Title - PROCESS FOR THE PREPARATION OF A NITRIC OXIDE DONOR PROSTAGLANDIN ANALOGUE

Abstract

Claim 1: A process for the preparation of (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of 6-(nitrooxy)hexanoic acid of formula (1), said process characterized in that it comprises the following steps: a) reacting the compound of formula (2) with 6-(nitrooxy)hexanoyl chloride of formula (4) in the presence of free 4-dimethylaminopyridine, to obtain the compound of formula (3); b) removing the boronate protecting group from the compound of formula (3) to obtain the compound of formula (1).

Description

PROCESS FOR THE PREPARATION OF A NITRIC OXIDE DONOR PROSTAGLANDIN ANALOGUE Field of invention The present invention relates to an improved process for the large-scale preparation of (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-l-yl]-3,55 dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6-(nitrooxy)-hexanoic acid of formula (I). Background of the invention The (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-l-yl]-3,5-dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6-(nitrooxy)hexanoic acid 10 of formula (I) is a prostaglandin analogue that has been shown to be effective as an IOP-lowering agent (Impagnatiello F, Toris CB, Batugo M, Prasanna G, Borghi V, Bastía E, Ongini E, Krauss ΑΗΡ; Invest Ophthalmol Vis Sci. 2015; 56:6558-64). A process for preparing compound of formula (I) is described in document WO 2009 / 136281. Document WO 2009 / 136281 specifically describes the synthesis of compound (I) and, in general, the preparation of 15-alkyl nitrate esters of IF-2019-42693428-APN-ANP#INPI Page 1 of 21 bimatoprost. WO 2009 / 136281 describes the synthesis of the compound of formula (I) (Example B1) by reacting bimatoprost in a boronate-protected form (compound of formula (II)) with 6-bromohexanoyl chloride to form the 15-(65 bromohexanoyl) ester of bimatoprost in a boronate-protected form (compound of formula (VIII)) which is converted to the nitrate derivative by silver nitrate in acetonitrile and deprotected / purified under reversed-phase chromatography to obtain the compound of formula (I). The main disadvantages of the above synthesis are the use of more than an equimolar amount of 6-bromohexanoyl chloride in the esterification reaction, which presents a structural alert for potential mutagenicity, and, in the final step, the use of silver nitrate, which generates a large amount of silver salts in wastewater. Another major disadvantage of this process is the formation of impurities and by-products such as 15-(6-bromohexanoyl) bimatoprost ester (compound (IX)) and 15-(6-chlorohexanoyl) bimatoprost ester (compound (X)), which are difficult to extract even after multiple purifications, as they have similar polarity in chromatography and lipophilicity and / or solubility to compound (I). IF-2019-42693428-APN-ANP#INPI Page 2 of 21 either According to the procedure described in WO 2009 / 136281, the 15-(6-bromohexanoyl) ester of bimatoprost (compound (IX)) is an impurity derived from the incomplete reaction of compound (VIII) with silver nitrate, after removal of the boronate protection. The 15-(6-Chlorohexanoyl) ester of bimatoprost (compound (X)) is a by-product formed by the halogen exchange reaction between the bromine atom of the 15-(6-bromohexanoyl) ester of bimatoprost in a boronate-protected form (compound (VIII)) and the free chlorine anion from the 4-dimethylaminopyridine hydrochloride formed during the esterification step. The 15-(6-chlorohexanoyl) ester of bimatoprost in a boronate-protected form (Villa) (Figure 3 - Scheme 3) does not react with silver nitrate and, after removal of the protecting group, yields compound (X). WO 2009 / 136281 also describes an alternative process for the preparation of 15-acylalkylnitrate bimatoprost derivatives (Examples N1 and 10). The synthesis comprises reacting bimatoprost in a boronate-protected form (compound of formula (II)) with an alkyl carboxylic acid nitrate chloride in the presence of resin-supported 4-dimethylaminopyridine (DMAP) (PS-DMAP), followed by removal of the boronate protecting group and purification using silica gel chromatography. IF-2019-42693428-APN-ANP#INPI Page 3 of 21 The above process avoids the use of 6-bromohexanoyl chloride and the removal of silver salts from the final product. However, this method has another major drawback: the use of resin-supported 4-dimethylaminopyridine, which makes the process unsuitable for commercial scaling and costly. Furthermore, the alkyl carboxylic acid nitrate chloride is added in two successive steps and in high excess with respect to the compound of formula (II); in fact, the alkyl carboxylic acid chloride is added in an amount of approximately 2 to 4 equivalents. WO 2009 / 136281 also describes another process (Examples Q1) for the preparation of 15-acylalkylnitrate bimatoprost derivatives. In this process, the compounds were obtained by esterification of bimatoprost in a boronate(II)-protected form with an excess of nitrate-alkyl-(p-nitrophenyl)-carboxylate in the presence of 4-dimethylaminopyridine. The main disadvantages of this process are the removal of unreacted nitrate-alkyl-(p-nitrophenyl)-carboxylate and the by-product p-nitrophenol, formed in equimolar amounts to the compound of formula (I), using chromatographic methods. WO 2016 / 155906 discloses 15-nitrooxy derivatives of fluprostenol and reports the synthesis of the 15-nitrooxy-hexyl ester of the isopropyl ester of fluprostenol. The compound was prepared by reacting the isopropyl ester of fluprostenol in a boronate-protected form with (4-nitrophenyl)-6-nitrooxyhexanoate in the presence of excess 4-dimethylaminopyridine. As previously reported, the removal of unreacted nitrate-alkyl-(p-nitrophenyl)carboxylate and, especially, the removal of the p-nitrophenol by-product by chromatographic methods are the main disadvantages of this process. In recent years, various regulatory authorities have emphasized purity requirements and the identification of impurities in active pharmaceutical ingredients (APIs). Currently, any impurity is IF-2019-42693428-APN-ANP#INPI Page 4 of 21. Considered as organic material, in addition to the active ingredient, impurities can influence the efficacy and safety of pharmaceutical products. Therefore, the identification and quantification of each impurity, especially those with structural mutagenic potential, have become mandatory regulatory requirements. Furthermore, since the products are intended for pharmaceutical use, the range of industrially acceptable reagents, solvents, catalysts, etc., that can be used in the synthesis of the active ingredient is limited to those acceptable within the pharmaceutical industry. The compound of formula (I) is an oil, and its purification on a large scale is difficult because it cannot be crystallized; therefore, the presence of impurities is a fundamental problem for large-scale production. Since the main sources of impurities are the intermediates and by-products of the synthesis, the purity of the intermediates and the control of the reaction conditions are important requirements for obtaining a final product with acceptable pharmaceutical purity. Prior art processes for the preparation of the compound of formula (I) have some disadvantages; e.g., the use of bromohexanoyl chloride and the reaction conditions lead to the formation of the by-product 15-(6-chlorohexanoyl)bimatoprost ester (compound (X)) which has a structural alert of potential mutagenicity; the use of silver nitrate for the preparation of the nitrate-alkyl carboxylic acid chloride intermediate or for the nitration of 15-(6-bromohexanoyl)bimatoprost ester in a boronate-protected form (compound (VIII)) leads to the handling of a large amount of wastewater with silver nitrate; in addition, the metal content in the active pharmaceutical ingredients must meet specific acceptance criteria. Therefore, there is a need to provide a compound of formula (I) with high purity and good performance. The compound of formula (I) has been found to be able to be prepared with high