Technical Field The invention relates to a process for preparing obeticholic acid with a reduced number of synthetic steps.

Background Art Obeticholic acid, i.e. (3a,53,6a,7<x)-6-ethyl-3,7-dihydroxycholanic acid, of structural formula VII

was first described in Yu, D. et al., Steroids 2012, 77(13): 1335, Pellicciari, R. et al., in J Med Chem 2002, 45(17): 3569 and in the international patent application WO 2002/072598 as an agonist of the farnesoid X receptor (FXR).

Synthesis of obeticholic acid has been described in literature as well as in numerous patent documents. The synthesis described in these documents is based on 3-a-hydroxy-7-keto^-cholanic acid of formula I

and proceeds via a protected derivative by alkylation, hydrogenation and reduction to the ethyl ester,

which is then hydrolyzed to obeticholic acid. However, this method provides a very low yield of the final compound.

Synthesis of obeticholic acid has been described in other literature as well as in numerous patent documents. The document WO 2006/122977 (Erregierre S.p.A.) discloses a synthetic method that starts with the same compound of formula I

and proceeds via the methyl ester and its silyl derivative and silyl enol ether,

from which the methyl ester is obtained by reaction with an aldehyde, catalyzed by a Lewis acid, which then provides obeticholic acid by hydrolysis, hydrogenation and reduction. The problem of alkylation of the methyl ester within this synthesis is that it partly provides the ethyl derivative in position a with respect to the carboxyl instead of the 6-ethyl derivative unless partial thermal desilylation in position a is conducted in the previous step. The same cannot be monitored well by chromatography or other analytic methods common in interoperational monitoring of technological processes. In addition, the yield is low again, which is also supported by easy hydrolysis of the 3-TMS protecting group during alkylation (quick hydrolysis by Lewis acids). Further, retroaldolization occurs during the alkylation - Lewis acids also catalyze the backward step of the alkylation balance.

Synthesis using the methyl ester as an intermediate is also described in the documents CN 105399793, CN 105646633, CN 105566429, WO 2017/027396, CN 106083971, CN 106279335 or WO 2013/192097.

Synthesis with the use of the benzyl ester as an intermediate is for example described in the documents CN 105481925, CN 104876995, CN 105585603 and CN 106083970.

It would be advantageous to have an efficient synthesis method making it possible to obtain obeticholic acid with high conversion, with a minimal or no share of retroaldolization and deprotection.

Disclosure of the Invention

It has now been found that the above-mentioned problems will be solved by a process, in which a suitable amide of 7-keto-lithocholic acid is used instead of the ester as the starting compound, said 7-keto-lithocholic acid amide being at the same time protected with a dialkylcarbamoyl group in position 3, wherein the alkyls in the amido and carbamyol groups are the same and the two groups are introduced in one step.

The essence of the present invention consists in conversion of the 7-keto-litocholic acid amide protected in position 3 of formula II, wherein R ¹ , R ² are a Ci - C ₆ n-alkyl, or together a C ₅ - C ₆ cycle (for example piperidide, pyrrolidide, morpholide), to 7-trimethylsilyl enol ether of formula III, which is then converted by reaction with an alkylation agent to the 6- ethylidene derivative of formula IV, which is reduced to the compound of formula V and then simultaneously hydrolyzed and deprotected in position 3 to the compound of formula VI and then reduced to obeticholic acid of formula VII.

The amide of formula II is prepared in such a way to protect position 3 by carbamoylation at the same time in one common step (which will shorten the process).

In particular, various steps of the process can be carried out for instance as follows:

Step 1 - The conversion of 7-keto-litocholic acid to the amide with simultaneous protection of the hydroxyl in position 3 can be carried out by reaction of this acid, after suitable activation, with the respective amine. The activated intermediate is prepared by reaction of 7-keto-lithocholic acid with, e.g., DCC-carbonyl diimidazole, hydroxybenzotriazole, or by another method described in the amide preparation literature (Houben-Weyl: Methoden der Organischen Chemie, Bd. E5, G. Thieme, Stuttgart 1985) under the conditions described therein. CDI and a suitable amine can be preferably used because the carbamate protection in position 3 can then be achieved in one step. Thus, one step is saved and the final deprotection is also carried out together with the hydrolysis of the amide. Step 2 -The preparation of 7-silyl enol ether is conducted via a lithio or sodio enolate by means of an alkali amide, while the basicity of the secondary amine used for preparing the amide must be lower than the basicity (pKa) of LDA and higher than pKa of the ketolithocholic acid 7-enolate itself. The absolute value of pKA depends on the environment (solvent used), advantageously ethers, especially THF. This criterion is met by, e.g., HMDS, diphenylamine, ditolylamine, dinaphthylamine, hexamethyldisilazane, /V-methylaniline, N- methyltoluidine, /V-isopropylaniline, /V-tertbutylaniline and other secondary aromatic and silylated amines, preferably HMDS and diphenylamine.

