable reduction of the ketone (MDP2P) to the corresponding alcohol l-(3’,4’-MDP)-2-propanol. Without wishing to be bound by theory it is postulated that two factors are important here. The first is driving of the Schiff’s base formation with excess amine. The cyanoborohydride acts upon the Schiff s base to reduce it to the corresponding amine and so the second fact is a consequential reduction reaction being driven to proceed after the formation of the Schiff s base. Typically, the reduction step is conducted in the same vessel but is intended to proceed after the formation of the Schiff s base or imine (the central structure in the Reaction Scheme 10). The alcohol is a likely competitive product if the sequence is compressed so that it does not reach equilibrium or if an excess of methylamine is not allowed (which would cause an equilibrium portion of the ketone to remain). Preparation of MDMA salt

[0072] The MDMA prepared according to the present invention may be in the form of a liquid and may optionally be converted to a desired salt. In particular, hydrochloride salt of MDMA free base is well known and characterised.

EXAMPLES

[0073] The present invention will be further described with reference to the following nonlimiting examples.

Analysis:

[0074] The examples include reference to use of ’ H and ¹³ C nuclear magnetic resonance spectroscopy (NMR) to establish structure of key intermediates and products and HPLC-UV-MS to test the purity and detect by-products.

[0075] HPLC-UV-MS has been used to further identify the structures of any products obtained.

[0076] Method of analysis for UHPLC with UV detection: Analysis of samples was performed using a LC-30 UHPLC with SPD M20A diode array detector in series with a LCMS-8O3O triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto Japan). The separation was achieved using a Luna Omega C18 column (150 x 2.1 mm.id., 1.6 micron particles) from Phenomenex (Torrence, CA, USA) operated at 50 °C. The mobile phase consisted of Solvent A (0.1%v/v formic acid in MilliQ water) and Solvent B (0.1 % v/v formic acid in acetonitrile). A linear solvent gradient was programmed from an initial composition of 0% B (0.0-5.0 min) to 100 % B (5.0-25.0 min) and held at the final composition (25.0-26.0 min) before returning to the starting composition (26.0-30.0 min). The flow rate was 0.300 mL/min. Solvents were membrane degassed using a LC-30 degasser (Shimadzu)

[0077] The injection volume was 10 pL using a needle in line SIL-30AC autosampler (Shimadzu). The UV detection wavelength was 2.54 nm. Samples for analysis were reconstituted in methanol at a concentration of 0.1 mg/mL immediately prior to analysis (standing time was less than 2 hours at ambient temperature). Data was collected and processed using LabSolutions software (Shimadzu).

[0078] The LCMS-83OO (Shimadzu Corporation, Kyoto, Japan) was operated in line with the UV detector with 100% of the sample flowing to the ion source. Ionisation was in electrospray mode using an ESI-8O3O source. The gas flows were nebulising gas flow (3 L/min) and drying gas (15 L/min). The DL temperature was 150 °C and the Heater Block was set to 400 °C. The interface voltage was 4.5 kV.

[0079] For scanning experiments, Q3 scan was used across three mass ranges of 100-200 mass units at a scan rate of 15000 micron/sec, 200-300 mass units at a scan rate of 15000 micron/sec, and 300-500 mass units at a scan rate of 15000 micron/sec. The different mass ranges were used to allow the management of chemical noise and the detection of by-products.

[0080] Collision experiments were carried out using precursor ions identified by correlation of significant peaks detected with the UV detector (>0.5% of the height of the peak of interest) and the corresponding significant masses detected at that retention time (corrected for the retention gap between detectors). Collision was at -10, -20, and -35 Volts acceleration potential difference and the product ions were scanned at 15000 micron/sec. In some cases, where the Q- Array resulted in the generation of significant fragmentation, MS ³ experiments were also performed using the MS ² conditions and selection of the Q-Array generated fragment as the Q2 precursor ion. Data was collected and processed using LabSolutions software (Shimadzu).

Example 1 - Preparation of MDP2P Intermediate

[0081] Example 1 illustrates a two-step reaction to form MDP2P according to Reaction Scheme 7 or Reaction Scheme 8.

[0082] The first step is the formation of 3’,4’-methylenedioxy-2-nitroprop-l-ene and two variations of the condensation reaction namely: a Henry reaction utilising cyclohexylamine as the catalysing base in the presence of acetic acid (Example 1A); or use of ethylenediamine as the catalysing base as a neat reaction (Example IB).

