Method for preparing N,N'-diacetyl-L-cystine disodium salt from cystine and acetyl chloride in methanol in the presence of sodium hydroxide

Abstract

A method for preparing N,N'-diacetyl-L-cystine disodium salt, the method comprising: (i) mixing hydroxyalkane (0.5-100 L, preferably 0.5-10 L, more preferably 1-3 L, most preferably 1 L / mole cystine) with sodium base (4.0 molar equivalents / mole cystine) to form a cold solution at a temperature of 5-10°C; (ii) adding cystine (1 molar equivalent) to the cold solution and stirring for a sufficient time to form an alkaline cystine solution; (iii) optionally cooling the cystine solution to 5°C; (iv) adding acetyl chloride (2 molar equivalents / mole cystine) in batches while maintaining the temperature at 3-50°C, preferably at 5-35°C, more preferably below 10°C, most preferably at 5°C, thereby obtaining a white suspension; (v) stirring the white suspension and warming the suspension to room temperature of 15°C-50°C, preferably at 20-35°C, more preferably 20°C, thereby obtaining N,N'-diacetyl-L-cystine disodium salt product dissolved in solution, and a sodium chloride byproduct precipitated solid in the suspension. This specification discloses an exemplary method on a small laboratory scale (500 mg cystine isolate, 688 mg product, 90% yield) (Specification page 13, Example 1).

Description

Technical Field

[0001] The present invention relates to an efficient method for preparing N,N'-diacetyl-L-cystine disodium salt ("NDAC disodium salt") directly from cystine using an acetylating agent. Background Art

[0002] Healthy-looking skin is a universal consumer desire that influences their personal care. Healthy-looking skin can be described based on appearance attributes (glow, radiance, evenness of hue, pigmentation spots), texture attributes (smooth, silky, free of bumps and pores), and age attributes (fine lines, wrinkles, elasticity, and sagging / looseness).

[0003] While different classes of compounds claim to have cosmetic benefits for skin appearance, cysteine ​​and cystine derivatives have not received much attention.

[0004] Many cysteine / cystine derivatives, including β-substituted cysteine / cystine, cystine diamide, cystine dialkyl esters and N-alkanoyl cysteine, have potential therapeutic benefits, for example in the prevention of kidney stones ( See also "Rational Design of Novel Crystal Growth Inhibitors for Treatment of Cystinuria Kidney Stones" by Zhu et al. 2013 ProQuest Dissertations and Theses ; CrystEngComm, 2016, 18, 8587). However, even the simplest cystine derivative, N,N'-diacetyl-L-cystine ("NDAC"), lacks industrial applications, especially in the beauty and personal care fields.

[0005] Due to almost no known use, NDAC and / or NDAC salts are not actually available on a commercial scale. It has been found that NDAC salts decompose into cystine, which is reduced to cysteine ​​in the cells of an organism, i.e., the intracellular formation of cysteine. Cysteine ​​and its dimer cystine are precursors of glutathione.

[0006] Glutathione (GSH) is a tripeptide composed of glutamate, cysteine ​​and glycine. Glutathione is present in all mammalian tissues. It is the main antioxidant in the living body and protects cells from oxidation by quenching reactive oxygen species. GSH is believed to play an important role in protecting cells from the cytotoxic effects of ionizing radiation, heat, certain chemicals and solar ultraviolet radiation. Although this is true in all parts of the body, this is particularly important in the skin because the skin is exposed to radiation, especially ultraviolet radiation, and the damaging effects of environmental pollutants. Reduced intracellular glutathione concentrations in the skin are associated with cell damage, inflammation, skin darkening, discoloration, spots or freckles caused by exposure to ultraviolet radiation, and overall physiological aging.

[0007] Compositions for enhancing intracellular glutathione production have been described. See, for example, U.S. Pat. No. 7,740,831 to Chiba et al., (USRE37934, USRE42645, WO2016 / 033183 and US20050271726) to Crum et al.; U.S. Pat. No. 6,149,925 to Mammone and US 20060063718 to Perricone. In, for example, U.S. published patent applications Nos. US20 / 9034, US20 / 16059 and US19 / 328631 to the applicant, local compositions and enhancement of glutathione production from their constituent amino acids (glutamate, cysteine ​​and / or glycine, i.e., glutathione precursors) in the skin are addressed for cellular uptake and synthesis of GSH tripeptides.

