Process for the preparation of n-retinoyl-l-cysteic acid methyl esters

Abstract

The invention relates to an improved process for the preparation of an N-retinoyl-L-cysteic acid methyl ester, or a pharmaceutically acceptable salt thereof. The improvements relate to the order in which the reagents are added during the reaction, the size of subsequent additions, and the choice of the solvent, allowing the product to be obtained in higher yields and at lower costs. The improved process also significantly simplifies the workup of the reaction product.

Description

[0001] PROCESS FOR THE PREPARATION OF N-RETINOYL-L-CYSTEIC ACID METHYL ESTERS CROSS-REFERENCE TO RELATED APPLICATIONS 5 This applicaƟon claims priority to Swedish applicaƟon No.2430369-5,filed July 11, 2024, the disclosure of which is incorporated herein by reference in its enƟrety. TECHNICAL FIELD 10 The invenƟon relates to an improved process for the preparaƟon of an N-reƟnoyl-L-cysteic acid methyl ester, or a pharmaceuƟcally acceptable salt thereof. The improvements relate to the order in which the reagents are added during the reacƟon, the size of subsequent addiƟons, and the choice of the solvent, allowing the product to be obtained in higher yields and at lower costs. The improved process also significantly simplifies the workup of the reacƟon product. 15 BACKGROUND WO 2004 / 009538 discloses reƟnol derivaƟves that are able to form micelles and enhance the potency of cytotoxic agents through solubilisaƟon. The two compounds N-(all-trans-reƟnoyl)-cysteic acid 20 methyl ester sodium salt (XMeNa) and N-(13-cis reƟnoyl)-L-cysteic acid methyl ester sodium salt (13XMeNa) are currently used as micelle-forming agents in the product Apealea®, which is a solvent- free formulaƟon of paclitaxel. WO 2017 / 099662 discloses a method for producing derivaƟves of N-reƟnoylaminoalkane sulfonic acid, 25 including N-(13-cis-reƟnoyl)-cysteic acid methyl ester sodium salt and N-(all-trans-reƟnoyl)-cysteic acid methyl ester sodium salt. This method comprises mixing a reƟnoic acid, a chloroformate, a cysteic acid methyl ester and a base in an organic solvent, so that the reacƟon mixture comprises a single liquid phase wherein the product is formed. The organic solvent is preferably an aproƟc solvent, and most preferably THF. A similar method for the preparaƟon of N-(all-trans-reƟnoyl)-cysteic acid methyl 30 ester sodium salt is disclosed in Yang et al. (Pharmaceut. Fronts 2022, vol.4, e188-196). The method of WO 2017 / 099662 is currently used for the manufacturing of the sodium salts of N-(all- trans-reƟnoyl)-cysteic acid methyl ester sodium salt and N-(13-cis reƟnoyl)-L-cysteic acid methyl ester, but gives the products at best in only about 70 to 75% yield. Despite an excess of coupling reagents applied, about 20 to 30% of unreacted reƟnoic acid is typically leŌ at the end of the reacƟon. Also, the workup of the reacƟon product is Ɵme-consuming and requires large volumes of solvent. There is therefore a need for an improved process that results in higher yields of the desired product, and that simplifies the workup of the reacƟon product. 5 DETAILED DESCRIPTION OF THE INVENTION The synthesis of N-reƟnoyl-L-cysteic acid methyl ester from reƟnoic acid and L-cysteic acid methyl ester is shown in scheme 1 below. It comprises the formaƟon of an amide bond, but technically consists of 10 two consecuƟve steps. In afirst step, the reƟnoic acid is acƟvated by reacƟon with a coupling reagent such as a chloroformate, resulƟng in the formaƟon of a mixed anhydride. The mixed anhydride then reacts with cysteic acid methyl ester to afford thefinal product. AŌer workup of the reacƟon mixture under basic condiƟons, the product is isolated as the corresponding sodium salt. 15 Scheme 1. Synthesis of N-reƟnoyl-L-cysteic acid methyl ester It was found that the addiƟon of a large excess of coupling reagent at the very beginning of the reacƟon did not lead to a higher conversion of the starƟng material. Surprisingly, however, it was discovered 20 that a considerably higher conversion (>95%) of the starƟng material can be obtained if the coupling reagent is added in two porƟons, i.e., when a second porƟon of the coupling reagent is added to the reacƟon mixture aŌer an iniƟal reacƟon period. AddiƟonally, it was found that the size of the second porƟon was important for the yield of the reacƟon, as the addiƟon of a smaller second porƟon generally led to higher yields than the addiƟon of a larger second porƟon. With these improvements, 25 the product may be isolated in yields of 90 to 95%. In afirst aspect, therefore, the invenƟon relates to a process for the preparaƟon of an