[0083] The second step is the oxidative hydrolysis of the nitropropene adduct (MDP-2- nitropropene) in the presence of iron dust and acetic acid to form MDP2P (Example 2).

Example 1A- Condensation of Piperonal and Nitroethane in the presence of a Cyclohexylamine Base to Form MDP-2-nitropropene

[0084] A nitroaldol reaction was carried out according to the following reaction:

Reaction Scheme 7

[0085] By comparison with other examples herein, Example 1A illustrates the surprising novelty of the reaction continuing in a non- acidic environment intended to protect the nitroethane from degradation.

[0086] The reaction was carried out by combining the following reagents in a round bottom flask and refluxing at approximately 120°C for 6-8 hours:

[0087] The progress of the reaction was monitored to completion using a TLC system (100% dichloromethane on silica). A by-product, piperonylonitrile, was identified.

[0088] NMR spectroscopy of the materials isolated from the reaction (FIG.l) shows the presence of both the desired product and piperonylonitrile. The nitrile is a by-product produced by the degradation of nitroethane. Conditions that prevent the breakdown of the nitroethane will prevent the formation of the nitrile.

[0089] High purity MDP-2-nitropropene product was crystallised from the reaction mixture however the yield was relatively low (50-60%). Poor yield, particularly on scale up can be explained by crystallisation during work up and a significant production of the by product, piperonylonitrile. An alternative workup involving a base wash to remove acetic acid may increase the yield, but concomitantly decrease purity.

[0090] Prior art wisdom as embodied in PiHKAL, A Chemical Love Story by Shulgin and Shulgin, (1991) Transform Press, at pages 298 and 320 is that the reaction of piperonal, nitroethane and cyclohexylamine should be carried out by reflux in glacial acetic acid solvent. Hitherto there has been no readily apparent reason to diverge from this practice. [0091] However, it was surprisingly found that refluxing in the absence of acetic acid solvent significantly improved suppression of by-product formation. Without wishing to be bound by theory it is believed that the formation of piperonylonitrile is acid catalysed - the result of conversion of the aldehyde to a corresponding oxime and subsequent dehydration of the oxime to the nitrile. The formation of hydroxylamine from nitroalkanes is well understood and proceeds in an acidic environment according to the following reaction: nitroethane

[0092] The formation of piperonylonitrile is therefore a competing acid-catalysed reaction that may be reduced by the elimination of acetic acid as the solvent as follows: piperonal MD-benzonitriie

[0093] Again, without wishing to be bound by theory, the reagents are liquids and the intended reaction kinetics are likely determined by bimolecular collision which is enhanced at higher concentrations.

Example IB- Condensation of Piperonal and Nitroethane in the Presence of Ethylenediamine

Base to Form MDP-2-nitropropene

[0094] The following nitroaldol condensation reaction was carried out:

Reaction Scheme 8

[0095] The reaction was carried out by combining the following reagents. Initial small scale experiments were successfully scaled up to 30 g.

[0096] The reagents were combined in a round bottom flask and the reaction performed under reflux at approximately 120°C for 12-16 hours. The progress of the reaction was monitored to completion using a TLC system (100% dichloromethane on silica). The desired product was detected as a bright yellow spot (RF = 0.91) and consumption of the starting material (RM = 0.71). The TLC did not indicate the presence of by-products.

[0097] Importantly, the reaction was monitored for end point. Allowing the reaction to continue beyond this point results in formation of a Michael addition by product. The amount of catalyst added is also critical, preferably 1-2% , in order to avoid formation of by-products.

[0098] The reaction slowed at around the 10 hour mark. Addition of an equal portion of ethylenediamine allowed the reaction to continue. Upon complete consumption of the starting material (monitored by the same TLC method) the reaction was allowed to cool.

[0099] The reaction mixture was worked up by removing half of the remaining nitroethane using rotary evaporation (ca. 40mbar @ 50°C). Upon cooling an orange slurry formed. To the slurry, a 1:2 mixture of methanol: water was added to remove the remaining nitroethane and promote precipitation of the desired MDP-2-nitropropene.

[00100] The precipitate was collected by filtration and the filtrate washed with a 1:1 methanol: water solution. The product was dried in a vacuum oven for 1-3 hours at 70°C. The bright yellow MDP-2-nitropropene product was recovered in high yield (90-95%).

[00101] FIG 2 incudes and ¹³ C NMR spectra, consistent with the structure of MDP-2- nitropropene with no significant evidence of impurities or by-products.