[0008] The applicant has now found that for enhancing glutathione synthesis in skin cells, the performance of NDAC is comparable to that of cystine, but NDAC is more soluble and can be better delivered to the skin, thereby producing better performance. Proteases in the body can cleave (or hydrolyze) the N-acetyl bonds of NDAC, ultimately generating cystine, so NDAC can also be used as a controlled release alternative source of cystine. Therefore, there is a demand for NDAC salt materials.

[0009] Examples of other cystine derivatives that are less favorable than NDAC are N-acetylcysteine ​​("NACys") containing a thiol group (SH) and L-cystine diethyl ester ("DEC") without SH. NACys ​​is a thiol drug that is commonly used as an expectorant (Cryst. Eng. Comm., 2016, 18, 8587, referred to as "NACe" therein). NACys ​​produces an unpleasant sulfur odor, which is unacceptable in cosmetics. In addition, the sulfur odor (monitored as hydrogen sulfide or H 2S) is consistent with NACys ​​decomposition, which is also unacceptable in commercial cosmetics. Similarly, DEC also produces a strong and unpleasant sulfur odor and is unstable in formulated cosmetics.

[0010] NDAC is an amide, i.e., an N,N'-diacetyl derivative of cystine. For the purposes of the present invention, NDAC has the following chemical structure:

[0011]

[0012] The molecular weight of NDAC = 324.4.

[0013] The NDAC stereoisomers of the present invention (referring to the stereoisomerism of the α-carbon atom between the nitrogen atom and the carbonyl group of the carboxylic acid moiety) include R,R (L-cystine), R,S, S,R and S,S (D-cystine). Preferably, the L stereoisomer is used, which is the most abundant and natural isomeric form found in nature.

[0014] NDAC is not readily available commercially, but can be obtained on a laboratory scale. NDAC can be synthesized directly from NACys ​​as described by Vandana Rathore et al. in Organic Letters, 20(19), 6274-6278; 2018 and Scott J. Pye et al. in Green Chemistry, 20(1), 118-124; 2018:

[0015]

[0016] This chemical method involving the oxidative dimerization of NACys ​​or other SH-containing raw materials to generate NDAC has significant disadvantages. First, any unreacted NACys ​​is difficult to remove from the desired NDAC product, and a special purification method is required, such as chromatography using a polar solvent system. Secondly, this purification method is impractical on a very large scale (e.g., in kg, tons). Third, even a very small amount of NACys ​​impurities in the final NDAC product will make it unusable for cosmetics, because SH-containing impurities such as NACys ​​will produce undesirable sulfur smell when formulated. Fourth, during the oxidation of NACys ​​to NDAC, premature oxidation of NACys ​​and peroxidation of NDAC may result, and become undesirable and difficult to remove sulfenic acid (sulfenic / sulfenic acid), sulfone and / or sulfoxide impurities. Therefore, it is very desirable to avoid using a chemical method containing thiol raw materials and / or oxidation conditions.

[0017] A sensible approach to avoid the use of thiol-containing starting materials that may produce cystine derivatives such as NDAC involves the use of cystine or cystine-derived starting materials. Practical methods involving the direct conversion of cystine to NDAC have not been reported, mainly due to the extremely low solubility of cystine in water and organic solvents (e.g., at 25° C., the solubility of cystine in water is 0.112 mg / mL; cystine is more soluble in aqueous solutions at pH less than 2 or pH greater than 8; cystine is almost insoluble in organic solvents, such as alcohols, ethers, esters, ketones, etc.).