N-reƟnoyl-L- cysteic acid methyl ester, or a pharmaceuƟcally acceptable salt thereof, comprising the steps of: a) providing a soluƟon of a coupling reagent, a reƟnoic acid and a base in afirst solvent; b) adding a soluƟon of L-cysteic acid methyl ester and a base in a second solvent to the soluƟon of 5 step a), followed by reacƟng for at least 15 minutes; c) adding addiƟonal base and an addiƟonal soluƟon of the coupling reagent in thefirst solvent to the soluƟon of step b), followed by reacƟng for at least 5 minutes; d) adding an addiƟonal soluƟon of L-cysteic acid methyl ester and a base in the second solvent to the soluƟon of step c), followed by reacƟng for at least 30 minutes; and 10 e) isolaƟng the N-reƟnoyl-L-cysteic acid methyl ester, or the pharmaceuƟcally acceptable salt thereof, from the reacƟon mixture of step d). The coupling reagent is preferably an acyl chloride or a chloroformate, such as a compound of formula Cl-C(O)-R or Cl-C(O)-OR wherein R is straight or branched C1-8 alkyl or C5-8 cycloalkyl. Examples of15 suitable acyl chlorides include, but are not limited to, acetyl chloride and pivaloyl chloride (2,2- dimethylpropanoyl chloride). Examples of suitable chloroformates include, but are not limited to, isobutyl chloroformate, sec-butyl chloroformate, tert-butyl chloroformate, cyclopentyl chloroformate and cyclohexyl chloroformate. In some embodiments, the coupling reagent provides steric bulk due to the volume of the R-group. As shown in the experimental secƟon, it has been observed that an 20 increase of steric bulk of the coupling reagent leads to a higher conversion of reƟnoic acid. In a preferred embodiment, the chloroformate is isobutyl chloroformate. The base is preferably an organic base, more preferably an amine such as triethylamine, N,N- dimethylbenzylamine, imidazole or N,N-dimethylaniline. In some embodiments, the base provides 25 steric bulk. It has been discovered that an increase of steric bulk of the base leads to a higher conversion of reƟnoic acid. In one preferred embodiment, the base is triethylamine. In another preferred embodiment, the base is N,N-dimethylbenzylamine. ReƟnoic acid has several carbon-carbon double bonds, each of which may be present in either trans- 30 or cis-configuraƟon. The present invenƟon encompasses all geometric isomers of reƟnoic acid. In some embodiments, the reƟnoic acid used in step a) of the process disclosed herein is all-trans-reƟnoic acid, which results in the formaƟon of N-(all trans-reƟnoyl)-L-cysteic acid methyl ester. In some embodiments, the reƟnoic acid used in step a) is 13-cis-reƟnoic acid, which results in the formaƟon of N-(13-cis reƟnoyl)-L-cysteic acid methyl ester. In some embodiments, the N-reƟnoyl-L-cysteic acid methyl ester is isolated as a pharmaceuƟcally acceptable salt thereof. Examples of suitable pharmaceuƟcally acceptable salts include base-addiƟon salts, such as an alkali metal salt (e.g., a lithium, sodium or potassium salt), an alkaline earth metal salt 5 (e.g., a magnesium or calcium salt), a transiƟon metal salt (e.g., an iron or zinc salt), an ammonium salt, or a salt with an organic base which affords a physiologically acceptable caƟon, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2- hydroxyethyl)amine. In a preferred embodiment, the N-reƟnoyl-L-cysteic acid methyl ester is isolated as the sodium salt thereof. 10 In some embodiments, the invenƟon relates to a process for the preparaƟon of N-(13-cis reƟnoyl)-L- cysteic acid methyl ester. In some embodiments, the invenƟon relates to a process for the preparaƟon of the sodium salt of N-(13-cis reƟnoyl)-L-cysteic acid methyl ester. In some embodiments, the invenƟon relates to a process for the preparaƟon of N-(all trans-reƟnoyl)-L-cysteic acid methyl ester. 15 In some embodiments, the invenƟon relates to a process for the preparaƟon of the sodium salt of N- (all trans-reƟnoyl)-L-cysteic acid methyl ester. Thefirst solvent is preferably an aproƟc solvent selected from the group consisƟng of ethers, esters, amides, nitriles, sulfoxides, or a mixture of two or more of such solvents. More preferably, thefirst 20 solvent is an ether, such as tetrahydrofuran (THF), diethyl ether or methyl tert-butyl ether (MTBE). It is further preferred that thefirst solvent is not miscible with water. It has been found that the use of a water-immiscible solvent drasƟcally simplifies the workup procedure, as the Ɵme-consuming evaporaƟon of water-miscible solvents (such as THF) from an aqueous phase can be avoided. In a more preferred embodiment, therefore, thefirst solvent is MTBE, even though the solubility of reƟnoic acid 25 in this solvent is lower than in, e.g., THF. The second solvent is preferably a proƟc solvent such as an alcohol. Examples of suitable alcohols include methanol, ethanol, propanol and butanol. In a preferred embodiment, the second solvent is methanol. 