Step 3 - The 6-ethylation can be conducted with common alkylation agents: ethyl bromide, ethyl iodide, ethyl triflate, ethyl mesylate, ethyl tosylate, diethyl sulphate, preferably EtJ and Et triflate with an auxiliary base, e.g. a tertiary amine, preferably HUnig's base. The working methods are known to experimenters skilled in the art.

An alternative comprises two-stage alkylation (aldolization with acetaldehyde and its derivatives) and subsequent reduction, which provides higher yields, does not work with alkylation agents, which are carcinogenic, but adds one step (reduction in the synthetic process). Acetaldehyde or its cyclic derivatives (metaldehyde, paraldehyde) in DCM or ethers are used, preferably in DCM, being catalyzed with strong Lewis acids (BF3 etherate, TiCl and SnCl ₄ ), preferably TiCI ₄ in DCM. The process is conducted at low temperatures, in the range of -80 to -20°C, preferably -60 to -40°C, to prevent retroaldolization. The aldolization is fast, and at the same time slow dehydration to the 6-ethylidene derivative proceeds, which is accelerated by addition of ACN, catalysis with J2, HCI, TMSCI, NaHS0 ₃ , ZnCI ₂ and other weak Lewis acids, preferably with a combination of ACN and ZnCI ₂ .

The reaction is stopped and back retroaldolization is blocked by complexation of TiCI ₄ by means of Et ₃ N or DMF, preferably Et ₃ N. Titanium salts soluble in the organic phase are removed by precipitation either as Ti0 ₂ or Ti ⁴⁺ -2DMF or by extraction with a-hydroxy acids into the aqueous phase (tartaric, hydroxyacetic, lactic, citric and other acids, best tartaric acid).

Step 4 - The hydrogenation is conducted in an autoclave on a Pd catalyst (5-10% Pd/C, with or without Cu modification) under the pressure of 1-10 atmospheres, best 5% Pd/C and 3 atmospheres at RT to 100°C, in a solution of methanol, HCI or HAC, best at 40 to 60°C in HAC. After completed hydrogenation the catalyst is filtered off and returned to recycling and the solution is evaporated or neutralized and extracted, after dilution with water, to the organic phase.

Step 5 - The hydrolysis of the amide and deprotection in position 3 is conducted by alkaline hydrolysis (KOH, NaOH, KOt-Bu, Cs ₂ C0 ₃ , KOEt and NaOEt). The hydrolysis is carried out in higher boiling alcohols miscible with water under reflux (n-butanol, amyl alcohol, hexanol, cyclohexanol, ethylene glycol, propylene glycol, ethyl cellosolve, best glycols, KOH, 120°C). After completion of the hydrolysis, it is carefully neutralized and acidified to the free acid and extracted with an organic solvent (DCM, toluene, MTBE, EtOAc). The final product (6-ethyl-7- ketoacid) can be purified by crystallization in the form of salts with amines (diethylamine, diisopropylamine, dibutylamine, te/t-butylamine, octylamine, tert-octylamine, cyclohexylamine, dicyclohexylamine, morpholine, benzylamine, dibenzylamine, N- benzylmethylamine, (S)-a-methylbenzylamine, (R)-a-methylbenzylamine, preferably t- octylamine, (S)-a-methylbenzylamine).

Step 6 - The process of final reduction with NaBH is identical with the basic patent (US7138390). The final product can be crystallized via a t-octylamminium salt in an environment of methanol or isopropanol.

The invention is clarified in more detail in the working examples below, which, however, do not restrict its scope.

