PROCESS OF MAKING N,N'-DIACETYL-L-CYSTINE

Abstract

An effective process of making N,N′-diacetyl-L-Cystine (“NDAC”), which process is fast, green, does not require labor-intensive isolation or purification of the product, by yielding products in desired ratio, and has improved yield and purity. The process comprising the steps of Forming a reaction mixture, starting with a cystine derivative di-tert- butyl-L-cystine as the dihydrochloride form; Acetylating said di-tert-butyl-L-cystine to obtain N,N′-diacetyl-di-tert- butyl-L-cystine; followed by Removing said tert- butyl groups from said N,N′-diacetyl-di-tert-butyl- L-cystine to obtain N,N′-diacetyl-L-cystine product; and Isolating said N,N′-diacetyl-L-Cystine product from said reaction mixture; wherein said acetylating agent is acetic anhydride.

Claims

1-13. (canceled)

14. A process of making N-N′-diacetyl-L-cystine comprising: (a) forming a reaction mixture with a cystine derivative, wherein the cystine derivative is di-tert-butyl-L-cystine as a dihydrochloride form; (b) adding an acetylating agent to the reaction mixture and acetylating the di-tert-butyl-L-cystine to obtain N,N′-diacetyl-di-tert-butyl-L-cystine; (c) removing tert-butyl groups from the N,N′-diacetyl-di-tert-butyl-L-cystine to obtain N,N′-diacetyl-L-cystine; and (d) isolating the N,N′-diacetyl-L-cystine from the reaction mixture, wherein the acetylating agent is acetic anhydride.

15. The process according to claim 14, wherein the reaction mixture of step (a) is at a temperature of 5 to 50° C.

16. The process according to claim 14, wherein the reaction mixture of step (a) is at a temperature of 5 to 35° C.

17. The process according to claim 14, wherein the reaction mixture of step (a) is at a temperature of 10 to 25° C.

18. The process according to claim 14, wherein step (b) further comprises adding a base, and stirring a mixture comprising the acetylating agent, the base and di-tert-butyl-L-cystine at a temperature from 5 to 50° C.

19. The process according to claim 18, wherein the stirring is at a temperature of 5 to 35° C.

20. The process according to claim 18, wherein the stirring is at a temperature of 10 to 25° C.

21. The process according to claim 18, wherein the mixture is stirred from 1 to 24 hours, and until N,N′-diacetyl-di-tert-butyl-L-cystine precipitates out of the mixture.

22. The process according to claim 14, wherein step (c) further comprises forming a suspension comprising N,N′-diacetyl-di-tert-butyl-L-cystine and an acid, and stirring the suspension at a temperature from 20 to 100° C.

23. The process according to claim 22, wherein the suspension is stirred from 1 to 24 hours, and until N,N′-diacetyl-L-cystine is formed.

24. The process according to claim 18, wherein the base is an inorganic base.

25. The process according to claim 18, wherein the base is selected from the group consisting of (i) an alkali metal bicarbonate or carbonate, (ii) alkaline earth metal bicarbonate or carbonate, (iv) alkali metal hydroxide, and (iii) mixtures thereof.

26. The process according to claim 18, wherein the base is selected from the group consisting of sodium bicarbonate, sodium carbonate, sodium hydroxide, calcium hydroxide, calcium oxide, and mixtures thereof.

27. The process according to claim 18, wherein the base is sodium bicarbonate.

28. The process according to claim 22, wherein the acid is selected from the group consisting of formic, sulfuric, phosphoric or hydrochloric acid, and mixtures thereof.

29. The process according to claim 22, wherein the acid is formic acid.

30. The process according to claim 14, wherein, step (d) further comprises adding a water-immiscible organic solvent to the N,N′-diacetyl-L-cystine and stirring vigorously to effect precipitation and/or solidification into a white solid.

31. The process according to claim 30, wherein the water-immiscible organic solvent is ethyl acetate.

32. The process according to claim 14, wherein step (d) comprises centrifugation or filtration.

33. The process according to claim 14, wherein the process is carried out in a single vessel.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0042] 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.”

[0043] 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.

[0044] 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.

