The invention relates to the field of medicinal chemistry. More in particular, it relates to methods for providing highly pure, pharmaceutical grade synthetic Palmitoylethanolamine (PEA), to pure PEA preparation obtainable thereby, and the uses thereof.

Palmitoylethanolamide (N-(2-hydroxyethyl)hexadecanamide,

PEA) (CAS 544-31-0; EINECS 208-867-9), also known as

palmitoylethanolamide or palmidrol, is a naturally occurring C16:0 fatty acid derivative wherein the carboxylate function is amidated by the primary amine of ethanolamine. N-acylethanolamine compounds are known to have anti-inflammatory and anti-nociceptive effects, as described in Lambert et al (Lambert DM, Vandevoorde S, Jonsson KO, Fowler CJ. Curr Med Chem. 2002;9:663-74); Lambert DM et al (Lambert DM, DiPaolo FG, Sonveaux P, Kanyonyo M, Govaerts SJ, Hermans E, Bueb J, Delzenne NM, Tschirhart EJ. Biochim Biophys Acta. 1999; 1440:266-74); Brown AJ (Br J Pharmacol. 2007; 152(5):567-75); and US 5,506,224 and US 2005/0054730.

A large number of scientific investigations on the effects of PEA and PEA-related compounds are available, and give rise to new therapeutic opportunities. Multiple synthetic procedures of PEA have been reported. PEA can for example be prepared via the coupling of palmitic acid and ethanolamine in the presence of a coupling reagent. Exemplary coupling routes for synthesis of PEA involve the use of l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI) in the presence of 4- dimethylaminopyridine (DMAP) in methylene chloride (see

WO2015/179190), or l-Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium

hexafluorophosphate (COMU) in the presence of Di-isopropylethylamine (DIPEA) in a mixture of methylene chloride and acetonitrile (Chemistry and physics or lipids 2012, 165, 705). Still further examples involve 2-(lH- Benzotriazole- l-yl)-l, l,3,3-tetramethylaminium tetrafluoroborate (TBTU) in the presence of triethylamine in acetonitrile (Bioorganic and Medicinal Chemistry 2011 , 19, 1520), Ν,Ν'-carbodiimide (CDI) in methylene chloride (Tetrahedron Letters 1980, 21, 841) or Ν,Ν'-dicyclohexylcarbodiimide (DCC) in the presence or DMAP in methylene chloride (Chemistry Letters; (1985); p. 701). The synthesis of PEA typically involves aqueous workup and

purification using flash chromatography, often in combination with crystallization.

Alternatively, PEA can be prepared by reacting palmitic acid with acetic anhydride followed by ethanolamine (J. Am. Chem. Soc, 1952, 74 (13), 3442), or via aminolysis of isopropyl palmitate and ethanolamine in the presence of sodium ethoxide/ethanol (see US2016/038443), via

aminolysis of ethyl, methyl, or vinyl palmitate or the reaction of palmitoyl chloride and ethanolamine in the presence of a base. Moreover, synthesis of PEA has been reported using activation of palmitic acid via the formation of an active ester (Archiv der Pharmazie 342 (2009) 34; WO2006/109321;

Chemistry Letters; (1985) 701; Chemical & Pharmaceutical Bulletin 32 (1984) 2687).

A procedure that is frequently used for large scale preparation of PEA for pharmaceutical use involves the direct heating of the palmitic acid in the presence of ethanolamine, followed by removal of excess of

ethanolamine to provide a crude PEA preparation.

It is known that crude PEA prepared from direct reaction of palmitic acid with ethanolamine is often contaminated with a number of impurities. The level of impurities can be reduced by PEA crystallization from a solvent, such as methanol. However, it was consistently observed by the present inventors that efficient removal of impurities to an acceptable level while maintaining acceptable yields failed. Therefore, the present inventors aimed at minimizing such impurities in crude PEA prepared from direct reaction of palmitic acid with ethanolamine. To that end, they first set out to chemically characterize the contaminants, and thereafter provide a method to minimize their presence.

Using HPLC-MS techniques, it was observed that crude PEA contains several contaminants. These were identified as N-(2-(2- aminoethoxy)ethyl)palmitamide, Myristoylethanolamide,

Stearoylethanolamide, N,N'-(oxybis(ethane-2, l-diyl))dipalmitamide and 2- Palmitamidoethyl palmitate. All impurities could be attributed either to those resulting from impurities in the starting material or to unwanted side reactions.

It was surprisingly found that heating a suspension of crude PEA in a solvent mixture in the presence of a strong base catalyst to reflux, thereby allowing the formation of crystalline PEA, is highly effective to isolate crystalline PEA that is essentially free from impurities which remain in solution. Without wishing to be bound be any theory, it is envisioned that the presence of the more labile ester functionality in PEAP compared to the amide functionality aids in the removal of PEAP from the crude product. By adding an base catalyst as additive to the crystallization medium to facilitate solvolysis of the ester functionality , PEAP could completely be ehminated to provide PEA and methyl (ethyl also possible?) palmitate. After crystallization, methyl palmitate remains in solution and PEA free from unwanted impurities is obtained.