Example 2 - oxidative hydrolysis of MDP-2-nitropropene with iron

[00102] The following oxidative hydrolysis was carried out: [00103] The reaction was carried out by combining the following reagents. Initial small scale experiments were successfully scaled up to 5 g and 10 g.

[0104] Very fine iron powder as to be interchangeable with ‘iron dust’ was combined with acetic acid in a round bottom flask and stirred with heating to 100°C. Immediately upon reaching this temperature, MDP-2 -nitropropene solution (~0.25g/mL) in acetic acid was added dropwise. Upon full addition, the solution was refluxed until the reaction reached completion after 1.5-2.0 hours.

[0105] The progress of the reaction was monitored to completion using a TLC system (100% dichloromethane on silica) for the formation of the desired product (RF = 0.61) and consumption of the starting material (RF = 0.91). No by-products were detected.

[0106] Advantageously, although published prior art methods teach 10-12 equivalents of iron, the present invention has been successfully conducted using no more than a 5-fold excess or iron which greatly simplifies the purification method over the prior art.

[0107] Two work up methods were carried out:

Work Up Method 1:

[0108] In this method, the 5g scale reaction was worked up. The reaction solution was filtered to remove undissolved salts. The precipitate was washed with dichloromethane to give a dark filtrate. Saturated sodium bicarbonate was added to the filtrate to neutralise excess acetic acid, creating gas and some precipitate, which was filtered off.

[0109] Further, saturated sodium hydrogen carbonate was added to the filtrate to fully neutralise the acid present. The organic layer was dried over magnesium sulphate, filtered and the solvent removed via rotary evaporation. The MDP2P produced was a light brown oil (3.247g, 18.22mmol, 76% yield).

Work Up Method 2:

[0110] In this work up method the reaction solution was added to a large volume of water to dissolve iron salts and residual acetic acid. The aqueous layer was extracted with dichloromethane (DCM). The combined organic fractions were washed with a base (saturated sodium bicarbonate/dilute (5%) sodium hydroxide solution) to neutralise the acid.

[0111] The organic layer was dried over magnesium sulphate, filtered and the solvent removed via rotary evaporation to yield the product as a light brown oil (7.525g, 42.33mmol, 87.48% yield).

[0112] Although recovery Method 2 provides a significantly higher product yield than Method 1, Method 2 requires a large volume of water to dilute the acetic acid and consequently presents more significant handling and waste disposal issues.

[0113] FIG 3 incudes ¹ H and ¹³ C NMR spectra, consistent with the structure of MDP2P with no significant evidence of impurities or by-products.

[0114] Accordingly, MDP2P can be generated easily and in high purity from MDP-2- nitropropene with little evidence of side reactions. The yield of recovered product was as high as 90% depending on the extraction (work up) method.

Example 3 - reductive amination of MDP2P with sodium cyanoborohydride in the presence of methylamine hydrochloride.

[0115] The following reductive amination reaction was carried out:

Reaction Scheme 10

[0116] The reaction was carried out by combining the following reagents. Initial small scale experiments were successfully scaled up to 5 g.

[0117] The reaction was carried out by combining the reagents at ambient temperature to create a suspended solution. The suspension was stirred at ambient temperature and the pH measured and adjusted to between 6 and 7 using methanoic HC1. The pH was measured and readjusted every 3-4 hours, the system slowly becoming basic over time, and the reaction becoming complete in 30-40 hours.

[0118] The progress of the reaction was monitored to completion using a TLC system (100% dichloromethane on silica) via consumption of the MDP2P (R = 0.61) and product formation (R = 0.14).

[0119] The reaction mixture was worked up by removing the methanol solvent using rotary evaporation. The evolved solids were redissolved in a volume of water approximately equal to the reaction volume. Concentrated HC1 (38%) was added to the crude material to make the solution strongly acidic (<pH 1) to decompose the excess borohydride. Significant gas was evolved from the crude material.

[0120] The aqueous layer containing the product was washed with dichloromethane, then the organic layer was re-extracted with IM HC1. Combined aqueous fractions were made basic (~pH 10) using a strong NaOH solution, causing a cloudy suspension to form. The final step was extraction with dichloromethane and removal of the solvent via rotary evaporator to isolate the MDMA product as a light brown oil in 70% yield. Re-extracting the organic layer with additional acid improved the yield of product by -10%.

[0121] FIG 4 incudes 1H and 13C NMR spectra, consistent with the structure of MDMA with no significant evidence of impurities or by-products.