[0045] The present invention provides a two-step process to prepare N,N′-diacetyl-L-Cystine (“NDAC”) from a cystine derivative. In a general aspect, the process comprises the steps: [0046] Starting with a cystine derivative di-tert-butyl-L-cystine ester (1) as the dihydrochloride form, [0047] Acetylating to obtain N,N′-diacetyl-di-tert-butyl-L-cystine (2), followed by [0048] Removing the tert-butyl groups and isolating N,N′-diacetyl-L-Cystine (“NDAC”) product, wherein said acetylating agent is acetic anhydride.

[0049] Firstly, the present invention obviates the needs of the prior art by providing a chemical process to prepare N,N′-diacetyl-di-tert-butyl-L-cystine (2) using water as the solvent, takes less than 16 hours, does not need any extractive workup or purification, the product is isolated by simple filtration and the yields are high ( greater than 88%). The starting material is di-tert-butyl-L-cystine (1), a commercially available white solid.

[0050] Additionally, the present invention obviates the needs of the prior art by providing a chemical process to prepare N,N′-diacetyl-L-cystine (NDAC) using formic acid as both the reagent and solvent that can be recycled. The reaction takes less than three hours, does not need any extractive workup or purification, the product is isolated upon solvent evaporation with no need for purification, and the yields are high (about 97%).

[0051] In another aspect, the present invention includes a chemical process of making N,N′-diacetyl-L-Cystine (“NDAC”), the process comprising the steps: [0052] Starting with a derivative of L-cystine, Di-tert-butyl Ester (1) as the dihydrochloride salt, which is commercially available; [0053] Acetylating the precursor in aqueous medium in the presence of acetic anhydride and sodium bicarbonate; [0054] Removing the tert-butyl groups using formic acid as both a reagent and a solvent under heating conditions; [0055] Isolating N,N′-diacetyl-L-Cystine (“NDAC”) product from the reaction mixture. [0056] All amino acids were L stereo isomers. L-Cystine (>98%) was purchased from Sigma. N,N′-diacetyl-L-cystine (95%) was purchased from CombiBlocks. Di-tert-butyl-L-cystine dihydrochloride (DTBC; 98%) was purchased from Bachem.

Di-Tert-Butyl-L-Cystine (“DTBC”)

[0057] As a starting material for the inventive process, , Di-tert-butyl-L-cystine ester (1 or “DTBC”), which is commercially available as the dihydrochloride form in large quantities as a white powder solid, is chosen. DTBC is itself a glutathione precursor. However, the increased reactivity and labile primary free amine functionality of DTBC over NDAC makes it a less suitable candidate for cosmetic use and more suitable as a reagent. This starting reagent is also referred to herein as di-tert-butyl-L-cystine (1 or “DTBC”) or di-tert-butyl-L-cystine dihydrochloride.

N,N′-Diacetyl-L-Cystine (“NDAC”)

[0058] Applicants have discovered that N,N′-diacetyl-L-cystine (Also referred to as N,N′-diacetyl-L-cystine or NDAC) and NDAC salts, such as disodium salts, are useful amides in personal care compositions. NDAC structure is shown below (C.sub.10H.sub.16N.sub.2O.sub.6S.sub.2):

##STR00008##

[0059] The molecular weight of NDAC = 324.4.

[0060] In skin cosmetic compositions, NDAC and its disodium salts, when applied to skin, are converted to cystine and cysteine intracellularly. Cysteine in turn has a number of cosmetic uses, including as a glutathione precursor. NDAC has superior functional benefits, including ultimate delivery of cysteine for cosmetic uses. Additionally, NDAC is more stable than simple alternative cystine esters such a DMC or DEC and does not result in unpleasant sulfur odor.

[0061] N,N′-diacetyl-L-Cystine ( “NDAC”) may be prepared according to the inventive process.

[0062] According to another aspect, in the first step DTBC (1) is diacetylated so that N,N′-diacetyl-di-tert-butyl-L-cystine (2) is formed as described above. In the second step, N,N′-diacetyl-di-tert-butyl-L-cystine (2) is heated in the presence of formic acid which serves both as a solvent and reagent, thereby removing the tert-butyl group and resulting in NDAC product.

