PROCESS OF MAKING N,N-DIACETYL-L-CYSTINE DISODIUM SALT FROM CYSTINE AND ACETYL CHLORIDE IN METHANOL IN THE PRESENCE OF SODIUM HYDROXIDE

Abstract

A process of making N,N′-diacetyl-L-Cystine disodium salt, the process comprising: (i) Mixing hydroxy alkane (between 0.5-100 L, preferably between 0.5-10 L, more preferably between 1-3 L, most preferably 1 L per mol of cystine) with a sodium base (4.0 molar equivalents per mole of cystine) to form a cold solution at a temperature of from 5 to 10° C.; (ii) Adding cystine (1 molar equivalent) to said cold solution and stirring, for a sufficient time, to form a basic cystine solution; (iii) Optionally, Cooling the cystine solution to 5° C.; (iv) Adding acetyl chloride (2 molar equivalents per mole of cystine) portionwise, while maintaining the temperature between 3 and 50° C., preferably between 5 and 35° C., more preferably below 10° C., most preferably 5° C., thereby resulting in a white suspension; (v) Stirring said white suspension and allowing said suspension to warm up to a room temperature of 15° C. to 50° C., preferably 20° C. to 35° C., more preferably 20° C., thereby resulting in N,N′-diacetyl-L-Cystine disodium salt product dissolved in solution and sodium chloride by-product precipitated solid in said suspension. The present description discloses an exemplary process on a small laboratory scale (500 mg cystine educt; 688 mg product; 90% yield) (page 16; example 1).

Claims

1. A process of making N,N′-diacetyl-L-Cystine disodium salt, the process comprising: (i) mixing a hydroxyalkane with a sodium base to form a cold solution at a temperature of from 5 to 10° C.; (ii) adding cystine to the cold solution and stirring for 5 minutes to 1 hour to form a basic cystine solution; (iii) optionally, cooling the basic cystine solution to 5° C.; (iv) adding acetyl chloride portionwise to the basic cystine solution, while maintaining the temperature between 3 and 50° C., thereby resulting in a white suspension; (v) stirring the white suspension and allowing the suspension to warm up to a room temperature of 15° C. to 50° C., thereby resulting in N,N′-diacetyl-L-Cystine disodium salt product dissolved in a solution and sodium chloride by-product precipitated solid, wherein the process includes 1 mol of cystine, between 0.5-100 L of the hydroxyalkane per mol of cystine, 4.0 molar equivalents of the sodium base per mol of cystine, and 2 molar equivalents of acetyl chloride per mol of cystine.

2. The process according to claim 1, further comprising separating the N,N′-diacetyl-L-Cystine disodium salt product from the solution.

3. The process according to claim 1, wherein, following step (v), the process further comprises: filtering off the sodium chloride by-product precipitated solid, thereby separating said precipitated solid away from N,N′-diacetyl-L-Cystine disodium salt product dissolved in the solution, washing the precipitated solid with anhydrous methanol and collecting a methanol filtrate, combining the methanol filtrate and the solution, and evaporating under reduced pressure and temperature of 40° C., resulting in N,N′-diacetyl-L-Cystine disodium salt product as white solid at 90% yield.

4. The process according to claim 1, wherein the hydroxyalkane is methanol, ethanol or isopropanol.

5. The process according to claim 1, wherein the sodium base is selected from the group consisting of alkali metal hydroxides, carbonates, and mixtures thereof.

6. (canceled)

7. The process according claim 1, wherein stirring the white suspension takes 10 minutes to 24 hours.

8. The process according to claim 1, wherein warming the white suspension to room temperature takes 10 minutes to 30 minutes.

9. The process according to claim 1, wherein total reaction completion takes from 15 minutes to 25 hours.

10. A chemical process of making N-N′-di-acetyl-cystine disodium salt, the process comprising the steps: forming a reaction mixture, starting with cystine in an alcohol solvent in the presence of sodium hydroxide; acetylating said cystine with an acetylating agent; and isolating said N,N′-diacetyl-L-Cystine disodium salt from said reaction mixture.