Example 4 - formation of hydrochloride salt from MDMA free base

[0122] MDMA produced according to the procedural steps set out in Examples 1 to 3 can be readily converted to the hydrochloride salt according to Reaction Scheme 11: Reaction Scheme 11

[0123] The method is based on the titration of methanolic hydrochloric acid up to a stoichiometric quantity in a solvent that is able to sequester water (from the hydrochloric acid) while also being a poor solvent for the MDMA hydrochloride. In this case, diethyl ether was used for convenience but could be replaced with acetone or a higher alcohol. [0124] The salt formation was carried out by combining the following reagents. Initial small scale experiments were successfully scaled up to 700mg.

[0125] MDMA was dissolved in diethyl ether at ambient temperature. Methanoic HC1 was added dropwise, resulting in precipitation of light brown to cream coloured MDMA.HC1 precipitate.

[0126] The solid was collected via centrifugation and supernatant decanted. The separated solid was placed in a vacuum oven at 70°C for 1 hour to provide light brown to cream coloured MDMA.HC1 crystalline solid in 52% yield. A further crop of crystalline solid was recovered upon concentration of the supernatant.

[0127] FIGs 5A and 5B include and ¹³ C NMR spectra obtained in CD3OD, consistent with the structure of MDMA.HC1 with no significant evidence of impurities or by-products. The peak (arrow) at ~5.0ppm in the 1H spectrum has an integral of two and is attributable to one water of hydration. This identifies the salt as a monohydrate. Microscopy shows the solid to be crystalline.

[0128] MDMA released from the hydrochloride salt yields identical chromatographic retention and LC-MS and LC-MS ² spectra to the free base.

Example 5 - Microwave Assisted Synthesis

[0129] Microwave assisted synthesis makes use of a microwave reactor system instead of using conductive heating from an electrical or gas-powered source. Use of a microwave source provides more uniform heating. In general, for every 10°C increase in temperature in a microwave reactor, the reaction time halves as compared with conductive heating. This is important because higher applied temperatures can cause unexpected or continued (secondary) reactions that generate different chemical species compared to conventional heating methods.

[0130] In the present case, microwave assisted synthesis is suitable for Step (a) - the preparation of MDP-2-nitropropene, such as using cyclohexylamine as the catalysing base according to Reaction Scheme 7,

Reaction Scheme 7 or ethylenediamine according to reaction Scheme 8.

Reaction Scheme 8

Example 5A - Step (a) using cyclohexylamine as catalysing base

[0131] Microwave assisted synthesis was investigated as a source of heating using the synthetic route described for Step (a) at Example 1A. Specifically, it was unclear whether the thermodynamics would favour production of the desired product MDP-2-nitropropene, or whether it would promote a Michael addition reaction, leading to an undesirable Michael addition byproduct according to Reaction Scheme 12:

Reaction Scheme 12

[0132] Reactions fully consumed the starting material after 20mins at 160°C, in contrast to the 6 to 8 hours at 110°C reported in Example 1 A when conventional heating was used.

[0133] Further analogous experiments were carried out with variations to the temperature, time, equivalents of nitroethane and volume of solvent (acetic acid). None of the experiments produced the desired product in excess of 70% yield.

Example 5B - Step (a) using ethylenediamine as catalysing base

[0134] As noted in Example IB conventional heating provided the desired product in very high purity, but the reaction is slow. Microwave assisted synthesis was investigated as a source of heating using the synthetic route described for Step (a) at Example IB to see if an increased reaction rate could be obtained. [0135] Reactions were carried out according to the synthesis steps reported in Example IB but using microwave heating at several temperatures (140°C, 160°C and 180°C).

[0136] In each case TLC monitoring indicated that significant amounts of impurities formed before all the starting material had been consumed. Furthermore, the reaction mixture developed a brown tint, instead of clear deep orange; a similar effect being noted during conventional heating if the reaction is heated for too long. However, in this case the impurity only appears after the starting material is fully consumed and it is postulated that the impurity may be the nitropropene product reacting with a second molecule of ethylenediamine to form the undesirable Michael addition by-product. The presence of the Michael addition product was confirmed by LCMSMS analysis and is a very minor product if the conventionally heated reaction is monitored and stopped as appropriate.

Preparation of a Medicament

[0137] MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof produced according to any of the aforementioned methods may be used in the preparation of a medicament for MDMA-assisted psychotherapy. The medicament may be used in combination with one or more other kinds of therapeutic agents and /or therapeutic methods for the treatment, amelioration and/or prevention of psychological disorders.