[0063] The inventive process is most useful for the synthesis of NDAC. As starting materials to prepare NDAC via its di-tert-butyl intermediate, DTBC (1) is used, which is a common and stable reagent available commercially in large quantities as the dihydrochloride salt. Upon reacting DTBC (1) with the acetylating agent acetic anhydride, N,N′-diacetyl-di-tert-butyl-L-cystine (2) is obtained. Next, N,N′-diacetyl-di-tert-butyl-L-cystine (2) is heated in the presence of formic acid which serves both as a solvent and reagent, thereby removing the tert-butyl group and resulting in NDAC product. The NDAC prepared according to the inventive process has superior functional benefits.

Inventive Procedure

[0064] As a general procedure, Step 1 of the inventive process is preparation of N,N′-diacetyl-di-tert-butyl-L-cystine (2), an intermediate for NDAC production.

[0065] Step 1 starts with preparing a solution of di-tert-butyl-L-cystine (1) (1 molar equivalent) as the dihydrochloride salt in water at temperatures ranging between 5-50° C. (preferably between 5-35° C., most preferably between 10-25° C.).

[0066] Step 1 includes adding the acetylating agent acetic anhydride (2-5 molar equivalents) to the solution..

[0067] Step 1 further includes adding an alkali metal or alkaline earth metal bicarbonate or carbonate (2-6 molar equivalents) to the solution, to form a mixture of di-tert-butyl-L-cystine (1), the acetylating agent, and alkali metal or alkaline earth metal bicarbonate or carbonate. Suitable are sodium bicarbonate or an alkali metal hydroxide such as sodium hydroxide (NaOH), or an alkaline earth metal hydroxide or oxide such as calcium hydroxide or calcium oxide. Preferred is sodium bicarbonate.

[0068] A reaction is allowed to occur by continuing to stir the mixture, at temperatures ranging between 5-50° C. (preferably between 5-35° C., most preferably between 10-25° C.). Stirring continues until all the di-tert-butyl-L-cystine (starting material) is consumed (typically between 1-24 h) and the desired N,N′-diacetyl-di-tert-butyl-L-cystine product precipitates out of solution.

[0069] The N,N′-diacetyl-di-tert-butyl-L-cystine (2) product (intermediate for NDAC) is filtered off and washed with water.

[0070] Step 2 of the inventive process is preparation of N,N′-diacetyl-L-Cystine (“NDAC”) , the final NDAC product.

[0071] Step 2 includes providing a suitable acid including, but are not limited to, for example, formic, sulfuric, phosphoric or hydrochloric acid, and mixtures thereof (preferably formic acid due to its non-toxic, biodegradable and reusable properties and suitability as both a solvent and reagent). Suitable concentration is between 0.5 ml-10 ml acid per mmol of the N,N′-diacetyl-di-tert-butyl-L-cystine (2).

[0072] Step 2 includes suspending N,N′-diacetyl-di-tert-butyl-L-cystine (2) (1 molar equivalent) in the acid to form a suspension.

[0073] Step 2 includes stirring the suspension at temperatures ranging between 20-100° C. (preferably between 40-80° C., most preferably between 50-70° C.) to generate a clear and colorless homogeneous solution. Stirring is continued until all the N,N′-diacetyl-di-tert-butyl-L-cystine (starting material for Step 2) is consumed (typically between 1-24 h) and the desired product N,N′-diacetyl-L-Cystine (“NDAC”) is generated.

[0074] The solvents are evaporated to give N,N′-diacetyl-L-Cystine (“NDAC”) as an amorphous solid.

[0075] Further, Step 2 may include purification and isolation of N,N′-diacetyl-L-cystine as a higher quality crystalline white solid, by adding a water-immiscible organic solvent such as for example ethyl acetate to the amorphous N,N′-diacetyl-L-cystine, stirring vigorously to effect precipitation/solidification into a white solid. The last step of the process, may include isolating the precipitated N,N′-diacetyl-L-Cystine (“NDAC”) product, by centrifugation or filtering, preferably by filtering.

[0076] All starting materials suitable for the inventive process are available from commercial sources, e.g. from Sigma-Aldrich, Bachem.

[0077] Particularly preferred in Step 1 is sodium bicarbonate, because it generates non-toxic sodium chloride as a by-product in solution which is easily separated from the product via filtration.

[0078] The relative amounts of the reagents and solvents are such as to not have excessive starting ingredients to minimize the amount of waste upon reaction completion.