11. The process according to claim 10, further comprising recycling said alcohol solvent.

12. The process according to claim 10, wherein said alcohol solvent is methanol.

13. The process according to claim 10, wherein said acetylating agent is acetyl chloride or acetic anhydride.

14. The process according to claim 10, wherein said acetylating agent is used in an amount of 2 to 4 molar equivalents with respect to said cystine.

15. The process according to claim 10, wherein said isolated N,N′-diacetyl-L-cystine disodium salt from said reaction mixture contains cystine disodium salt, N-acetyl-L-cystine sodium salt or sodium acetate, or a mixture thereof.

16. The process according to claim 1, wherein the hydroxyalkane is methanol.

17. The process according to claim 1, wherein the sodium base is sodium hydroxide.

18. The process according to claim 1, wherein stirring the white suspension takes 15 minutes to 24 hours.

19. The process according to claim 1, wherein stirring the white suspension takes about 20 minutes.

20. The process according to claim 1, wherein warming the white suspension to room temperature takes about 10 minutes.

21. The process according to claim 1, wherein total reaction completion takes about 35 minutes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0047] Except in the examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts of material or conditions of reaction, physical properties of materials and/or use are to be understood as modified by the word “about.”

[0048] It should be noted that in specifying any range of concentration or amount, any particular upper concentration can be associated with any particular lower concentration or amount.

[0049] For the avoidance of doubt, the word “comprising” is intended to mean “including” but not necessarily “consisting of” or “composed of.” In other words, the listed steps or options need not be exhaustive.

[0050] The present invention obviates the needs of the prior art by providing a chemical process to specifically prepare N,N′-diacetyl-L-cystine (NDAC) disodium salt (2) directly from L-cystine (1) using an acetylating agent (e.g. acetyl chloride or acetic anhydride).

[0051] The chemical process of the present invention is quick and is performed in a single vessel in one step, when a batch process is used. With either batch or continuous process, the inventive reaction advantageously starts with cystine, and uses alcohol-based solvents which can be recycled. There are no undesirable cystine-derived by-products generated and any by-products generated consists of innocuous salts (e.g. NaCl) that can be conveniently filtered off and recycled or small amounts of acceptable by-products consisting of cystine disodium salt (from unreacted cystine), N-acetyl-L-cystine sodium salt (resulting from monoacetylation of cystine) and sodium acetate. The N,N′-diacetyl-L-cystine (NDAC) disodium salt (2) product is isolated upon solvent evaporation, with no purification needed.

N,N′-diacetyl-L-cystine Disodium Salt (“NDAC Disodium Salt”)

[0052] Applicants have discovered that N,N′-diacetyl-L-Cystine (“NDAC”) and NDAC disodium salts are useful amides in personal care compositions. In skin cosmetic compositions, NDAC and its disodium salts, when applied to skin, are converted to cysteine intracellularly. Cysteine in turn has a number of cosmetic uses, including as a glutathione precursor. Applicants have discovered that NDAC disodium salt has superior functional benefits for ultimate delivery of cysteine for cosmetic uses.

[0053] NDAC salt is a disodium salt of N,N-diacetyl cystine (Also referred to as N,N′-diacetyl-L-cystine or NDAC). NDAC structure is shown below (C.sub.10H.sub.16N.sub.2O.sub.6S.sub.2):

##STR00006##

The molecular weight of NDAC = 324.4.

[0054] N,N′-diacetyl-L-Cystine (“NDAC”) disodium salt of Formula (2), prepared according to the inventive process, has the following general structure (C.sub.10H.sub.14N.sub.2Na.sub.2O.sub.6S.sub.2):

##STR00007##

The molecular weight of NDAC disodium salt = 368.3.

[0055] NDAC or its disodium salt is not readily commercially available in large scale. N,N′-diacetyl-L-cystine disodium salt (“NDAC disodium salt”) may be prepared according to the inventive process. The inventive process is most useful for the synthesis of NDAC disodium salt by using L-cystine (1), which is readily available commercially in bulk quantities.

Inventive Process

[0056] According to the inventive process, N,N′-diacetyl-L-Cystine salts (NDAC salts) are prepared in a single step.