Examples

Example 1: - Preparation of the 3-dimethylcarbamoyl-7-keto-lithocholic acid dimethylamide

In a 500 ml flask annealed in vacuo and purged with nitrogen, 16.6 g (102.4 mmol, 2 equiv.) of I, -carbonyldiimidazole was slowly added to 20 g (51.2 mmol, 1 equiv.) of 7-keto- litocholic acid (I) in 120 ml of dichloromethane. The reaction was conducted in an inert atmosphere (N ₂ ), C0 ₂ being vigorously generated during the reaction. After 1 hour (monitoring with LC/MS) 12.5 g of dried dimethylamine hydrochloride (153.6 mmol, 3 equiv.) was added to the reaction mixture, rinsed with a small amount of dichloromethane, and 21.4 ml of triethylamine (153.6 mmol, 3 equiv.) was added dropwise. The reaction mixture was stirred overnight (16 hours; the reaction half-life is ca. 2 hours). The reaction with dimethylamine can be accelerated by heating under reflux.

After 16 hours, the reaction mixture was stirred with 150 ml of distilled water (5 min), the phases were separated. The organic layer was then shaken with 150 ml of 1M HCI, 150 ml of H ₂ 0, 150 ml of saturated NaHC0 ₃ , 150 ml H ₂ 0 and 150 ml of brine. The clear solution was dried with IMa ₂ S0 ₄ , filtered and the filtrate was concentrated using a rotary vacuum evaporator (hereinafter RVE only), which provided a white amorphous product (lie). The crude reaction yield was almost quantitative. Crystallization of the intermediate (lie) from 60 ml of cyclohexane and 6 ml of toluene. Yield 20.5 g (82%) - white crystalline substance. HRMS (ESI+) calculated for C ₂₉ H ₄₉ N ₂ 0 ₄ [M+H] ⁺ 489.3692, found 489.3688. Example 2 - Preparation of 7-keto-lithocholic acid B- /V ^' -pentamethylenocarbamoyl piperidide

In a 500 ml flask annealed in vacuo and purged with nitrogen, 16.6 g (102.4 mmol, 2 equiv.) of I, -carbonyldiimidazole was slowly added to 20 g (51.2 mmol, 1 equiv.) of the 7-keto- litocholic acid (I) in 120 ml of dichloromethane. The reaction was conducted under an inert atmosphere (N ₂ ), C0 ₂ being vigorously generated during the reaction. After 1 hour (LC/MS check) 15.2 ml (153.6 mmol, 3 equiv.) of piperidine was added to the reaction mixture, and rinsed with a small amount of dichlormethane. The reaction mixture was shaken with 3x 150 ml of distilled water after 1 hour, and the phases were separated. The organic phase was shaken with 150 ml of brine; the resulting clear solution was dried with Na ₂ S0 ₄ , filtered, and the filtrate was concentrated using a rotatory vacuum evaporator (hereinafter RVE only), affording a white amorphous solid (lid). The crude yield of the reaction was almost quantitative. Crystallization of the intermediate (lid) from 60 ml of cyclohexane and 6 ml of toluene. Yield 26.8 g (92%) - white crystalline solid. HRMS (ESI+) calculated for C _2g H ₄₉ lM ₂ 0 ₄ [M+H] ⁺ 568.424, found 568.431.

Example 3 - Preparation of the TMS-enol ether

In a 500 ml flask annealed in vacuo and purged with nitrogen, 100 ml of dry THF was charged through a septum together with 20.59 ml of hexamethyl disilazane (98.2 mmol, 2.4 equiv.). The mixture was cooled down to -70°C, and at this temperature, 39.3 ml (98.2 mmol 2.4 equiv.) of n-BuLi was added dropwise (reaction mixture temperature < -60°C) in an inert atmosphere. After completion of the addition of butyllithium, the stirring and cooling continued for 30 minutes. A solution of 20 g (40.92 mmol, 1 equiv.) of the 7-keto-litocholic acid amide (lie) in 100 ml of dry THF was added dropwise to lithium bis(trimethylsilyl) amide in THF at the temperature of -80°C. Then, 12.47 ml of trimethyl silyl chloride (98.2 mmol, 2.4 equiv.) was added dropwise to the mixture and the stirring and cooling continued for 60 minutes.

200 ml of water was poured to the mixture, the mixture was removed from the bath and stirred for 10 minutes. The layers were separated, the aqueous layer was shaken with 3x 50 ml of toluene. The combined organic fractions were shaken with 150 ml of a solution of NaHC0 ₃ (saturated) : H ₂ 0 (1:1), 150 ml of H ₂ 0 and 150 ml of brine. Dried with Na ₂ S0 ₄ , the desiccant was filtered off and the filtrate was concentrated using an RVE at T _max = 30°C to obtain an oily evaporation product. The crude reaction yield was almost quantitative. The product (lllc) is a white amorphous substance. Crystallization of the intermediate from 65 ml of cyclohexane. Yield 21.1 g (92%) - white crystalline substance. HRMS (ESI+) calculated for C ₃₂ H ₅₇ N ₂ 0 ₄ Si [M+H] ⁺ 561.4088, found 561.4094.