[0079] Preferably the inventive process also comprises optional steps. For example optional step (1-A), wherein the water filtrate solution containing sodium acetate and sodium chloride by-products generated upon filtration of intermediate N,N′-diacetyl-di-tert-butyl-L-cystine (2) can be reused for additional cycles of step 1 or processed to recycle the water and independently isolate sodium acetate (or acetic acid) and sodium chloride for other uses. Optional step (2-A), wherein the formic acid used in step 2 is collected and recycled for additional cycles of step 2. Both of these optional steps allow for improved product turnover and reduced waste.

[0080] The inventive process is advantageous, at least because it does not use any toxic, hazardous or flammable organic solvents, results in the minimal formation of by-products, if any, does not generate product with undesirable sulfurous odor, both steps can be carried out in a single vessel and is relatively fast. It also results in improved purity of from 90% to 99.9%, preferably from 95 to 99.5%, and most preferably at least 95%, and improved overall yield from 85% to 95%, preferably from 90%, and most preferably at least 90% to 95%.

EXAMPLES

Experimental Methods

[0081] All reagents and solvents were obtained from commercial sources and used without further purification. All amino acids purchased were L stereo isomers. L-Cystine (>98%) was purchased from Sigma. N,N′-diacetyl-L-cystine (NDAC; 95%) was purchased from CombiBlocks. Di-tert-butyl-L-cystine dihydrochloride (DTBC; 98%) was purchased from Bachem.

[0082] 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 methods.

Example 1

[0083] An example of the process within the scope of the invention was performed.

[0084] A specific nonexclusive representative experimental procedure was carried out in two steps as follows.

Step 1 - N,N′-Diacetyl-Di-Tert-Butyl-L-Cystine (2)

[0085] Acetic anhydride (0.55 ml, 5.9 mmol) was added to a solution of di-tert-butyl-L-cystine (1) dihydrochloride (1 g, 2.4 mmol) in water (10 ml), followed by sodium bicarbonate (790 mg, 9.4 mmol) and the mixture stirred @ R.T. for 16 h at which point the product precipitated as a white solid.

[0086] At this time, TLC (100% ethyl acetate elution) showed the clean formation of a major product.

[0087] The product was filtered off and washed with water (3 X 10 ml) and dried under vacuum to give pure N,N′-diacetyl-di-tert-butyl-L-cystine as a white solid which was pure enough for the next step (905 mg, 88% yield). Other trials of this same process at various scales were completed in only 1.5 hours and gave yields of up to 95%. Product identity and purity (>95%) was confirmed by 1H NMR and LCMS.

Step 2 - N,N′-Diacetyl-L-Cystine (NDAC)

[0088] N,N′-diacetyl-di-tert-butyl-L-cystine (2) (500 mg, 1.15 mmol) was suspended in formic acid (1 ml) and heated at 60° C. for 3 h to generate a clear and colorless homogeneous solution.

[0089] At this time, TLC (30:40:30 ethyl acetate:isopropyl alcohol:water) showed the clean formation of a single product and no reactant/starting material.

[0090] The solvents were removed under reduced pressure at 60° C. to give a colorless glassy gel.

[0091] Optionally, the product was precipitated by adding ethyl acetate, filtered off and dried under high vacuum to give pure N,N′-diacetyl-L-Cystine (“NDAC”) as a white solid (350 mg, 97% yield). Acidic reagents other than formic acid (sulfuruc acid, acetic acid and Amberlyst® 15 hydrogen form) were also tested and gave successful conversion of NDAC. Product identity and purity (>95%) was confirmed by 1H NMR and LCMS via comparison with an NDAC (95% purity) authentic standard.

[0092] The specific experimental procedure is summarized in the chemical diagram below:

##STR00009##

Example 2

[0093] An example of the process within the scope of the invention was performed.

[0094] A specific nonexclusive representative experimental procedure was carried out in one step in a single vessel as follows.

##STR00010##

N,N′-Diacetyl-L-Cystine (NDAC)

[0095] Acetic anhydride (0.28 ml, 2.9 mmol) was added to a solution of di-tert-butyl-L-cystine (1) dihydrochloride (0.5 g, 1.2 mmol) in water (5 ml), followed by sodium bicarbonate (395 mg, 4.7 mmol) and the mixture stirred @ R.T. for 1.5 h at which point the product precipitated as a white solid.