[0057] The process starts with dispensing a solution of hydroxyalkane (about 0.5 ml to about 100 ml hydroxyalkane per mmol of L-cystine), such as for example methanol, ethanol or isopropanol (preferably methanol, MeOH) or a dihydroxyalkane such as for example propylene glycol, or a trihydroxyalkane such as for example glycerol.

[0058] The process includes adding an alkali metal hydroxide, or alkaline earth hydroxides or oxides (4-6 molar equivalents relative to L-cystine) to the solution, thereby producing a mixture of hydroxyalkane and alkali metal hydroxide and/or alkaline earth hydroxides or oxides in solution. Alkaline earth hydroxides or oxides include for example group II alkali earth metals such as (Mg, Ca and Ba), preferably calcium hydroxide or calcium oxide. Other bases that could be used are the hydroxides, carbonates, bicarbonates and oxides of selected group I alkali metals (such as Li, K and Cs). Alkali metal hydroxide preferably include halide metal hydroxide, most preferably for example sodium hydroxide or potassium hydroxide. Particularly preferred are sodium hydroxide, sodium carbonate and sodium bicarbonate, because they generate non-toxic sodium chloride as the only by-product in solution which is easily separated from the product via filtration. Sodium hydroxide is most preferred because it is the strongest base of the three, leading to more efficient reaction rates.

[0059] The process includes adding Cystine (1 molar equivalent, i.e., 500 mg, 2.1 mmol) to the solution mixture.

[0060] The process includes maintaining the solution at temperatures ranging between 3-50° C. (preferably between 5-35° C., more preferably between 5-25° C., even more preferably less than about 10° C., most preferably at about 5° C. At this point, the process includes stirring the solution mixture between 5 min to 2 h.

[0061] Subsequently, the process includes cooling the solution to between about 5-25° C., depending on the initial temperature referenced above.

[0062] Next, the process includes adding an acetylating agent to the cooled solution mixture slowly or in portions. An acetylating agent includes for example acetyl chloride or acetic anhydride (preferably acetyl chloride, AcCI) (2-4 molar equivalents with respect to L-cystine).

[0063] The process includes maintaining the temperature below 50° C. (preferably below 30° C., more preferably below 20° C., most preferably less than about 10° C.), depending on the type of acetylating agent used to achieve the chemical transformation on the nitrogen groups of L-cystine. In one embodiment, acetyl chloride (AcCI) is added at room temperature (R.T.) of about 15° C. to about 20° C.

[0064] The process includes stirring the reaction mixture until all the cystine is consumed (typically between 15 min-24 h) and the desired N,N′-diacetyl-L-cystine disodium salt is generated. Upon reacting cystine with acetyl chloride, in the presence of sodium hydroxide in methanol, N,N′-diacetyl-L-cystine disodium salt, i.e., compound of formula (2) is obtained as a solution in methanol and the innocuous by-product sodium chloride (“NaCl”) precipitates out of the reaction mixture. Any small amounts of unreacted cystine, monoacetylated cystine (N-acetyl-L-cystine) or sodium acetate impurities will remain in the methanol solution.

[0065] Upon completion of the reaction, the process includes filtering off the precipitated NaCl solids and washing them with a hydroxyalkane (preferably an alcohol, most preferably methanol), followed by allowing the combined filtrates to evaporate, to yield N,N′-diacetyl-L-cystine disodium salt as a solid. Small amounts of impurities that may be present in the N,N′-diacetyl-L-cystine disodium salt include cystine disodium salt, N-acetyl-L-cystine sodium salt and sodium acetate.

[0066] All reagents suitable for inventive process are available from commercial sources.

[0067] The relative amounts of cystine and a base are such as to not have excessive starting ingredients or by-product salts upon reaction completion. According to the inventive process, about 0.9 to 1 molar equivalent of cystine to 4 to 6 molar equivalents of a base are preferred, preferably 1.0 molar equivalents of cystine to 4 molar equivalents of base.

[0068] Sufficient hydroxyalkane and base are used in the inventive process to generate a basic solution [as determined by measuring the pH of a solution prepared using an aliquot of said hydroxyalkane/base solution (2 parts) with water (8 parts)] with an apparent pH of between 9-15. Enough equivalents of base are added to 1) deprotonate the amine group of cystine, thereby making it nucleophilic enough, to allow it to react with acetyl chloride and 2) neutralize the HCl generated from the reaction of amine with acetyl chloride. The stronger the base, the higher the pH and the faster the reaction time.