[0018] The higher molecular weight N,N'-dialkanoyl cystine, which is more complex than NDAC, is chemically prepared directly from cystine and a higher molecular weight acyl chloride ( See also For example, Journal of Chromatography (1989) 483, 239-252; Journal of Materials Chemistry B (2015) 3, 7262-7270; Applied Materials and Interfaces (2016) 8, 21155-21162; Organic and Biomolecular Chemistry (2017) 15, 3840–3842; Journal of Dispersion Science and Technology (2014) 35, 1051-1059; Journal of Medicinal Chemistry (2008) 51, 684-688; Invention Patent Application 102093468). Higher molecular weight anhydrides can also be used as reagents ( See also For example, invention patent application publication specification 1810769). In addition, high molecular weight activated carboxylic acids can also be used as reagents ( See also For example, Bioconjugate Chemistry (2015) 26, 145152; ACS Chemical Biology (2009) 4, 547-556; US 20120232120; Bio-macromolecules (2015) 16, 2436-2443). Although the yields are acceptable, unfortunately, most of these methods use undesirable organic solvents such as tetrahydrofuran, N,N-dimethylformamide, dichloromethane and / or mixed solvent systems (e.g., Schotten-Baumann conditions) on a commercial scale. These organic solvents are toxic and / or flammable and need to be removed at the end of the reaction in any case. In addition, existing methods often lead to the formation of by-products, which also require complex and labor-intensive removal, such as by multi-step extraction or chromatography.

[0019] On the other hand, chemical preparations of N,N'-dialkanoylcystine products from low molecular weight acid chlorides (e.g., acetyl chloride), anhydrides (e.g., acetic anhydride), or other activated carboxylic acids have low yields and require complex purification methods. This is due in part to the higher reactivity of low molecular weight acid chlorides and anhydrides (and their higher likelihood of decomposition when exposed to the alkaline aqueous media required to solubilize cystine to effectuate its chemical reaction in solution).

[0020] Therefore, there is a commercial need for a chemical process to prepare N,N'-diacetylcystine ("NDAC") disodium salt (2) directly from cystine (1) using low molecular weight acetylation agents such as acetyl chloride or acetic anhydride.

[0021]

[0022] There is a need in the art for a practical chemical process for forming NDAC disodium salt directly from cystine using low molecular weight acid chlorides or anhydrides, which process is also "green" or does not require the generation of organic or other hazardous byproducts, generates no (or minimal) waste, and the reagents / solvents used in the process can be recovered and recycled. Summary of the invention

[0023] The present invention eliminates the above needs in the art by providing a chemical process for preparing N,N'-diacetyl-L-cystine ("NDAC") disodium salt (2) directly from cystine (1) using an acetylating agent such as acetyl chloride or acetic anhydride.

[0024]

[0025] When using a batch process, the chemical method of the present invention is rapid and is completed in a single container in one step. For batch or continuous processes, the reaction of the present invention advantageously starts with cystine and uses a recyclable alcohol-based solvent. No unwanted cystine-derived by-products are produced, and any by-products produced consist of harmless salts (e.g., NaCl) that can be easily filtered out and recycled, or a small amount of acceptable by-products (which consist of cystine disodium salt (from unreacted cystine), N-acetyl-L-cystine sodium salt (produced by monoacetylation of cystine) and sodium acetate). The N, N'-diacetyl-L-cystine ("NDAC") disodium salt (2) product is isolated by solvent evaporation without purification.

[0026] The present invention comprises a single-step process for preparing N-diacetyl-L-cystine disodium salt ("NDAC disodium salt") comprising:

[0027] L-cystine was added to a cold (~10°C) methanol (5ml) solution containing sodium hydroxide (333mg, 8.3mmol) and stirred for 5 minutes to form a solution;

[0028] The solution was cooled to 5°C and acetyl chloride (0.3 ml, 4.2 mmol) was added portionwise while keeping the temperature below 10°C to give a white suspension;

[0029] The resulting white suspension was stirred at 5°C for 20 minutes and allowed to warm to room temperature (~20°C) over 10 minutes to afford the N,N'-diacetyl-L-cystine disodium salt product.