30 Step a) It is preferred that of the coupling reagent and the base each are present in the soluƟon of step a) in slight excess to the reƟnoic acid, such as about 1.05 equivalents, about 1.1 equivalents, about 1.15 equivalents or about 1.2 equivalents relaƟve to the amount of reƟnoic acid. Step a) is preferably performed at a temperature of between about 0 and about 20 °C, and more preferably at a temperature of between about 5 and about 15 °C, such as between about 5 and about 10 °C, or such as between about 10 and about 15 °C. In some embodiments, step a) is performed at 5 about 5 °C, or at about 10 °C, or at about 15 °C. The soluƟon of step a) is preferably reacted for a period of about 5 to 60 minutes before the reagents of step b) are added, to allow the formaƟon of the mixed anhydride. In some embodiments, the soluƟon of step a) is reacted for at least about 5 minutes, such as at least about 10 minutes, such as at 10 least about 20 minutes, or such as at least about 30 minutes before the reagents of step b) are added. In some embodiments, the soluƟon of step a) is reacted for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes or about 30 minutes before the reagents of step b) are added. 15 Step b) The L-cysteic acid methyl ester is preferably added in slight excess to the reƟnoic acid, such as about 1.1 to 1.5 equivalents, e.g., about 1.1 equivalents, about 1.2 equivalents, about 1.3 equivalents, about 1.4 equivalents or about 1.5 equivalents relaƟve to the amount of reƟnoic acid. The base should also be added in excess to the reƟnoic acid, such as about 1.5 to 2 equivalents, e.g., about 1.5 equivalents, 20 about 1.6 equivalents, about 1.7 equivalents, about 1.8 equivalents, about 1.9 equivalents or about 2 equivalents relaƟve to the amount of reƟnoic acid. The soluƟon of step b) is preferably reacted for a period of about 15 minutes to 2 hours minutes before the addiƟonal reagents of step c) are added. In some embodiments, the soluƟon of step b) is reacted 25 for at least about 15 minutes, such as at least about 30 minutes, such as at least about 45 minutes, or such as at least about 1 hour before the addiƟonal reagents of step c) are added. In some embodiments, the soluƟon of step b) is reacted for about 30 minutes to 2 hours, about 30 minutes to 1.5 hours, about 30 minutes to 1 hour, about 1 to 2 hours, about 1 to 1.5 hours, or about 1.5 to 2 hours before the addiƟonal reagents of step c) are added. In some embodiments, the soluƟon of step b) is 30 reacted for about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours or about 2 hours before the addiƟonal reagents of step c) are added. Step c) The addiƟonal coupling reagent is preferably added in about 0.3 to 0.7 equivalents, more preferably about 0.4 to 0.6 equivalents, e.g., about 0.4 equivalents, about 0.5 equivalents or about 0.6 equivalents relaƟve to the amount of reƟnoic acid. The addiƟonal base is preferably added in about 5 0.3 to 0.7 equivalents, more preferably about 0.4 to 0.6 equivalents, e.g., about 0.4 equivalents, about 0.5 equivalents or about 0.6 equivalents relaƟve to the amount of reƟnoic acid. The soluƟon of step c) is preferably reacted for a period of about 5 to 30 minutes before the addiƟonal reagents of step d) are added. In some embodiments, the soluƟon of step c) is reacted for at least 10 about 5 minutes, such as at least about 10 minutes, such as at least about 15 minutes, or such as at least about 20 minutes, before the addiƟonal reagents of step d) are added. In some embodiments, the soluƟon of step c) is reacted for about 5 to 15 minutes, or about 15 to 30 minutes before the addiƟonal reagents of step d) are added. In some embodiments, the soluƟon of step c) is reacted for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 15 30 minutes before the addiƟonal reagents of step d) are added. Step d) The addiƟonal L-cysteic acid methyl ester is preferably added in about 0.3 to 0.7 equivalents, more preferably about 0.4 to 0.6 equivalents, such as about 0.4 equivalents, about 0.5 equivalents or about 20 0.6 equivalents relaƟve to the amount of reƟnoic acid. The addiƟonal base is also preferably added in about 0.3 to 0.7 equivalents, more preferably about 0.4 to 0.6 equivalents, such as about 0.4 equivalents, about 0.5 equivalents or about 0.6 equivalents relaƟve to the amount of reƟnoic acid. The soluƟon of step d) is preferably reacted for a period of about 30 minutes to 4 hours, to allow