[0069] In the penultimate step of the process, the precipitated by-product salts (e.g. “NaCl”) are separated and isolated from the filtrate containing the NDAC disodium salt product, by centrifugation or filtering, preferably by filtering. The isolated by-product salts are optionally washed with additional hydroxyalkane and all filtrates containing the NDAC disodium salt product are combined.

[0070] In the last step of the process, the combined filtrates are evaporated, leaving the desired NDAC disodium salt product as a solid in pure form. Preferably the inventive process also comprises a further step, wherein the evaporated alcohol/methanol solvent from the combined filtrates is condensed and reused and the isolated by-product salts are recycled

[0071] The inventive process is advantageous, at least because it uses greener solvents relative to other solvents used in the industry to carry out amidations using these specific types of reactants, results in no undesirable cystine-derived by-products and only innocuous by-products salts (NaCl) that can be recycled or acceptable by-products (cystine disodium salt, N-acetyl-L-cystine sodium salt and sodium acetate), the solvents can be fully reused, and is relatively fast. It also results in improved purity of from 90% to 99%, preferably from 95% to 99%, and most preferably at least 98% to 99%, and improved yield from 85% to 99%, preferably from 90% to 99%, and most preferably at least 95% to 99%.

EXAMPLES

Experimental Methods

[0072] All reagents and solvents were obtained from commercial sources and used without further purification. L-Cystine (>98%), acetyl chloride (98%), sodium hydroxide (97%) and anhydrous methanol (99.8%) were purchased from Sigma. N,N′-diacetyl-L-cystine (NDAC; 95%) was purchased from CombiBlocks..

Reaction Monitoring Methods

[0073] Reaction monitoring was performed using thin layer chromatography (TLC) on silica gel using mixtures of ethyl acetate, isopropyl alcohol and water. Visualization of TLC plates was performed by subjecting TLC plates to 2% ninhydrin in ethanol followed by heat. Qualitative analysis and confirmation of reaction products was performed using 1H nuclear magnetic resonance (1H NMR) and liquid chromatography mass spectrometry (LCMS). Purity of reaction products was assessed via comparison of pure authentic standards using a combination of TLC, 1H NMR and LCMS. Product identity was confirmed via comparison with an authentic NDAC disodium salt reference standard using 1H NMR, TLC and LCMS.. A reference standard of NDAC disodium salt was prepared by dissolving NDAC (100 mg, 0.3 mmol) in water (1 ml), adding sodium bicarbonate (51 mg, 0.6 mmol) and stirring for 10 min. The solvent was removed under reduced pressure at 50° C. to give pure NDAC disodium salt as a white powder (111 mg, 98%, >95% purity).

Example 1

[0074] An example of the process within the scope of the invention was performed. The chemical reaction scheme is shown below.

##STR00008##

[0075] A specific experimental procedure was carried out in one step to prepare N,N′-diacetyl-L-cystine disodium salt, as follows.

[0076] Cystine (1) (500 mg, 2.1 mmol) was added to a cold (~10° C.) solution of methanol (5ml) containing sodium hydroxide (333 mg, 8.3 mmol) and stirred for 5 min.

[0077] The solution was cooled to 5° C. and acetyl chloride (0.3 ml, 4.2 mmol) added portionwise, maintaining the temperature below 10° C.

[0078] The resulting white suspension was stirred at 5° C. for 20 min and allowed to warm up to R.T. (~20° C.) over 10 min.

[0079] At this time, TLC (3:4:3 ethyl acetate:isopropyl alcohol:water) showed the clean formation of a major product.

[0080] The precipitated solids were filtered off, washed with anhydrous methanol (4 × 1 ml) and the combined filtrates evaporated under reduced pressure @ 40° C. to give N,N′-diacetyl-L-cystine disodium salt (2) as white solid (688 mg, 90% yield).

[0081] Product identity and purity (>95%) was confirmed by 1H NMR, TLC and LCMS via comparison with an authentic NDAC disodium salt reference standard.