[0030] In one aspect, a method for preparing N,N'-diacetyl-L-cystine disodium salt comprises:

[0031] The hydroxyalkane (5 ml) was mixed with sodium base (4 mmol equivalent / mmol cystine) and cooled at 10°C to form a cold solution;

[0032] Cystine (1 mmol equiv.) was added to the cold (~10°C) solution and stirred for 5 minutes to form a cystine alkaline solution;

[0033] The cystine alkaline solution was cooled to 5°C, and acetyl chloride (2.0 mmol equivalent / mmol cystine) was added portionwise while keeping the temperature below 10°C to obtain a white suspension;

[0034] The white suspension was stirred at 5°C for 20 minutes and the suspension was allowed to warm to room temperature (~20°C) over 10 minutes until the reaction was complete, yielding the N,N'-diacetyl-L-cystine disodium salt product dissolved in solution and a precipitated solid of the sodium chloride byproduct in the suspension.

[0035] In a larger scale alternative method for preparing N,N'-diacetyl-cystine disodium salt, the method comprises:

[0036] (i) mixing hydroxyalkane (0.5-100 L, preferably 0.5-10 L, more preferably 1-3 L, most preferably 1 L per mole of cystine) with sodium base (4.0 molar equivalents per mole of cystine) to form a cold solution at a temperature of 5-10° C.;

[0037] (ii) adding cystine (1 molar equivalent) to the cold solution and stirring for a sufficient time to form an alkaline cystine solution;

[0038] (iii) optionally, cooling the cystine solution to 5° C.;

[0039] (iv) adding acetyl chloride (2 molar equivalents per mole of cystine) in portions while maintaining the temperature at 3-50°C, preferably at 5-35°C, more preferably below 10°C, most preferably at 5°C, to obtain a white suspension;

[0040] (v) stirring the white suspension and warming the suspension to room temperature of 15°C to 50°C, preferably 20°C to 35°C, more preferably 20°C,

[0041] This results in the N,N'-diacetyl-L-cystine disodium salt product dissolved in solution, and a precipitated solid of the sodium chloride byproduct in the suspension.

[0042] In addition, the method may include separating the NDAC disodium salt (2) product from the reaction mixture. In particular, the method may include:

[0043] filtering out the sodium chloride byproduct precipitated solid, thereby separating the precipitated solid from the N,N'-diacetyl-L-cystine disodium salt product dissolved in the filtrate,

[0044] Wash the precipitated solid with anhydrous methanol and collect the methanol filtrate,

[0045] Combine all filtrates,

[0046] The combined filtrates were evaporated under reduced pressure at 40° C. to give N,N′-diacetyl-L-cystine disodium salt (2) as a white solid in 90% yield.

[0047] The method of the present invention is rapid, does not require labor-intensive product separation or purification, and by removing organic solvents or by-products, NDAC has an improved yield and purity, and the by-products and solvents can be recovered and reused.

[0048] The general procedure for the preparation of N,N'-diacetyl-L-cystine salt is as follows.

[0049] L-cystine (1 molar equivalent, 500 mg, 2.1 mmol) is added to a solution of a hydroxyalkane or a dihydroxyalkane or a trihydroxyalkane, preferably to a solution of a hydroxyalkane, wherein the concentration is 0.5 ml to 100 ml of hydroxyalkane (or dihydroxyalkane or trihydroxyalkane) / mmol of L-cystine, containing 4 to 6 molar equivalents of an alkali metal hydroxide such as sodium hydroxide (preferably an alkali metal hydroxide, most preferably sodium hydroxide), or an alkaline earth metal hydroxide, or an oxide such as calcium hydroxide or calcium oxide, relative to cystine, at a pH in the range of 9 to 15 (preferably 10 to 14, most preferably less than 14), at a temperature in the range of 5 to 50° C. (preferably 5 to 35° C., more preferably 5 to 25° C., most preferably less than about 10° C.), and stirred for 5 minutes to 2 hours, depending on the scale of the process or the amount of reagents, and the corresponding mixing time required to produce a homogeneous solution / mixture.

[0050] Suitable hydroxyalkanes include, for example, methanol, ethanol or isopropanol (preferably methanol). Suitable dihydroxyalkanes include, for example, propylene glycol. Suitable trihydroxyalkanes include, for example, glycerol.