the 25 reacƟon to go to compleƟon. In some embodiments, the soluƟon of step d) is reacted for at least about 30 minutes, such as at least about 1 hour, such as at least about 1.5 hours, such as at least about 2 hours, such as at least about 3 hours or such as at least about 4 hours. In some embodiments, the soluƟon of step d) is reacted for about 1 to 4 hours, about 1 to 3 hours, about 1 to 2 hours, about 1 to 1.5 hours, about 1.5 to 4 hours, about 1.5 to 3 hours, about 1.5 to 2 hours, about 2 to 4 hours, about 30 2 to 3 hours, or about 3 to 4 hours. In some embodiments, the soluƟon of step d) is reacted for about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours or about 4 hours. It has been found that the presence of oxygen and light leads to the formaƟon of certain oxidaƟon and / or reducƟon products, resulƟng in lower yields of the desired product. In some embodiments, therefore, the process described herein is performed under inert condiƟons, such as under a nitrogen or argon atmosphere. In some embodiments, the process is protected from light. In a more preferred embodiment of the invenƟon, the process comprising the steps of: 5 a) providing a soluƟon of isobutyl chloroformate, a reƟnoic acid and triethylamine in MTBE; b) adding a soluƟon of L-cysteic acid methyl ester and triethylamine in methanol to the soluƟon of step a), followed by reacƟng for at least 30 minutes; c) adding addiƟonal triethylamine and an addiƟonal soluƟon of isobutyl chloroformate in MTBE to the soluƟon of step b), followed by reacƟng for at least 5 minutes; 10 d) adding an addiƟonal soluƟon of L-cysteic acid methyl ester and triethylamine in methanol to the soluƟon of step c), followed by reacƟng for at least 30 minutes; and e) isolaƟng the sodium salt of the N-reƟnoyl-L-cysteic acid methyl ester from the reacƟon mixture of step d). 15 As also shown in the appended examples, the process disclosed herein allows thefinal products to be obtained in yields of up to 95%, which is significantly higher than what can be obtained using the process of WO 2017 / 099662. AddiƟonally, because of the lower amount of unreacted reƟnoic acid leŌ in the reacƟon mixture, the workup is considerably simplified. The use of MTBE as the aproƟc solvent instead of THF further simplifies the workup, as the solvent evaporaƟon step is completely removed, 20 thereby drasƟcally reducing the workup Ɵme. The enƟre preparaƟon of XMeNa or 13XMeNa (reacƟon and workup) according to the process disclosed herein therefore only takes 1 day, instead of 2-3 days according to previous processes. DefiniƟons 25 Unless otherwise defined, all technical and scienƟfic terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invenƟon belongs. Methods and materials are described herein for use in the present invenƟon; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustraƟve only and not 30 intended to be limiƟng. All publicaƟons, patent applicaƟons, patents, sequences, database entries, and other references menƟoned herein are incorporated by reference in their enƟrety. In case of conflict, the present specificaƟon, including definiƟons, will control. As used herein, the term “pharmaceuƟcally acceptable” refers to those compounds, materials, composiƟons and / or dosage forms that are suitable for human or veterinary pharmaceuƟcal use and that are generally safe, non-toxic and neither biologically nor otherwise undesirable. 5 As used herein, the term “reacƟng” is to be interpreted as allowing the starƟng materials and / or reagents to react with each other, thereby transforming the starƟng materials into intermediates or the desired products. The term “reacƟng” should furthermore be interpreted to include condiƟons such as “agitaƟng”, “sƟrring”, “shaking”, “cooling”, “heaƟng” or “refluxing” the reacƟon mixture. 10 As used herein, the term “comprising” is to be interpreted as including, but not being limited to. As used herein, the term “about” refers to a value or parameter herein that includes (and describes) embodiments that are directed to that value or parameter per se. For example, descripƟon referring to “about 20” includes descripƟon of “20”. Numeric ranges are inclusive of the numbers defining the 15 range. Generally speaking, the term "about” refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. The invenƟon will now be described by the following examples which do not limit the invenƟon in any 20 respect. All cited documents and references menƟoned herein are incorporated by reference in their enƟreƟes. EXPERIMENTAL SECTION 25 AbbreviaƟons aq aqueous BnNMe2N,N-dimethylbenzylamine eq equivalent(s) HPLC high-performance liquid chromatography 30 