The preparation of the lithioenolate can also be carried out with the commercially available lithium bis(trimethylsilyl) amide.

Example 4 - Alkylation of the TMS-enol ether

A 1L sulfonation flask with a mechanical stirrer was annealed in vacuo and purged with nitrogen. After cooling off the flask, 120 ml of dried dichloromethane (dried using an A4 molecular sieve) and 1.75 ml (13.2 mmol, 0.37 equiv.) of paraldehyde were charged in a nitrogen atmosphere. The solution was cooled down to -60°C. Then, 19.55 ml of TiCI ₄ (178.3 mmol, 5 equiv.) was added dropwise and a solution of 20 g (35.66 mmol, 1 equiv.) of the TMS-enol ether (lllc) in 120 ml of dichloromethane (R.m. T maintained at -60 to -55°C) was added dropwise to the slightly yellowish suspension. The primarily produced suspension got dissolved, providing an orange to brown solution.

After 10 min from the addition of the TMS-enol ether (lllc), the reaction was stopped by adding a solution of 39.76 ml (285.27 mmol, 8 equiv.) of triethylamine in 200 ml of acetonitrile.

The temperature of the reaction mixture was adjusted to the room temperature within 15 min and then 1.93 ml of distilled water (106.97 mmol, 3 equiv.) and 486 mg (3.57 mmol, 10 mol %) of zinc chloride were added. In 30 min after the addition of ZnCI ₂ , the product got completely dehydrated to (IVb). 300 ml of MTBE and 300 ml of water were poured to the mixture, stirred, the phases were separated and the organic layer was stirred with 14.12 ml (106.97 mmol, 3 equiv.) of triethanolamine for 10 min. 200 ml of water were then added to the mixture, and stirred for 10 min. The phases were separated, the organic layer was stirred with 4x 350 ml of 20% tartaric acid (4x 20 min). Then, the organic phase was shaken with 300 ml of water, 300 ml of a saturated solution of NaHC0 ₃ , 300 ml of water and 300 ml of brine. Dried with Na ₂ S0 . The desiccant was filtered off and the filtrate was concentrated in an RVE. Crude reaction yield 15.2 g (83%) of (IVb). The crude intermediate was purified by stirring with Al ₂ 0 ₃ and then with active charcoal. Crystalization was made from the mixture diisopropylether : cyclohexane (10 : 1) in the ratio of 3 ml of solvent per 1 g of the substance. HRMS (ESI+) calculated for C ₃ IH ₅ IN ₂ 0 ₄ [M+H] ⁺ 515.3849, found 515.3855.

Example 5 - Alkylation of the TMS-enol ether

lllc IVb

120 ml of dry DCM and 1.75 ml (13.2 mmol, 0.37 equiv.) of paraldehyde were charged in an annealed 1L sulfonation flask in a nitrogen atmosphere. The solution was cooled down to

-60°C. Then, 47.3 ml of BF ₃ Et ₂ 0 (>46.5 BF ₃ basis, 178.3 mmol, 5 equiv.) was added and subsequently a solution of 20 g (35.66 mmol, 1 equiv.) of the TMS-enol ether (lllc) in 120 ml of DCM was added dropwise. After stirring for 30 min at T = -60°C, the temperature of the mixture was adjusted to the room temperature and the mixture was stirred for another 1 hour. The mixture was cooled down to 0°C, diluted with 150 ml of distilled water and extracted with toluene (3x 100 ml). The combined organic fractions were shaken with 150 ml of water, 150 ml of a saturated solution of NaHC0 ₃ , 150 ml of water and 150 ml of brine. Dried with Na ₂ S0 ₄ , the desiccant was filtered off and the filtrate was concentrated in an RVE. The yield was 15 g (82%) of crude (IVb). The crude intermediate was purified by stirring with Al ₂ 0 ₃ and then with active charcoal. Crystallization was made from the mixture diisopropylether : cyclohexane (10 : 1) in the ratio of 3 ml of solvent per 1 g of the substance. HRMS (ESI+) calculated for C ₃ IH ₅ IN ₂ 0 ₄ [M+H] ⁺ 515.3849, found 515.3855. Example 6 - Hydrogenation of the 6-ethylidene amide (IVb)