[0051] The solution is cooled to 5-25°C, and an acetylating agent such as acetyl chloride or acetic anhydride (preferably acetyl chloride, most preferably acetyl chloride) (2-4 molar equivalents, preferably 2-3 molar equivalents, most preferably 2 molar equivalents relative to cystine) is added slowly or in portions, keeping the temperature below 50°C (preferably below 30°C, more preferably below 20°C, most preferably below about 10°C). In one embodiment, acetyl chloride (AcCl) is added at room temperature (RT) of about 15°C to about 20°C.

[0052] The reaction mixture is stirred at a temperature below 50°C (preferably below 30°C, more preferably below 20°C, most preferably below about 10°C) until all the cystine is consumed (usually between 15 minutes and 24 hours) and the desired N,N'-diacetyl-L-cystine salt is produced.

[0053] The precipitated solid is filtered off, washed with a hydroxyalkane (preferably an alcohol, most preferably methanol) and the combined filtrates are evaporated to give N,N'-diacetyl-L-cystine salt as a solid.

[0054] The reaction scheme according to the present invention is as follows:

[0055]

[0056] In a specific embodiment, N,N'-diacetyl-L-cystine disodium salt is prepared as follows. L-cystine (1) (500 mg, 2.1 mmol) is added to a cold (~10°C) solution of methanol (5 ml) containing sodium hydroxide (333 mg, 8.3 mmol) and stirred for 5 minutes. The solution is cooled to 5°C and acetyl chloride (0.3 ml, 4.2 mmol) is added in portions, keeping the temperature below 10°C. The resulting white suspension is stirred at 5°C for 20 minutes and allowed to warm to RT (room temperature, about 20°C) within 10 minutes. At this point, TLC (3:4:3 ethyl acetate:isopropanol:water) shows clean formation of the main product. The precipitated solid was filtered off, washed with anhydrous methanol (4×1 ml), and the combined filtrates were evaporated under reduced pressure at 40° C. to give N,N′-diacetyl-L-cystine disodium salt (2) as a white solid (688 mg, 90% yield, >95% purity). The TLC and 1H NMR of the product were consistent with those of the N,N′-diacetyl-L-cystine disodium salt reference standard.

[0057] In an alternative embodiment, the N,N'-diacetyl-L-cystine disodium salt product isolated from the reaction mixture contains cystine disodium salt, N-acetyl-L-cystine sodium salt or sodium acetate, or a mixture thereof. DETAILED DESCRIPTION OF THE INVENTION

[0059] Except in the examples or where otherwise explicitly indicated, all numbers in this description indicating amounts of material or reaction conditions, physical properties of materials and / or use are to be understood as modified by the word "about".

[0060] It should be noted that in specifying any range of concentrations or amounts, any particular upper concentration can be associated with any particular lower concentration or amount.

[0061] For the avoidance of doubt, the word "comprising" means "including" but not necessarily "consisting of" or "composed of." In other words, the listed steps or options do not have to be exhaustive.

[0062] The present invention provides a chemical method for preparing N,N'-diacetyl-L-cystine (NDAC) disodium salt (2) directly from L-cystine (1) by using an acetylating agent (such as acetyl chloride or acetic anhydride), thereby eliminating the need for the prior art.

[0063] When a batch process is used, the chemical method of the present invention is rapid and is completed in a single container in one step. For batch or continuous processes, the reaction of the present invention advantageously starts with cystine and uses a recyclable alcoholic solvent. No unwanted cystine-derived by-products are produced, and any by-products produced consist of harmless salts (e.g., NaCl) that can be conveniently filtered out and recycled, or consist of a small amount of acceptable by-products consisting of cystine disodium salt (from unreacted cystine), N-acetyl-L-cystine sodium salt (produced by monoacetylation of cystine) and sodium acetate. The N,N'-diacetyl-L-cystine (NDAC) disodium salt (2) product is isolated by solvent evaporation without purification.

[0064] N,N'-diacetyl-L-cystine disodium salt ("NDAC disodium salt")

[0065] Applicants have found that N, N'-diacetyl-L-cystine ("NDAC") and NDAC disodium salt are useful amides in personal care compositions. In skin cosmetic compositions, when applied to the skin, NDAC and its disodium salt are converted into cysteine ​​in cells. Cysteine ​​has many cosmetic uses, including as a glutathione precursor. Applicants have found that NDAC disodium salt has excellent functional benefits for the final delivery of cysteine ​​for cosmetic purposes.