Herewith, the invention provides a method for providing pure palmitoyolethanolamide (PEA), comprising the steps of (i) heating a suspension of crude PEA in a solvent mixture comprising an alcohol to reflux in the presence of a base catalyst having a pKA (in water) of at least 13, thereby allowing the formation of crystalhne PEA; and (ii) isolating said crystalline PEA.

A schematic outline of the synthesis and subsequent refining of PEA according to the invention is exemplified in Scheme 1.

Palmitic acid

HO.

NH ₂

Et anolamine

Palmitoylethanolamine

2-Palmitamidoet yl palmitate

W,/V'-(oxybis(ethane-2,1-diyl))dipalmitamide

Solvent mixture

Base catalyst

Palmitoylethanolamine

Methyl palmitate (remains in solution)

O O

X

N,W-(oxybis(ethane-2,1-diyl))dipalmitamide (remains in solution)

Scheme 1 The first step in a method of the invention comprises preparing a

suspension of crude PEA in a suitable solvent mixture. Typically, a method of the invention finds its use for purifying crude PEA that is obtained from a direct reaction of palmitic acid and ethanolamine. For example, the crude PEA for use as starting material in a method provided herein comprises one or more unwanted substances selected from the group consisting of N-(2-(2- aminoethoxy)ethyl)palmitamide Myristoylethanolamide,

Stearoylethanolamide, N,N'-(oxybis(ethane-2, l-diyl))dipalmitamide and 2- Palmitamidoethyl palmitate. The suspension may comprise about 0.1- 1 kg per liter, preferably 0.2 to 0.4 kilogram of PEA per liter of solvent mixture.

The solvent mixture preferably comprises an ahphatic alcohol, more preferably a C 1-C3 alcohol, in combination with a suitable co-solvent.

Preferred co-solvents include aromatic hydrocarbons like toluene, mesitylene, benzene, xylene and ethylbenzene. Good results were obtained using toluene as co-solvent. In a preferred embodiment, the solvent mixture comprises or consists of a combination of toluene and an alcohol, preferably toluene and methanol or toluene and ethanol. For example, the solvent mixture consists of toluene and methanol in a relative ratio in the range of 1.4 : 1 to 1.8 : 1, preferably 1.5: 1 to 1.7: 1, by volume.

The suspension of crude PEA is heated, preferably while stirring, to reflux in the presence of a strong base catalyst. In one embodiment, the base catalyst has a pKa (in H2O) of at least 13.5, preferably at least 14.

Good results are obtained wherein the base catalyst is an amidine base or a strong guanidine base. Such base catalysts are known in the art, see for example "Superbases for Organic Synthesis: Guanidines, Amidines, Phosphazenes and Related Organocatalysts, Amidines" in Organic

Synthesis 50-91 . In one embodiment, the base catalyst is an amidine base.

Amidines are strong bases (pKa ranges from 5-12). The protonation occurs on the imino nitrogen leading to symmetrical amidinium ion which is stabilized by resonance as in the isoelectronic carboxylate anion. Preferably, the amidine base for use in a method of the invention is selected from the group consisting of l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5- diazabicyclo[4.3.0]non-5-ene (DBN), 2,3,4,6,7,8,9, 10-octahydropyrimido[l, 2- a]azepine), NaHCOa and NaOC¾.

In another embodiment, the base catalyst is a strong guanidine base, preferably triazabicyclodecene (l,5,7-triazabicyclo[4.4.0]dec-5-ene; TBD).

The reaction is allowed to proceed for a time period sufficient to achieve solvolysis of the impuritie(s), typically at least 0.5 hour, preferably at least 1 hour. In one embodiment, the suspension is heated to reflux for about 1-2 hours. Thereafter, the reaction mixture is allowed to cool to room temperature, resulting in the formation of pure, crystalline PEA. The crystalline PEA is suitably isolated by methods known in the art, e.g.

comprising the filtering and washing crystalline PEA. Also provided herein is a pure PEA preparation comprising at least 98%, preferably at least 99%, more preferably at least 99.5 % by weight PEA, obtainable by a method according to the invention. This PEA preparation is typically characterized by being essentially devoid (e.g. less than 2%, preferably less than 1.5%, more preferably less than 1% by weight in total) of unwanted substances selected from the group consisting of N-(2-(2- aminoethoxy)ethyl)palmitamide Myristoylethanolamide,

Stearoylethanolamicle, N,N'-(oxybis(ethane-2, l-diyl))dipalmitamide and 2- Palmitamidoethyl palmitate (PAEP). A pure PEA preparation according to the invention is advantageously used as human or veterinary supplement or medicament. Therapeutic

applications of PEA include those already known in the art, as well as any application yet to be discovered. For example, provided herein is the a pure PEA preparation for use in a method of reducing or preventing pain in a mammalian subject, preferably chronic pain, neuropathic pain or a related disorder based on overactivation of glia and glia-related cells, such as in diabetes and glaucoma. Also provided is a pure PEA preparation according to the invention for use in a method of reducing or preventing symptoms associated with influenza, common cold, infections, auto-immune disease, atopic or contact dermatitis, depression, epilepsy and chronic inflammatory diseases.