Example 6a - Capsules

[0138] In one embodiment, the medicament would be in the form of capsules, preferably two- piece capsules telescoping gelatin capsules containing a powder mix.

[0139] During manufacture, typically the MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof combined with any excipients, such as binders, disintegrants, fillers, glidant and preservatives would be loaded as a powder in one half of the capsule. The other of the capsule would then be pressed on to close the powder inside the capsule.

[0140] In a preferred embodiment the excipient would be a filler such as dextrose, lactose, glucose or sucrose.

[0141] In one embodiment, a #3 capsule would be filled with 60 to 120 mg of active produced according to the present invention and 80 to 140 mg of excipient. In another embodiment, a #3 capsule would be filled with 20 to 60 mg of active produced according to the present invention and 140 to 180 mg of excipient. Example 6b - Tablets

[0142] In one embodiment, the medicament would be in the form of tablets, typically pressed tablets. The press mix for a tablet would typically include MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof combined with excipients, such as binders, disintegrants, fillers, glidant, lubricant and preservatives.

[0143] A preferred direct compression formulation would typically include;

40 to 60 wt%, preferably 55 wt% of MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt

30 to 50 wt%, preferably 41 wt% binder,

0.01 to 5 wt%, preferably 1 wt% lubricant,

1 to 5 wt%, preferably 3 wt% disintegrant.

[0144] The exact proportions of component would be varied according to the desired dissolution profile, tablet hardness, friability, disintegration profile and dissolution profiles. The preferred proportions mentioned above would be likely to have low hardness (soft) but would probably have a desirable disintegration profile.

[0145] In one embodiment, the tablet would contain 60 to 120 mg of active produced according to the present invention and 80 to 140 mg of excipients. In another embodiment, the tablet would contain 20 to 60 mg of active produced according to the present invention and 140 to 180 mg of excipients.

Treatment

[0146] Selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine are Food and Drug Administration (FDA)-approved first-line therapeutics for the treatment of PTSD, but their efficacy is limited. Approximately 40-60% of patients do not respond to these therapeutics.

[0147] Evidence-based traumatic psychotherapies such as prolonged exposure and cognitive behavioural therapy are the gold standard treatments for PTSD, yet many participants fail to respond, continue to have significant symptoms, or dropout. Novel cost-effective therapeutics are therefore still being sought.

[0148] MDMA induces serotonin release by binding primarily to presynaptic serotonin transporters and has been shown to enhance fear memory extinction, modulate fear memory reconsolidation (possibly through an oxytocin-dependent mechanism), and bolster social behaviour in animal models. Accordingly, MDMA has been investigated in clinical settings for enhancing psychotherapy.

[0149] A combined treatment of MDMA administration and psychotherapy may be effective for treating trauma relates conditions such as post-traumatic stress disorder (PTSD). MDMA appears to diminish some patient’s fear response and decrease the patient’s defensiveness without blocking memories. During a psychotherapy session, a patient may be better able to remain emotionally engaged with traumatic memories without being overwhelmed by anxiety or other painful emotions or avoiding them by dissociation or emotional numbing

[0150] In an eighth aspect of embodiments described herein there is provided a method of treating, ameliorating and/or preventing a psychological disorder comprising administering to a subject in need thereof, a therapeutically effective amount of MDMA, (R)-MDMA or (S)-MDMA or a pharmaceutically acceptable salt thereof, produced according to any of the aforementioned methods. The method may be used in combination with one or more other kinds of therapeutic agents and /or therapeutic methods for the treatment, amelioration and/or prevention of psychological disorders.

A typical treatment regimen would include a patient receiving an initial dose during a clinically supervised psychotherapy session, of 60 or 150 mg, preferably 80 to 120 mg, the exact amount depending on body mass. If the patient and clinician believe it would be helpful, after a couple of hours the patient may receive a subsequent dose of between 20 to 80 mg, 40mg to 60mg. Typically, the dose would be delivered in the form of a capsule or tablet as described herein.