[0066] NDAC salt is the disodium salt of N,N'-diacetylcystine (also known as N,N'-diacetyl-L-cystine or NDAC). The structure of NDAC is shown below (C 10 H 16 N 2 O 6 S 2 ):

[0067]

[0068] The molecular weight of NDAC = 324.4.

[0069] The disodium salt of N,N'-diacetyl-L-cystine ("NDAC") of formula (2) prepared according to the process of the present invention has the following general structure (C 10 H 14 N 2 Na 2 O 6 S 2 ):

[0070]

[0071] The molecular weight of NDAC disodium salt = 368.3.

[0072] NDAC or its disodium salt is not readily available commercially on a large scale.

[0073] N,N'-diacetyl-L-cystine disodium salt ("NDAC disodium salt") can be prepared according to the method of the present invention. The method of the present invention is most useful for synthesizing NDAC disodium salt by using L-cystine (1), which is easily commercially available in large quantities.

[0074] Method of the present invention

[0075] According to the process of the present invention, N,N'-diacetyl-L-cystine salt (NDAC salt) is prepared in a single step.

[0076] The method begins with dispensing a solution of a hydroxyalkane such as methanol, ethanol or isopropanol (preferably methanol, MeOH), or a dihydroxyalkane such as propylene glycol, or a trihydroxyalkane such as glycerol (about 0.5 ml to about 100 ml hydroxyalkane per millimole of L-cystine).

[0077] The method comprises adding alkali metal hydroxide or alkaline earth metal hydroxide or oxide (4-6 molar equivalents relative to L-cystine) to the solution, thereby generating a mixture of hydroxyalkane and alkali metal hydroxide and / or alkaline earth metal hydroxide or oxide in solution. Alkaline earth metal hydroxide or oxide include, for example, Group II alkaline earth metals such as (Mg, Ca and Ba), preferably calcium hydroxide or calcium oxide. Other alkalis that can be used are hydroxides, carbonates, bicarbonates and oxides of selected Group I alkali metals (such as Li, K and Cs). Alkali metal hydroxides preferably include halogenated metal hydroxides, most preferably, for example, sodium hydroxide or potassium hydroxide. Particularly preferred are sodium hydroxide, sodium carbonate and sodium bicarbonate, because they can produce nontoxic sodium chloride as the only by-product in the solution, which is easy to separate from the product by filtration. Sodium hydroxide is most preferred, because it is the strongest alkali in the three alkalis, and can result in a more effective reaction rate.

[0078] The method involves adding cystine (1 molar equivalent, ie, 500 mg, 2.1 mmol) to the solution mixture.

[0079] The method includes maintaining the solution at a temperature range of 3-50°C (preferably 5-35°C, more preferably 5-25°C, even more preferably below about 10°C, most preferably about 5°C). At this point, the method includes stirring the solution mixture for 5 minutes to 2 hours.

[0080] Subsequently, the method includes cooling the solution to about 5-25°C, depending on the above initial temperature.

[0081] Next, the method includes slowly or batchwise adding an acetylating agent to the cooled solution mixture. The acetylating agent includes, for example, acetyl chloride or acetic anhydride (preferably acetyl chloride, AcCl) (2-4 molar equivalents relative to L-cystine).

[0082] The method comprises maintaining the temperature below 50° C. (preferably below 30° C., more preferably below 20° C., and most preferably below about 10° C.), depending on the type of acetylating agent used to achieve the chemical transformation on the nitrogen group of L-cystine. In one embodiment, acetyl chloride (AcCl) is added at room temperature (RT) of about 15° C. to about 20° C.

[0083] The method comprises stirring the reaction mixture until all the cystine is consumed (usually 15 minutes to 24 hours) and the desired N,N'-diacetyl-L-cystine disodium salt is produced. Cystine is reacted with acetyl chloride in methanol in the presence of sodium hydroxide to obtain a solution of N,N'-diacetyl-L-cystine disodium salt, i.e., a compound of formula (2), in methanol, and a harmless byproduct, sodium chloride ("NaCl"), is precipitated from the reaction mixture. Any small amount of unreacted cystine, monoacetylated cystine (N-acetyl-L-cystine) or sodium acetate impurity will remain in the methanol solution.

