WO2025120626A2

WO2025120626A2 - Synthetic method

Info

Publication number: WO2025120626A2
Application number: PCT/IB2025/053902
Authority: WO
Inventors: Larisa ANDREEVA
Original assignee: Mitocholine Ltd
Current assignee: Mitocholine Ltd
Priority date: 2025-04-14
Filing date: 2025-04-14
Publication date: 2025-06-12
Anticipated expiration: 2027-10-14
Also published as: WO2025120626A3

Abstract

The present invention relates to a two-step method of synthesis of di-choline succinate (DISU) from trimethylamine, succinic acid and ethylene oxide suitable for a large-scale production of the compound.

Description

Synthetic method Field of the Invention The present invention relates to a simple and robust method of synthesis of di-choline succinate (DISU) suitable for a large-scale production of the compound. Background of the invention Food supplementation with choline formulated as choline salts has been proven to be beneﬁcial for health of both humans and domesticated mammals. Production of several salts of choline suitable as food supplements, like choline chloride, choline bitartrate or choline citrate/dihydrogen citrate, is well established and food supplements comprising these salts are commercially available. Methods of preparation of crystalline pharmaceutical grade DISU at lab scale have been described in WO2001076583 and in WO2009022933, and synthesis of food grade DISU has been described in WO2024231663. However, all these methods have drawbacks to be applied for a large scale manufacturing of DISU as they all comprise multiple steps of synthesis of the compound and its puriﬁcation from undesirable contaminants of choline production. Di-choline salt of succinic acid (di-choline succinate or DISU), has been shown to be a potent sensitizer of the neuronal insulin receptor (Storozhevykh T., et al (2008) BCM Neurocsi. doi:10.1186/1471-2202- 8-84). Further, compositions of di-choline succinate with nicotinamide are synergistically eﬀective for increasing the levels of both NAD, ATP, and phosphocreatine in the brain cells (WO2019002858). Recently, it has been reported that DISU is also potent enhancer of skeletal muscle function (WO2022254185) and it has also been shown that DISU is the most potent in supporting one carbon metabolism in humans, compared to two other choline salts, choline bitartrate and choline chloride (WO2023026043). Methods of preparation of DISU at lab scale have been described in WO2001076583 and in WO2009022933, and, more recently, it has been described an industrial method for production of food grade DISU. Still, all these methods have their limitations, like use of excessive amounts of organic solvents or other means to purify the produced DISU from undesirable by-products of choline production. The later are probably the reason for DISU products are not yet commercially available on the market. Accordingly, a simple and robust industrial method for the large-scale production of food and pharmaceutical grade DISU would provide a great opportunity to satisfy the market needs in the compound. Summary of the invention The present invention relates to a method of synthesis of di-choline succinate (DISU) from trimethylamine, succinic acid and ethylene oxide, comprising (a) reacting succinic acid with trimethylamine and thereby forming di-trimethyl amine succinate salt, wherein said reacting is performed in an aqueous medium or a water miscible organic solvent, and (b) adding ethylene oxide in the reaction media of (a), and thereby obtaining a solution of di-choline succinate, wherein - the molar ratio trimethylamine to succinic acid in the aqueous medium of (a) is around 2:1, preferably between around 2:1,01 and around 2:1,1; - the molar ratio of ethylene oxide to trimethylamine in the reaction medium of (b) is 1:1, and - the di-choline succinate solution contains one or more o-ethoxylated choline derivative in the amount of at most around 0,1-0,2 wt%. The obtained DISU is of food grade quality. Another aspect of the invention relates to a food grade DISU preparation obtained by the method of the invention. Still another aspect of the invention relates to a pharmaceutical, nutritional supplement, food or beverage product comprising DISU preparation obtained by the method pf the invention. Description of Drawings Figure.1 presents is ¹HNMR spectrum of di-choline succinate (DISU) (obtained as described in Example 1) dissolved in D₂O. Figure 2 presents HPLC elution proﬁle of di-choline succinate (DISU) (obtained as described in Example 1) dissolved in methanol. Detailed description of the invention Synthesis of a food grade di-choline succinate (DISU) has recently been described in WO2024231663. According to WO202423166, to obtain DISU preparation of food grade quality, in particular, a DISU product that comprise at most 0,3 wt% o-ethoxylated choline derivative(s), the synthesis has to use either a food grad quality starting materials, e.g. salts of choline, like choline chloride, choline carbonate, etc, or choline hydroxide that is puriﬁed and does not contain these undesired contaminants. Both these steps make production of a food grade DISU complicated and laborious, which is undesirable in view of large scale manufacturing. Surprisingly, a more simple and robust synthetic procedure according to this invention can provide DUSU of food grade quality that comprises very little amounts, if any, of these undesired o-ethoxylated choline derivative(s), such as a DISU preparation that comprises undetectable amounts of one or more o-ethoxylated choline derivatives or at most around 0,1-0,2 wt% thereof. By “food grade” is meant a quality of a compound or preparation that meets requirements by Food Regulation Authorities for the quality of a product intended for oral consumption by a human as a food or food supplement product. The method of invention basically includes two steps: theﬁrst step of initial formation of a succinic salt of trimethylamine (TMA) from succinic acid and TMA, followed by the second step of forming DISU by the reaction of this TMA salt of succinic salt with ethylene oxide (EO). The advantages of the method, besides that the DISU preparation is very pure, include low material use for the synthesis, time, energy and cost eﬃcacy, and, also, environmental friendliness - the synthesis has low or no organic solvent use and is preferably carried out in water. Accordingly, the present invention relates to a method of synthetic production of DISU, comprising (a) reacting succinic acid with trimethylamine and thereby forming di-trimethyl amine succinate salt, wherein said reacting is performed in an aqueous medium or a water miscible organic solvent, and (b) adding ethylene oxide in the reaction media of (a), and thereby obtaining a solution of di-choline succinate. According to the invention the molar ratio of TMA to succinic acid in the aqueous medium of step (a) is around 2:1, preferably between around 2:1,01 and around 2:1,1, and the molar ratio of EO to TMA in the reaction medium of (b) is 1:1. The molar ratio of TMA to succinic acid of around 2:1 allows for formation of di-TMA salt of succinic acid/Bis(Trimethylammonium) succinate:

This salt is kept stable in the reaction medium due to the presence of a little molar access of succinic acid over TMA, i.e. it is advantageous, if there is 1,01-1,1 molar of succinic acid per 2 molar of TMA in the reaction medium of step (a). Without being bound to a theory, the inventors suggest that the excess of free succinic acid in the reaction medium of step (a) prevents formation of o-ethoxylated choline derivative(s) (as shown below) in reaction of step (b):

The compounds (1), (2), and/or (3) identiﬁed above are commonly mentioned through the speciﬁcation and claims as “one or more o-ethoxylated choline derivatives”. These o-ethoxylated choline derivative(s) (1), (2) and/or (3) are typical by-products of the synthesis of choline by reaction of TMA with EO. To further reduce the probability of formation of these undesirable compounds in the process of DISU synthesis according to the method of the invention, the temperature of the reactions of step (a) and/or step (b), preferably of both (a) and (b) steps, is hold below ambient, preferably between around 0⁰C and 20⁰C, preferably between around 2-10⁰C, such as around 5⁰C. At these conditions, the yield of o- ethoxylated choline derivative(s) in the reaction medium comprising dissolved DISU is practically diminished or is at most around 0,1-0,2 wt%. The term “around” means a range of values that includes the indicated value and values that from this value by 0,1-10 %. TMA used for synthesis could be, most preferably, a water solution, e.g. around 25-45 wt% TMA solution or it could be a 25-45 wt% TMA solution in a water miscible solvent, such as C1-C3 alcohol. Preferred C1-C3 alcohols are methanol, ethanol, propanol/propanol-1 or iso-propanol/propanol-2. The obtained DISU dissolved in the reaction medium of step (b) is preferably dried to remove residual TMA and EO, and also the solvent. Drying of DISU may done by e.g. heating the reaction medium up to 100⁰C or higher, such as 110-120⁰C, depending on the solvent, i.e. alcohols may be evaporated at lower temperatures than water. Any conventional means to prepare a dry matter form a liquid solution and to make the drying process more eﬃcient could be applied, like pressure, vacuum, etc. The drying may also be done by spray or freezes drying, or other suitable drying techniques. Any conventional drying techniques can be applied. The dried DISU preparation is preferably tightly sealed after drying to avoid hydration. This DISU product is a ready-to use product for food and pharmaceutical applications, as it does not contain any ingredients than DISU and about 0,1-2% of free succinic acid. If neat DISU material is needed, the access of succinic acid could be removed by DISU recrystallisation of the dry DISU material obtained by the method of the invention as described in Example 2 of the present patent application. The obtained by the method of the invention DISU material, both water solution (around 25-50 wt% DISU) and dried preparation of DISU, has a purity that is suitable for both dietary use as, e.g., a dietary supplement or part of a food/beverage preparation, or pharmaceutical use as, e.g., a medicament or adjuvant (by “ adjuvant” is meant a substance that is used to increase the eﬃcacy or potency of a drug). Some preferred, but not limiting, embodiments are described below: 1. A method of synthesis of di-choline succinate, comprising (a) reacting succinic acid with trimethylamine and thereby forming di-trimethyl amine succinate salt, wherein said reacting is performed in an aqueous medium or a water miscible organic solvent, and (b) adding ethylene oxide to the reaction media of (a) and thereby obtaining a solution of di-choline succinate, wherein - the molar ratio trimethylamine to succinic acid in the aqueous medium of (a) is around 2:1, preferably between around 2:1,01 and around 2:1,1; - the molar ratio of ethylene oxide to trimethylamine in the reaction medium of (b) is 1:1, and - the di-choline succinate solution contains one or more o-ethoxylated choline derivative in the amount of at most around 0,1-0,2 wt%. 2. The method of embodiment 1, wherein trimethylamine is a 25-50 wt% solution in water or C1-C3 alcohol selected from methanol, ethanol, propanol or isopropanol. 3. The method of embodiments 1 or 2, wherein succinic acid is a crystalline matter or a solution in water or C1-C3 alcohol selected from methanol, ethanol, propanol or isopropanol. 4. The method of any of embodiments 1-3, wherein the reactions of steps (a) and (b) are carried out at a temperature of around 0-25⁰C. 5. The method of any of embodiments 1-4, further comprising a step of precipitation of di- choline succinate. 6. The method of embodiment 5, wherein the precipitation includes evaporation, freeze drying or spray-drying of the liquid medium. 7. The method of embodiment 6, wherein the method further comprises (i) dissolving the dry matter comprising di-choline succinate and around 0,1- 0,2 wt% or less of one or more o-ethoxylated choline derivatives in isopropanol, and (ii) adding acetone to the solution of (i), thereby precipitating crystalline preparation of di-choline succinate. 8. A di-choline succinate preparation obtained by the method according to any of embodiments 1-6 or 7. 9. A dietary or pharmaceutical product comprising di-choline succinate of embodiment 8. Examples. The examples below are only for illustration of the invention and are not limiting the invention in any way. Example 1: Synthesis of di-choline succinate Adding 26,27 g of 45 wt% TMA in water (Thermo Scientiﬁc) into a reaction kettle, then adding 12,40 g of crystalline succinic acid (Sigma-Aldrich) in the TMA, keeping the temperature while dissolving succinic acid at around 25 ℃. After succinic acid is fully dissolved, cooling the mixture to 0-10 ℃, and holding the mixture at this temperature at continuous stirring for 1-3 hours. Then, adding (continuously) 8,81 g of ethylene oxide (Sigma-Aldrich) into the reaction mixture, succinic acid (0,1-2 g), holding the pH value around 8-9 and the temperature around 0-10℃, and continuously stirring the mixture for 1-2 h. After completion of synthesis, carrying out suctionﬁltration, heating theﬁltrate under reduced pressure at a temperature of 110 - 120℃ to complete evaporation of liquid (and remove occasional residual TMA and EO). Collecting the dry matter and analyzing the content by HPLC and NMR spectrometry. Theﬁltrate optionally can be treated with active carbon, in case some yellowish coloration is observed. The HPLC and MRI data are presented inﬁgure 1 (NMR) and 2 (HPLC) Example 2: Preparation of crystalline dicholine succinate Dry di-choline succinate prepared as described in Example 1 is dissolved and re-crystalized following the below procedure. 27 g of 98,5% (26,59 g of 100%) dry di-choline succinate is placed in a 0,5 l three-necked flask equipped with reflux condenser, agitator and thermometer, and 81 ml of 2-propanol added and mixed at 70-75 ⁰С until complete dissolution of the introduced product dry matter. To the solution, 2,7 g of activated carbon is added and the mixture is stirred at 70-75⁰С for 10-15 min. Then the mixture is filtrated using glass filter into a 0,5 l three-necked flask equipped with agitator and thermometer. The flask and carbon on the filter is rinsed with 8 ml of 2-propanol. The filtrate from the flask is added to 162 ml of acetone and keep at 10-15⁰С while stirring for 3 hours. Formed sediment is filtered using Buchner funnel, then pressed and rinsed 2 times with 21 ml of 2:1 propanol:acetone mixture. The moist sediment with residual solvent is dried in a drying chamber at 95-100⁰С for 2 hours until the constant weight is obtained.