Efficacy and Safety of MDMA

[0151] The efficacy and safety of MDMA-assisted therapy in individuals with severe PTSD has been assessed in a randomized, double-blind, placebo-controlled, multi-site phase 3 clinical trial (NCT03537014) reported in Mitchell et al., Nature Medicine, vol.27, June 2021, 1025-1033, https://doi.ort/10.1038/s41591-021-01336-3. The patients with severe PTSD, included some with common comorbidities such as dissociation, depression, a history of alcohol and substance use disorders, and childhood trauma. Participants were given three doses of MDMA or placebo in a controlled clinical environment and in the presence of a trained therapy team. Primary and secondary outcome measures (CAPS-5 and SDS, respectively) were assessed by a centralized pool of blinded, independent diagnostic assessors. MDMA-assisted therapy for PTSD was granted an FDA Breakthrough Therapy designation, and the protocol and statistical analysis plan (SAP) were developed in conjunction with the FDA [0152] A total of 345 participants were assessed for eligibility, 131 were enrolled, 91 were confirmed for randomization (United States, n=77; Canada, n=9; Israel, n=5), and 46 were randomized to MDMA and 44 to placebo. Study arms were not significantly different in terms of race, ethnicity, sex, age, dissociative subtype, disability or CAPS -5 score. The mean duration of PTSD diagnosis was 14.8 (s.d. 11.6) years and 13.2 (s.d. 11.4) years in the MDMA and placebo groups, respectively. Of note, six participants in the MDMA group and 13 participants in the placebo group had the dissociative sub-type according to CAPS-5 score

[0153] After psychiatric medication washout, participants (n= 90) were randomized 1 : 1 to receive manualized therapy with MDMA or with placebo, combined with three preparatory and nine integrative therapy sessions. PTSD symptoms, measured with the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5, the primary endpoint), and functional impairment, measured with the Sheehan Disability Scale (SDS, the secondary endpoint) were assessed at baseline and at 2 months after the last experimental session. Adverse events and suicidality were tracked throughout the study. MDMA was found to induce significant and robust attenuation in CAPS-5 score compared with placebo (P<0.0001, d=0.91) and to significantly decrease the SDS total score (P= 0.0116, d= 0.43). The mean change in CAPS-5 scores in participants completing treatment was -24.4 (s.d. 11.6) in the MDMA group and -13.9 (s.d. 11.5) in the placebo group. MDMA did not induce adverse events of abuse potential, suicidality or QT prolongation.

[0154] These data indicate that, compared with manualized therapy with inactive placebo, MDMA-assisted therapy is highly efficacious in individuals with severe PTSD, and treatment is safe and well-tolerated, even in those with comorbidities. Three doses of MDMA given in conjunction with manualized therapy over the course of 18weeks results in a significant and robust attenuation of PTSD symptoms and functional impairment as assessed using the CAPS-5 and SDS, respectively. MDMA also significantly mitigated depressive symptoms as assessed using the BDI- II.

[0155] The Phase III trial indicates that MDMA-assisted therapy represents a potential breakthrough treatment. The data illustrates the potential benefit of MDMA-assisted therapy for PTSD over the current FDA-approved first-line pharmacotherapies sertraline and paroxetine, which have both exhibited smaller effect sizes in pivotal studies. (Feduccia, A. A. et al., Breakthrough for trauma treatment: safety and efficacy of MDMA-assisted psychotherapy compared to paroxetine and sertraline. Front. Psychiatry 10, 650 (2019)). [0156] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification(s). This application is intended to cover any variations uses or adaptations of the invention following in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

[0157] As the present invention may be embodied in several forms without departing from the spirit of the essential characteristics of the invention, it should be understood that the above described embodiments are not to limit the present invention unless otherwise specified, but rather should be construed broadly within the spirit and scope of the invention as defined in the appended claims. The described embodiments are to be considered in all respects as illustrative only and not restrictive.

[0158] Various modifications and equivalent arrangements are intended to be included within the spirit and scope of the invention and appended claims. Therefore, the specific embodiments are to be understood to be illustrative of the many ways in which the principles of the present invention may be practiced.

[0159] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0160] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. The broad term "comprising" is intended to encompass the narrower "consisting essentially of and the even narrower "consisting of." Thus, in any recitation herein of a phrase "comprising one or more claim element" (e.g., "comprising A), the phrase is intended to encompass the narrower, for example, "consisting essentially of A" and "consisting of A" Thus, the broader word "comprising" is intended to provide specific support in each use herein for either "consisting essentially of or "consisting of." The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0161] One of ordinary skill in the art will appreciate that materials and methods, other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by examples, preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0162] Each references cited herein is incorporated by reference herein in their entirety. Such references may provide sources of materials; alternative materials, details of methods, as well as additional uses of the invention.