[0084] After the reaction is complete, the process includes filtering off the precipitated NaCl solid and washing it with a hydroxyalkane (preferably an alcohol, most preferably methanol), and then evaporating the combined filtrates to obtain a solid N,N'-diacetyl-L-cystine disodium salt. Minor impurities that may be present in N,N-diacetyl-L-cystine disodium salt include cystine disodium salt, N-acetyl-L-cystine sodium salt, and sodium acetate.

[0085] All reagents suitable for use in the methods of the present invention are available from commercial sources.

[0086] The relative amounts of cystine and base should be such that there is no excess starting component or by-product salt when the reaction is complete. According to the process of the present invention, about 0.9-1 molar equivalent of cystine to 4-6 molar equivalents of base is preferred, preferably 1.0 molar equivalent of cystine to 4 molar equivalents of base.

[0087] Sufficient hydroxyalkane and base should be used in the process of the present invention to produce an alkaline solution with an apparent pH between 9-15 [determined by measuring the pH of a solution prepared using aliquots of the hydroxyalkane / base solution (2 parts) and water (8 parts)]. Sufficient equivalents of base are added to 1) deprotonate the amine group of cystine, thereby making it sufficiently nucleophilic to react with acetyl chloride, and 2) neutralize the HCl generated by the reaction of the amine with acetyl chloride. The stronger the alkalinity, the higher the pH and the faster the reaction time.

[0088] In the penultimate step of the process, the precipitated by-product salt (e.g., "NaCl") is separated and isolated from the filtrate containing the NDAC disodium salt product by centrifugation or filtration, preferably by filtration. The separated by-product salt is optionally washed with additional hydroxyalkane, and all filtrates containing the NDAC disodium salt product are combined.

[0089] In the final step of the process, the combined filtrates are evaporated, leaving the desired NDAC disodium salt product as a solid in pure form. Preferably, the process of the present invention further comprises a further step wherein the evaporated alcohol / methanol solvent from the combined filtrates is condensed and reused and the separated by-product salt is recycled.

[0090] The process of the present invention is advantageous at least because it uses a greener solvent relative to other solvents used in the industry to carry out the amidation reaction with these specific types of reactants, does not produce unwanted cystine-derived byproducts, produces only harmless byproduct salts (NaCl) that can be recycled, or acceptable byproducts (cystine disodium salt, N-acetyl-L-cystine sodium salt and sodium acetate), the solvent can be completely reused, and is relatively fast. It also results in an improved purity of 90%-99%, preferably 95%-99%, most preferably at least 98%-99%, and an improved yield of 85%-99%, preferably 90%-99%, most preferably at least 95%-99%. DETAILED DESCRIPTION

[0091] Example

[0092] Experimental methods

[0093] 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.

[0094] Reaction Monitoring Methods

[0095] Using a mixture of ethyl acetate, isopropanol and water, thin layer chromatography (TLC) on silica gel is used for reaction monitoring. By placing the TLC plate in an ethanol solution of 2% ninhydrin, then heating, visualization of the TLC plate is performed. 1H nuclear magnetic resonance (1H NMR) and liquid chromatography mass spectrometry (LCMS) are used to qualitatively analyze and confirm the reaction product. By using a combination of TLC, 1HNMR and LCMS, the purity of the reaction product is evaluated by comparing with pure reliable standards. By using 1HNMR, TLC and LCMS to compare with reliable NDAC disodium salt reference standards, product identity is confirmed. By dissolving NDAC (100mg, 0.3 mmol) in water (1ml), adding sodium bicarbonate (51mg, 0.6 mmol) and stirring for 10 minutes to prepare the reference standard of NDAC disodium salt. The solvent is removed under reduced pressure at 50°C to obtain the pure NDAC disodium salt (111mg, 98%,>95% purity) of white powder.

[0096] Example 1

[0097] An example of a method for carrying out the present invention is shown below.