IVb V

A solution of 20 g (38.85 mmol, 1 equiv.) of (IVb) in 180 ml of MeOH and 40 ml H ₂ 0 was charged in an autoclave, 3.4 ml of 35% HCI (38.85 mmol, 1 equiv.) and 2 g of Pd/C (5% by weight) were added. The autoclave was pressurized 3 times and purged with N ₂ (10 atm), 3x

H ₂ (10 atm). At the pressure of 10 atm, the mixture was hydrogenated for 3 hours at T = 40°C. The catalyst was filtered off on a paper filter, rinsed with 20 ml of MeOH. The filtrate was heated at T = 50°C for 1 hour until the "undesired" b-epimer disappeared. MeOH was evaporated from the mixture in an RVE and then 100 ml of MTBE and 100 ml of H ₂ 0 were added to the mixture and the mixture was extracted. The aqueous phase was shaken with 2x

50 ml of MTBE, the combined organic phases were washed with 100 ml of water, 100 ml of brine, dried with Na ₂ S0 ₄ , the desiccant was filtered off and the filtrate was concentrated in an RVE. Reaction yield 14.5 g (72%) of (V). HRMS (ESI+) calculated for C3iH _{53 2} 04 [M+H] ⁺ 517.3994, found 517.4000.

Example 7 - Hydrogenation of the 6-ethylidene amide (IVb)

A solution of 20 g (38.85 mmol, 1 equiv.) of (IVb) in 140 ml of 80% AcOH was charged in an autoclave together with 2 g of 5% Pd/C (10% by weight). The autoclave was pressurized 3 times and purged with N ₂ (3 atm), 3x H ₂ (3 atm). At the pressure of 3 atm, the mixture was hydrogenated at T = 50°C for 2 hours. The catalyst was filtered off on a paper filter, rinsed with 20 ml of 80% AcOH. The filtrate was diluted with water, neutralized with a 2M solution of NaOH to pH 5-6, then extracted 3 times with 70 ml of MTBE. The combined organic phases were washed with 100 ml of water, 2x 100 ml of a 2M solution of NaOH, 100 ml of water and 100 ml of brine. Dried with Na ₂ S0 , the desiccant was filtered off and the filtrate was concentrated in an RVE. Reaction yield 15.9 g (79%) of (V). HRMS (ESI+) calculated for C ₃₁ H5 ₃ N2O ₄ [M+H] ⁺ 517.4000, found 517.4004.

Example 8 - Hydrolysis of the amidic groups of (V)

V VI

20 g (38.7 mmol, 1 equiv.) of (V) in 200 ml of ethylene glycol was dissolved in a 500ml flask and then 13 g (232.2 mmol, 6 equiv.) of KOH was added. The mixture was heated under reflux at 120°C for 5 hours, then cooled down to r.t. and neutralized with 2M HCI to pH = 7. The reaction mixture was extracted with 3x 60 ml of EtOAc. The combined organic fractions were washed with 2x 100 ml of water, 100 ml of brine and dried with Na ₂ S0 ₄ . The desiccant was filtered off and the filtrate was concentrated using an RVE. Reaction yield 14.1 g (87%) of crude (VI). Crystallization of the intermediate via the amminium salt: 10 ml MeOH, 1.02 equiv. of (S)-a-methylbenzylamine for 1 g of (VI); 10 ml of H ₂ 0 added under reflux. Then, the mixture was refluxed for 30 minutes, and left to cool down freely. The product after isolation was converted back to the free acid via the sodium salt with NaOH, then extracted, using 1M HCI, into EtOAc. The solvent was concentrated to the ratio 3 g of EtOAc : 1 g of (VI); after cooling down, a white crystalline substance was isolated. HRMS (ESI+) calculated for C ₂₆ H ₄₃ O ₄ [M+H] ⁺ 419.3156, found 419.3161.