IBCF isobutyl chloroformate LAME L-cysteic acid methyl ester MTBE methyl tert-butyl ether SM starƟng material TEA triethylamine General methods All solvents used were of analyƟcal grade. Commercially available anhydrous solvents were rouƟnely 5 used for reacƟons. StarƟng materials were available from commercial sources or prepared according to literature procedures. Room temperature refers to 20 - 25 °C. AnalyƟcal HPLC was performed using a Chromaster HPLC-system. Analyses were performed using a equipped with a Hypurity C18 column, 250 x 4.6 mm, 3 µm (Thermo ScienƟfic). Mobile phase: solvent 10 A – 10% NH4OAc (aq), 10 % SDS (aq) and 80% acetonitrile; solvent B – 10% NH4OAc (aq), 10 % SDS (aq) and 80% water. Flow rate: 1 mL / min. Column temperature: 20 °C. UV detecƟon: 350 nm and 310 nm. Run Ɵme: 65 minutes. Pump program: Time (min) Solvent A % Solvent B % 0 42.5 57.5 45 82 18 46 42.5 57.5 65 42.5 57.5 EXAMPLES 15 Example 1 EvaluaƟon of different coupling reagents A set of reacƟons was performed using different coupling reagents. In a typical experimental run, all 20 trans-reƟnoic acid (1.0 eq) was dissolved in THF in the presence of triethylamine (1.1 eq). The soluƟon was cooled to 10-15 °C and treated with a slight excess (1.1 eq) of a coupling reagent. The obtained mixture was then reacted with 1.3 eq cysteic acid methyl ester at room temperature, and the reacƟon was allowed to proceed for about 2 to 3 hours. A sample was then taken from the reacƟon mixture and the composiƟon was determined by HPLC. The amount of product (XMeNa) and unreacted starƟng 25 material (all trans-reƟnoic acid) are shown in Table 1. Table 1. ComposiƟon* of the reacƟon mixture, area % Entry Coupling reagent XMeNa all trans-reƟnoic acid 1 Isobutyl chloroformate 74.1 25.5 2 Sec-butyl chloroformate 87.5 10.8 3 Cyclopentyl chloroformate 56.7 15.1 4 Cyclohexyl chloroformate 86.0 13.6 5 Pivaloyl chloride 69.3 30.0 * As determined by HPLC. Results are the average values of 2 or 3 experiments. The results show that increasing of steric hindrance of the coupling reagent provides noƟceable 5 improvements in the conversion of reƟnoic acid. In parƟcular, the use of reagents with bulky groups such as sec-butyl and cyclohexyl proved to be beneficial for the formaƟon of XMeNa. Example 2 EvaluaƟon of different bases 10 A set of reacƟons was performed using two different coupling reagents (isobutyl chloroformate or sec- butyl chloroformate) and two different bases (TEA or BnNMe2). ReacƟons were performed on a 1.8 g scale, and conducted at room temperature without protecƟon from oxygen. 15 In a typical experimental run, all trans-reƟnoic acid (1.0 eq) was dissolved in THF in the presence of the base (1.1 eq). The soluƟon was treated with a slight excess (1.1 eq) of the coupling reagent. The obtained mixture was then reacted with 1.3 eq L-cysteic acid methyl ester and base at room temperature, and the reacƟon was allowed to proceed for about 2 to 3 hours. A sample was then taken from the reacƟon mixture and the composiƟon was determined by HPLC. The amount of product 20 (XMeNa) and unreacted starƟng material (all trans-reƟnoic acid) are shown in Table 2. Table 2. ComposiƟon* of the reacƟon mixture, area % Entry Coupling reagent Base XMeNa all trans-reƟnoic acid 1 Isobutyl chloroformate TEA 70.1 29.5 2 Sec-butyl chloroformate TEA 82.7 16.5 3 Isobutyl chloroformate BnNMe2 78.9 21.0 4 Sec-butyl chloroformate BnNMe2 89.0 10.4 * As determined by HPLC. Results are the average values of 2 experiments. The results show that increasing of steric hindrance of the base resulted in higher conversion of the 5 starƟng material and a higher yield of the product. Example 3 EvaluaƟon of excess of isobutyl chloroformate 10 Several reacƟons were performed using different amounts of reagents in steps b) to d), and with different delays before addiƟon of the reagents of step c). ReacƟons were performed on a 1.8 g scale, and conducted at room temperature without protecƟon from oxygen. In a typical experimental run, all trans-reƟnoic acid (1.0 eq) was dissolved in THF in the presence of 15 triethylamine (1.1 eq). The soluƟon was treated with a slight excess (1.1 eq) of the coupling reagent. The obtained mixture was then reacted with L-cysteic acid methyl ester and base at room temperature, and addiƟonal amounts of coupling reagent, base and L-cysteic acid methyl ester were added as indicated in Table 3. The reacƟon was allowed to proceed for about 2 to 3 hours. A sample was taken from the reacƟon mixture and the composiƟon was determined by HPLC. The amounts of product 20 (XMeNa) and unreacted starƟng material (all trans-reƟnoic acid) are shown in Table 3.