[0098]

[0099] As described below, a specific experimental procedure was performed in one step to prepare N,N'-diacetyl-L-cystine disodium salt.

[0100] Cystine (1) (500 mg, 2.1 mmol) was added to a cold (~10°C) solution of methanol (5 ml) containing sodium hydroxide (333 mg, 8.3 mmol) and stirred for 5 minutes.

[0101] The solution was cooled to 5°C and acetyl chloride (0.3 ml, 4.2 mmol) was added portionwise, keeping the temperature below 10°C.

[0102] The resulting white suspension was stirred at 5°C for 20 min and allowed to warm to room temperature (~20°C) over 10 min.

[0103] At this time, TLC (3:4:3 ethyl acetate:isopropanol:water) showed clean formation of major product.

[0104] The precipitated solid was filtered off, washed with anhydrous methanol (4 x 1 ml) and the combined filtrates were evaporated under reduced pressure at 40 °C to give N,N'-diacetyl-L-cystine disodium salt (2) (688 mg, 90% yield) as a white solid.

[0105] Product identity and purity (>95%) were confirmed by 1H NMR, TLC and LCMS by comparison with an authentic NDAC disodium salt reference standard.

Claims

1. A method for preparing N,N'-diacetyl-L-cystine disodium salt, the method comprising: (i) mixing 0.5-100 L of hydroxyalkane / mole of cystine with 4.0 molar equivalents of sodium base / mole of cystine to form a cold solution at a temperature of 5-10° C.; (ii) adding 1 molar equivalent of cystine to the cold solution and stirring for a sufficient time to form an alkaline cystine solution; (iii) optionally, cooling the cystine solution to 5° C.; (iv) adding 2 molar equivalents of acetyl chloride per mole of cystine in portions while maintaining the temperature at 3-50° C. to obtain a white suspension; (v) stirring the white suspension and warming the suspension to room temperature of 15° C. to 50° C., This results in the N,N'-diacetyl-L-cystine disodium salt product dissolved in solution and a precipitated solid of sodium chloride byproduct in the suspension.

2. The method of claim 1, further comprising isolating the N,N'-diacetyl-L-cystine disodium salt product from the reaction mixture.

3. The method according to claim 1, wherein the method further comprises: filtering out the sodium chloride byproduct precipitated solid, thereby separating the precipitated solid from the N,N'-diacetyl-L-cystine disodium salt product dissolved in the filtrate, The precipitated solid was washed with anhydrous methanol, and the methanol filtrate was collected. Combine all filtrates, The combined filtrates were evaporated under reduced pressure at 40°C to give N,N'-diacetyl-L-cystine disodium salt product as a white solid in 90% yield.

4. The method according to any one of the preceding claims 1 to 3, wherein the hydroxyalkane is methanol, ethanol or isopropanol. The method according to claim 4 , wherein the hydroxyalkane is methanol.

6. The process according to any one of the preceding claims 1 to 3, wherein the sodium base is selected from the group consisting of sodium hydroxide, sodium carbonate, sodium bicarbonate and mixtures thereof.

7. The method of claim 6, wherein the sodium base is sodium hydroxide.

8. The method according to any one of the preceding claims 1-3, wherein said stirring of cystine in the solution for said sufficient time is carried out for 5 minutes to 1 hour.

9. The method according to claim 8, wherein: The sufficient time for stirring the cystine in the solution was 5 minutes.

10. The process according to any one of the preceding claims 1 to 3, wherein the white suspension is stirred for 10 minutes to 24 hours.

11. The method according to claim 10, wherein the white suspension is stirred for 15 minutes to 24 hours.

12. The method according to claim 10, wherein the white suspension is stirred for 20 minutes.

13. The method according to any one of the preceding claims 1 to 3, wherein warming the white suspension to room temperature is performed for 10 to 30 minutes.

14. The method according to claim 13, wherein the white suspension is warmed to room temperature for 10 minutes.

15. The process according to any one of claims 1 to 3, wherein the total reaction takes from 15 minutes to 25 hours to complete.

16. The process of claim 15, wherein the total reaction takes 35 minutes to complete.

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