Claims

Claims 1. A method of synthesis of di-choline succinate, comprising (a) reacting succinic acid with trimethylamine and thereby forming di-trimethylamine succinate salt, wherein said reacting is performed in an aqueous medium or a water miscible organic solvent, and (b) adding ethylene oxide in the reaction media of (a), and thereby obtaining a solution of di-choline succinate, wherein - the molar ratio trimethylamine to succinic acid in the aqueous medium of (a) is around 2:1, preferably between around 2:1,01 and around 2:1,1; - the molar ratio of ethylene oxide to trimethylamine in the reaction medium of (b) is 1:1, and - the di-choline succinate solution contains one or more o-ethoxylated choline derivative in the amount around 0,1-0,2 wt%. 2. The method of claim 1, wherein trimethylamine is a 25-50 wt% solution in water or C1-C3 alcohol selected from methanol, ethanol, propanol or isopropanol. 3. The method of claim 1 or 2, wherein succinic acid is crystalline matter or a solution in water or in a C1-C3 alcohol selected from methanol, ethanol, propanol or isopropanol. 4. The method of any of claims 1-3, wherein the reactions of steps (a) and (b) are carried out at a temperature of around 0-25⁰C. 5. The method of any of claims 1-4, further comprising a step of precipitation of di-choline succinate. 6. The method of claim 5, wherein the precipitation includes evaporation, freeze drying or spray- drying of the liquid medium. 7. The method of claim 6, wherein the method further comprises (i) dissolving the dry matter comprising di-choline succinate in isopropanol, and (ii) adding acetone to the solution of (i), thereby obtaining crystalline preparation of di-choline succinate. 8. A di-choline succinate preparation obtained by the method according to any of claims 1-6. 9. A dietary or pharmaceutical product comprising di-choline succinate of claim 8.