[0096] At this time, TLC (100% ethyl acetate elution) showed the clean formation of intermediate N,N′-diacetyl-di-tert-butyl-L-cystine (2).

[0097] Formic acid (3 ml) was added and heated at 80° C. for 5 h to generate a clear and colorless homogeneous solution.

[0098] At this time, TLC (30:40:30 ethyl acetate:isopropyl alcohol:water) showed the clean formation of NDAC.

[0099] The solvents were removed under high vacuum at between 70-80° C. and the solid residue suspended in ethanol (6 mL), stirred for 10 min, filtered off and washed with ethanol (2 X 3 ml).

[0100] The combined ethanol filtrates were evaporated at 50° C. under high vacuum to give pure NDAC as a white solid (270 mg, 71% yield). Product identity and purity (>95%) was confirmed by TLC, 1H NMR and LCMS methods.

[0101] As demonstrated from the above procedure, the 2-step inventive process can be further simplified and conveniently carried out in a single vessel without the isolation of intermediate N,N′-diacetyl-di-tert-butyl-L-cystine (2). In this case isolation of the final product NDAC can be conveniently carried out by adding alcohol (to precipitate NaCl and sodium acetate salts) and simple filtration.

Comparative Example A

[0102] The following out-of-scope example illustrates criticality of the acetylating reagent under the inventive conditions. In this example acetyl chloride (an acid chloride) was used instead of acetic anhydride, resulting in no intermediate N,N′-diacetyl-di-tert-butyl-L-cystine product formation.

##STR00011##

Comparative Example A

[0103] Sodium bicarbonate (158 mg, 1.9 mmol) was added to a solution of di-tert-butyl-L-cystine (1) dihydrochloride (0.2 g, 0.5 mmol) in water (2 ml), followed by acetyl chloride (134 uL, 1.9 mmol) and the mixture stirred @ R.T. for 1.5 h at which point the solution remained a clear and colorless homogeneous solution.

[0104] At this time, TLC (100% ethyl acetate elution) did not show the formation of intermediate N,N′-diacetyl-di-tert-butyl-L-cystine (2) and only di-tert-butyl-L-cystine (1) dihydrochloride starting material was present.

[0105] As demonstrated from the example above, the nature of the acetylating agent is absolutely critical for the success of the inventive process. Equally important, this out-of-scope example further illustrates the uniqueness of the inventive process, considering that acid chlorides have been used as acetylating reagents to prepare other N,N′-dialkanoyl-di-tert-butylcystines from di-tert-butylcystine (see for example Liebigs Annalen der Chemie (1987) 895-9; Journal of Inorganic Biochemistry (2011) 105, 880-886).

Comparative Example B

[0106] For comparison purposes, a process was carried out in accordance with European Journal of Organic Chemistry (2008) 26, 4417-4425.

Comparative Example B - N,N′-Diacetyl-Di-Tert-Butyl-L-Cystine (2)

[0107] Acetic anhydride (2.7 mL, 29 mmol) was added to a suspension of di-tert-butyl-L-cystine dihydrochloride (2.48 g, 5.83 mmol) in pyridine (25 mL; anhydrous) and cooled to 0° C. After stirring the mixture to generate a homogeneous solution and allowing the exothermic reaction to subdue, the reaction mixture was placed at 4° C. for 2d. Ice was added, followed by portionwise addition of hydrochloride acid (12.1 M,25 mL) to maintain the temp below 20° C. and the pH between 4-5. The reaction mixture was extracted with ether (4 X 100 mL) and the combined organic layers washed with aqueous sulfuric acid (0.5 M) until pH between 2-3, followed by saturated sodium bicarbonate. After drying the organic layer and removing the solvent, the residue was dissolved in chloroform (20 mL) and hexane added until cloudiness persisted. The product was allowed to crystallize at 4° C. overnight, filtered and dried to give 2.16 g (85%).

[0108] As can be seen from the procedure above, the non-aqueous process to generate N,N′-diacetyl-di-tert-butyl-L-cystine (2) intermediate took much longer (3 days total) than the inventive process (1.5 to 16 hours), involved the use of toxic and flammable organic solvents (pyridine, ether, chloroform and hexane) versus the inventive process (water), resulted in slightly decreased yield (85%) versus the inventive process (88 to 95%) and was harder to purify (extractive work-up and crystallization) versus inventive process (filtration and water washing).