LEGEND TO THE FIGURES

Figure 1: HPLC chromatogram of crude PEA

Figure 2: Ή-NMR spectrum of crude PEA

Figure 3: HPLC chromatogram of purified PEA

Figure 4: Ή-NMR spectrum of purified PEA

EXPERIMENTAL SECTION

Example 1: Analysis of impurities in crude PEA Materials: crude PEA prepared by direct reaction of palmitic acid and ethanolamine was obtained from Zschimmer & Schwarz GmbH, Germany.

The crude preparation was analyzed using HPLC according to the following procedure:

Mobile phase A: mix 1.0 mL of formic acid with 1000 mL of Milli-Q.

Mobile phase B: mix 1.0 mL of formic acid with 1000 mL of ACN.

Column: Water Xbridge C18 5μιη 250x3.0 mm (part number: 186003133) Column temperature: 40 °C

Autosampler temperature: 18 °C

Injection volume: 20 μΐ^

Detection: UV 200 nm Gradient elution:

Time Flow Mobile phase A Mobile phase B

0,01 min. 1.00 mL/min 40 % 60 %

11,00 min. 1.00 mL/min 0 % 100 %

25,00 min. 1.00 mL/min 0 % 100 %

25,01 min. 1.00 mL/min 40 % 60 %

30.00 min. 1.00 mL/min 40 % 60 %

Test solution (0.5 mg/mL): weigh 25.0 mg of PEA and dissolve in 50.0 mL of MeOH. If necessary, use ultrasonication and gently warm the solution until all PEA is dissolved. Reference solution A (0.5 mg/mL): Weigh 25.0 mg of reference and dissolve in 50.0 mL of MeOH. If necessary, use ultrasonication and gently warm the solution until all PEA is dissolved.

Inject blank MeOH, reference solution A and test solution.

A representative HPLC chromatogram is given in Figure 1 and the proposed structures of impurities indicated therein are given in table 1. All impurities were confirmed by independent synthesis.

Table 1 List of impurities

The Ή-NMR spectrum in Figure 2 clearly shows the presence of impurities in crude PEA. The presence of myristoylethanolamide (cas # 142-58-5) and stearoylethanolamide (cas # 111-57-9) in the crud can be explained by the presence of myristic acid and stearic acid in the palmitic acid starting material. The presence of 2-palmitamidoethyl palmitate (PAEP) cas # 55349-67-2 can be explained by unwanted ester formation under the reaction conditions. The presence of N,N'-(oxybis(ethane-2, l- diyl))dipalmitamide (Ν,Ν-DPEA) and N-(2-(2- aminoethoxy)ethyl)palmitamide can be explained by the presence of 2,2'- oxybis(ethan- 1 -amine) in the crude ethanolamine starting material. Example 2: Purification of crude PEA using DBU To a stirred suspension of crude PEA (1 kg, 3.34 mol) in a mixture of toluene (2.4 L) and methanol (1.6 L) 10 g DBU (1,8- Diazabicydo[5.4.G]imdec-7-ene 64 mmol (ca 20 mol %) was added. The reaction mixture was heated to reflux for 1 h and the mixture was allowed to cool overnight. The resulting white crystals were isolated over a Buchiier funnel, washed with methanol and dried overnight at 35°C to give PEA as white crystals.

Figure 3 shows the HPLC chromatogram of purified PEA. Figure 4 shows the Ή-NMR spectrum of purified PEA.

Example 3: Large scale purification of crude PEA using DBU

To a stirred suspension of crude PEA (324 kg) in toluene (800 L) and methanol (500 L) was added DBU (5 kg). The reaction mixture was heated to reflux for 2.5 h and the mixture was allowed to cool overnight. The resulting white crystals were isolated over a Nutsche filter and. dried using a floating bed. dryer to give PEA as white crystals in ca. 75 % yield, with an assay of > 99.5 %.

Example 4 : Purification of crude PEA using NaHCOs

To a stirred suspension of crude PEA (5 g) in toluene (12 niL) and methanol (8 mL) NaHCO >, (100 mg) was added as base catalyst. The reaction mixture was heated to reflux for 1 h and the mixture was allowed to cool overnight. The resulting white crystals were isolated over a glass filter and dried overnight at 35°C to give PEA as white crystals in ca. 65 % yield.

Example 5 : Purification of crude PEA using NaOMe To a stirred suspension of crude PEA (50 g) in toluene (125 niL) and methanol (75 mL) was added NaOMe (135 mg). The reaction mixture was heated to reflux for 2 h and the mixture was allowed to cool overnight. The resulting white crystals were isolated over a glass filter and dried overnight at 35°C to give pure PEA as white crystals in ca 70 % yield.

Example 6 : Purification of crude PEA using TBD

To a stirred suspension of crude PEA (50 g) in toluene (125 mL) and methanol (75 mL) was added TBD (450 mg). The reaction mixture was heated to reflux for 2 h and the mixture was allowed to cool overnight. The resulting white crystals were isolated over a glass filter and dried overnight at 35°C to give pure PEA as white crystals in ca 68 % yield.