Example 9 - Hydrolysis of the amidic groups of (V)

V VI 20 g (38.7 mmol, 1 equiv.) of (V) was dissolved in 150 ml of 1-butanol in a 500ml flask and then 13 g (232.2 mmol, 6 equiv.) of KOH was added. The mixture was refluxed under a condenser for 10 hours, then cooled down to r.t.. 330 ml of distilled water and 200 ml of cyclohexane were added, shaken, phases separated and the aqueous layer was additionally shaken with 2x 100 ml of cyclohexane. Then the aqueous layer was neutralized with 2M HCI to pH = 2-3. The reaction mixture was extracted with lx 150 ml and 2x 50 ml of EtOAc. The combined organic fractions were washed with 2x 100 ml of water, 100 ml of brine and dried with Na ₂ S0 ₄ . The desiccant was filtered off and the filtrate was concentrated using an RVE. Reaction yield 13.5 g (83%) of crude (VI). Crystallization of the intermediate via the amminium salt: 10 ml MeOH, 1.02 equiv. of (S)-a-methylbenzylamine for 1 g of (VI), 10 ml of H ₂ 0 added under reflux. Then, the mixture was refluxed for 30 minutes, and left to cool down freely. The product after isolation was converted back to the free acid via the sodium salt with NaOH, and then extracted, using 1M HCI, into EtOAc. The solvent was concentrated to the ratio of 3 g of EtOAc : 1 g of (VI); after cooling down, a white crystalline substance was isolated. HRMS (ESI+) calculated for C ₂₆ H430 ₄ [M+H] ⁺ 419.3156, found 419.3162.

Example 10 - Hydrolysis of the amidic groups of (V)

V VI

20 g (38.7 mmol, 1 equiv.) of (V) was dissolved in 150 ml of 1-pentanol in a 500 ml flask and then 13 g (232.2 mmol, 6 equiv.) of KOH was added. The mixture was refluxed under a condenser for 3 hours, then cooled down to r.t., diluted with 50 ml of heptane and 50 ml of water. A heptane layer, product layer and aqueous layer were formed. The oily product was diluted with 150 ml of EtOAc, washed with 2x 100 ml of water, 100 ml of brine, dried with Na ₂ S0 ₄ . The desiccant was filtered off and the filtrate was concentrated using an RVE. Reaction yield 14.6 g (90%) of crude (VI). Crystallization of the intermediate via the amminium salt: 10 ml MeOH, 1.02 equiv. of (S)-a-methylbenzylamine for 1 g of (VI); 10 ml of H ₂ 0 added under reflux. Then, the mixture was refluxed for 30 minutes, and left to cool down freely. The product after isolation was converted back to the free acid via the sodium salt with NaOH, then extracted, using 1M HCI, into EtOAc. The solvent was concentrated to the ratio 3 g of EtOAc : 1 g of (VI); after cooling down, a white crystalline substance was isolated. HRMS (ESI+) calculated for C ₂₆ H ₄₃ 0 ₄ [M+H] ⁺ 419.3156, found 419.3159. Example 11 - Reduction of the 7-keto group of (VI) with NaBH ₄

VI VII

20 g (47.78 mmol, 1 equiv.) of (VI) was dissolved in 200 ml of a 50% solution of ethanol with water in a 500 ml round flask. 3.82 g (95.55 mmol, 2 equiv.) of NaOH was added. Then, 7.23 g (191.11 mmol, 4 equiv.) of NaBH ₄ was added in parts. The mixture was refluxed at the temperature of 80°C for 3 hours. Then it was cooled down to r.t., acidified with 2 M HCI to pH = 2-3, the temperature of the reaction mixture being kept below 30°C all the time. Then 100 ml of / ^' - PrOAc was added, shaken, phases separated; the aqueous layer was extracted with 2x 50 ml of / ^' -PrOAC. The combined organic extracts were washed with 4x 50 ml of distilled water and dried with Na ₂ S0 ₄ . After filtration, 3x 50 ml of MaOH was gradually added during evaporation of the filtrate in order to azeotropically remove excess /-PrOH in an RVE (T = 40°C). Subsequently, the mixture was additionally diluted with methanol such that 60 ml of

MeOH come to theoretical 20.1 g. Purification of the final product was made by crystallization in the form of a salt with 1,1,3,3-tetramethylbutylamine. 3 ml of MeOH, 1.02 equiv. of 1, 1,3,3- tetramethylbutylamine (tert-octylamine) for 1 g of (VII); 10 ml of H ₂ 0 added under reflux. Then, the mixture was refluxed for 30 minutes, and left to cool down freely. The product after isolation was converted back to the free acid via the sodium salt with NaOH, then extracted into /-PrOAc using 1M HCI. The organic phase was washed with 3x 100 ml of water, lx 100 ml of brine, and dried with Na ₂ S0 ₄ . The resulting filtrate was concentrated in an RVE to produce a dense oily evaporation residue, which was used for preparation of an amorphous product. HRMS (ESI+) calculated for C ₂₆ H ₄₅ 0 ₄ [M+H] ⁺ 421.3312 found 421.3316.