[0002] Based on the results of these experiments, it was concluded that a second addiƟon of base (0.6 eq) and coupling reagent (0.5 eq) aŌer 1 hour was most effecƟve. 5 Example 4 PreparaƟon of XMeNa and 13XMeNa on semi-large scale Three batches of XMeNa and one batch of 13XMeNa were synthesized on a 36 g scale. The reacƟons were protected from light and oxygen during the whole process. 10 A soluƟon of isobutyl chloroformate (17.2 mL, 1.1 eq) in MTBE (30 mL) was dropwise added to a chilled (5-10°C) soluƟon of the appropriate reƟnoic acid (36.1 g) and TEA (18.4 mL, 1.1 eq) in MTBE (400 mL) under inert condiƟons (N2) and the resulƟng mixture was sƟrred for 30 minutes at 5-10 °C. The resulƟng mixed anhydride was used directly into the next step. 15 A soluƟon of L-cysteic acid methyl ester (29.29 g, 1.3 eq) and TEA (33 mL, 2.0 eq) in methanol (90 mL) was added to the mixed anhydride and the resulƟng soluƟon was sƟrred under nitrogen at room temperature for 1 hour. An addiƟonal TEA (10 mL, 0.6 eq) was slowly added to the soluƟon, followed by a soluƟon of isobutyl chloroformate (8 mL, 0.5 eq) in MTBE (30 mL). The resulƟng mixture was 20 sƟrred for 15 minutes at room temperature. An addiƟonal soluƟon of L-cysteic acid methyl ester (13.5 g, 0.6 eq) and TEA (10 mL, 0.6 eq) in methanol (90 mL) was then added to the mixture. The mixture was sƟrred for 1.5 to 2 hours under nitrogen at room temperature. ExtracƟon 1 25 AceƟc acid (10 mL) was carefully added to the resulƟng soluƟon. The organic layer was extracted with water (500 mL) and the aqueous layer was separated. The extracted organic layer was extracted again with 200 mL water and 50 mL of methanol. AŌer layer separaƟon. the organic layer was discarded and the combined aqueous layers were washed with MTBE (200 mL). The aqueous layer was kept for the next extracƟon. 30 ExtracƟon 2 and washing Sodium hydrogen carbonate (42g) was added to the aqueous layer from extracƟon 1. When effervescence had stopped, brine (25% NaCl-soluƟon, 200 mL) was added and the resulƟng aqueous soluƟon was extracted with ethyl acetate (400 mL). The organic layer was separated, and the water phase was discarded. The organic layer was washed twice with a mixture of brine (12.5% NaCl-soluƟon, 400 mL) and methanol (50 mL). The aqueous layers were then discarded, and the organic layer was evaporated under reduced pressure (40-70 mbar at max-jacket temperature 39 °C). The release of the vacuum was done with nitrogen gas. The residue containing crude product was dissolved in methanol 5 (160 mL). Chromatographic purificaƟon of the crude product was performed using a LaPrep HPLC E-318 instrument equipped with stainless-steel columns (E-474 / E-475), and the purified material was concentrated using a Büchi rotary evaporator (E-301, E-302, E-303, Bergman-Labora). 10 The composiƟon of the crude reacƟon mixtures (before workup) and the isolated yields are shown in Table 4. Table 4. Entry ComposiƟon* of the reacƟon mixture, StarƟng materialarea %Isolated yield (%) Product StarƟng material 1 All trans-reƟnoic acid 98.0 0.26 93.2 (XMeNa) 2 ** All trans-reƟnoic acid 97.0 0.84 84.0 (XMeNa) 3 All trans-reƟnoic acid 98.6 0.43 93.0 (XMeNa) 4 13-cis-reƟnoic acid 98.8 0.18 93.0 (13XMeNa) 15 * As determined by HPLC. ** 250 instead of 90 mL methanol used forfirst addiƟon of L-cysteic acid methyl ester and TEA

Claims

CLAIMS 1. A process for the preparaƟon of an N-reƟnoyl-L-cysteic acid methyl ester, or a pharmaceuƟcally acceptable salt thereof, comprising the steps of: 5 a) providing a soluƟon of a coupling reagent, a reƟnoic acid and a base in afirst solvent; b) adding a soluƟon of L-cysteic acid methyl ester and a base in a second solvent to the soluƟon of step a), followed by reacƟng for at least 15 minutes; c) adding addiƟonal base and an addiƟonal soluƟon of the coupling reagent in thefirst solvent to the soluƟon of step b), followed by reacƟng for at least 5 minutes; d) adding an addiƟonal soluƟon of L-cysteic acid methyl ester and a base in the second solvent to the soluƟon of step c), followed by reacƟng for at least 30 minutes; and e) isolaƟng the N-reƟnoyl-L-cysteic acid methyl ester, or the pharmaceuƟcally acceptable salt thereof, from the reacƟon mixture of step d).

2. The process according to claim 1, wherein the coupling reagent is a chloroformate.

3. The process according to claim 2, wherein the chloroformate is isobutyl chloroformate.

4. The process according to any one of claims 1 to 3, wherein the base is triethylamine.

5. The process according to any one of claims 1 to 4, wherein the addiƟonal coupling reagent in step c) is added in about 0.3 to 0.7 equivalents, more preferably about 0.4 to 0.6 equivalents relaƟve to the amount of reƟnoic acid.

6. The process according to any one of claims 1 to 5, wherein the addiƟonal L-cysteic acid methyl ester in step d) is added in about 0.3 to 0.7 equivalents, more preferably about 0.4 to 0.6 equivalents relaƟve to the amount of reƟnoic acid.

7. The process according to any one of claims 1 to 6, wherein the reƟnoic acid is all-trans-reƟnoic acid.

8. The process according to any one of claims 1 to 6, wherein the reƟnoic acid is 13-cis-reƟnoic acid.

9. The process according to any one of the preceding claims, wherein the N-reƟnoyl-L-cysteic acid methyl ester is isolated as the sodium salt thereof.

10. The process according to any one of the preceding claims, wherein the sodium salt of N-(13-cis 5 reƟnoyl)-L-cysteic acid methyl ester or the sodium salt of N-(all trans-reƟnoyl)-L-cysteic acid methyl ester is prepared.

11. The process according to any one of the preceding claims, wherein thefirst solvent is MTBE.

12. The process according to any one of the preceding claims, wherein the second solvent is methanol.

13. The process according to any one of the preceding claims, wherein step a) is performed at a temperature of between about 0 and about 20 °C, preferably at a temperature of between about 5 and about 15 °C.

14. The process according to any one of the preceding claims, wherein the soluƟon of step a) is reacted for at least 30 minutes.

15. The process according to any one of the preceding claims, wherein the soluƟon of step b) is reacted for at least 60 minutes.

16. The process according to any one of the preceding claims, wherein the soluƟon of step c) is reacted for at least 15 minutes.

17. The process according to any one of the preceding claims, wherein the soluƟon of step d) is reacted for at least 1.5 hours.

18. The process according to any one of the preceding claims, wherein the process is performed under inert condiƟons.

19. The process according to any one of the preceding claims, wherein the process is protected from light.

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