IF-2019-42693428-APN-ANP#INPI Page 5 of 21 purity using the intermediate 6-(nitrooxy)hexanoyl chloride prepared efficiently by means of caprolactone ring-opening reaction followed by nitration. The present invention provides a large-scale production process that yields a compound of formula (I) having high chemical purity and, in particular, with an impurity content of 15-(6-chlorohexanoyl) bimatoprost ester below the safety level. Description of the invention The object of the present invention is a process for the preparation of (1S,2E)-310[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1(2-phenylethyl)-2-propen-1-yl ester of 6-(nitrooxy)hexanoic acid of formula (I): where said process comprises the following steps: a) react the compound of formula (II): IF-2019-42693428-APN-ANP#INPI Page 6 of 21 (Π) with 6-(nitrooxy)hexanoyl chloride of formula (IV): or (IV) in the presence of free 4-dimethylaminopyridine, to obtain the compound of formula (III): b) removing the boronate protecting group from the compound of formula (III) to obtain the compound of formula (I). 4-Dimethylaminopyridine (DMAP) in free form means DMAP not bound to a resin. Step a) is preferably carried out in an aprotic organic solvent, preferably selected from methyl tert-butyl ether, N,N-dimethylformamide or IF-2019-42693428-APN-ANP#INPI Page 7 of 21 dichloromethane. Preferably, the organic solvent is methyl tert-butyl ether. The molar ratio between the compound of formula (II) and the 6(nitrooxy)hexanoyl chloride of formula (IV) preferentially ranges from 1:1.4 to 1:1.6. The molar ratio between the compound of formula (II) and 4-dimethylaminopyridine preferentially varies from 1:2.0 to 1:2.4. The reaction in step a) is carried out at a temperature ranging from 0 °C to room temperature. The removal of the boronate protecting group (step b) is preferably carried out by reaction with methanol at a temperature of 17 °C to 24 °C. 6-(nitrooxy)hexanoyl chloride of formula (IV) is preferably obtained by a process comprising the following steps: i) react 2-caprolactone of formula (V): (V) with an inorganic base selected from KOH, NaOH and LiOH to obtain the salt of 6-hydroxyhexanoic acid of formula (VI): (VI) where M is K, Na or Li. ii) nitrate the compound of formula (VI) with a mixture of HNO3 and H2SO4 to obtain 6-(nitrooxy)hexanoic acid of formula (VII) ONE. IF-2019-42693428-APN-ANP#INPI Page 8 of 21 (VII) iii) convert 6-(nitrooxy)hexanoic acid of formula (VII) with a chlorinating reagent into 6-(nitrooxy)hexanoyl chloride of formula (IV). The 6-(nitrooxy)hexanoyl chloride of formula (IV) obtained in step iii) can be reacted directly with the compound of formula (II) in step a) without further purification. The inorganic base used in step i) is preferably potassium hydroxide. Step i) is preferably carried out in a solvent selected from methanol, ethanol or isopropanol; most preferably methanol. Steps ii) and iii) are carried out in dichloromethane. The chlorination reagent used in step iii) is oxalyl chloride. The compound of formula (II) is obtained by reacting bimatoprost with butylboronic acid. Preferably, the reaction is carried out in methyl tert-butyl ether as the solvent. A preferred process for the preparation of compound (I) is described in more detail in Schemes 1 and 2 (Figures 1 and 2), wherein said process comprises: step 1) react 2-caprolactone of formula (V) with potassium hydroxide in methanol to obtain potassium salt of 6-hydroxyhexanoic acid (compound of formula (VI) where M is potassium); step 2) react the potassium salt of 6-hydroxyhexanoic acid with a mixture of HNO3 and H2SO4 in dichloromethane to obtain 6-(nitrooxy)hexanoic acid (compound of formula (VII)); step 3) react 6-(nitrooxy)hexanoic acid with oxalyl chloride to obtain 6-(nitrooxy)hexanoyl chloride (compound of formula (IV)) which is used without further purification; Step 4) React bimatoprost with butylboronic acid (1.1-1.8 eq) in IF-2019-42693428-APN-ANP#INPI Page 9 of 21 methyl tert butyl ether (MTBE) at a temperature of approximately 40 °C; then remove the water by azeotropic distillation to obtain bimatoprost boronate (compound of formula (II)); Step 5) react bimatoprost boronate (compound of formula (II)) with 6-(nitrooxy)hexanoyl (IV) chloride, (1.4-1.6 equivalents) in methyl tert-butyl ether in the presence of 4-dimethylaminopyridine (2.0-2.4 equivalents) at a temperature ranging from 0 °C to approximately room temperature, to obtain the compound of formula (III); step 6) react the compound of formula (III) with methanol at room temperature to remove the protecting group to obtain the crude compound of formula (i)); Step 7) Purify the crude compound of formula (I) to obtain compound (I) having a chemical purity greater than 99%. The process of the invention is characterized in that the intermediate 6(nitrooxy)hexanoyl chloride (compound (IV)) is prepared with high chemical purity and high yield by means of the ring-opening reaction of 2-caprolactone. The experimental procedures for the steps of the invention process are described in detail below. All steps are performed under a nitrogen atmosphere. 6-(nitrooxy)hexanoyl(IV) chloride is prepared with high purity and high yield starting from 2-caprolactone (Figure 1 - Scheme 1); the synthesis comprises the following steps: Step 1) A solution of potassium hydroxide (1 equivalent) in methanol is added dropwise to a solution of 2-caprolactone (1 equivalent) in methanol; the mixture is cooled to approximately 5°C to 20°C and stirred for approximately 5 hours at 15°C to 20°C after the addition is complete; the IF-2019-42693428-APN-ANP#INPI Page 10 of 21 solvent is extracted (temperature equal to or less than 40 °C), the crude product is diluted in methyl tert-butyl ether, the potassium salt of 6-hydroxyhexanoic (VI) acid is filtered, washed with methyl tert-butyl ether and dried. The potassium salt of 6-hydroxyhexanoic (VI) acid was obtained with a yield of 95% and a purity of 98.5% (H-NMR and HC1 assay); Step 2) Potassium salt of 6-hydroxyhexanoic (VI) acid (1 eq) is added in portions to a mixture of HNO3 (4.6 eq) and H2SO4 (3.1 eq) in dichloromethane cooled to a temperature of 0 °C to 5 °C under nitrogen for approximately 30 min, maintaining the temperature below 10 °C; the resulting mixture is stirred for about 2-3 hours at a temperature of 0 °C to 10 °C, monitoring the end of the reaction by 'H-NMR analysis; the mixture is cooled to a temperature of 0 °C to 5 °C and added dropwise with a saturated aqueous solution of sodium chloride for approximately 20 min. The reaction mixture is maintained at a temperature below 10 °C; The organic layer is dried in anhydrous sodium sulfate, the solvent is extracted to obtain 6-(nitrooxy)hexanoic (VII) acid with a yield of 86-88% and an HPLC purity of 97%; Step 3) N,N-dimethylformamide and oxalyl chloride are added dropwise to a solution of 6-(nitrooxy)hexanoic (VII) acid in dichloromethane, keeping the solution temperature from 0 °C to 5 °C for 1 hour, then the mixture is stirred at a temperature of 15 °C to 30 °C for 24 hours; the solvent is evaporated to obtain 6-(nitrooxy)hexanoyl (IV) chloride in a yield of 88-97% which is used without further purification. The esterification process between bimatoprost and 6-(nitrooxy)hexanoyl chloride comprises the synthesis steps described below: Step 4) Bimatoprost is added to methyl tert-butyl ether, and the resulting solution is cooled to a temperature of approximately 15 °C to 18 °C; successively, n-Butylboronic acid (1.11–1.18 equivalents) is added in a portion, and the mixture is stirred for approximately 1–1.5 hours at 40 °C. The end of the reaction is monitored. IF-2019-42693428-APN-ANP#INPI Page 11 of 21 by *H NMR analysis; the reaction mixture is cooled to a temperature of approximately 20 °C to 25 °C, filtered, and the water formed is removed by azeotropic distillation of methyl tert-butyl ether at a temperature equal to or less than 40 °C until the water content is less than or equal to 0.25%, to obtain crude bimatoprost (II) boronate which is used directly in the next step; Step 5) Crude bimatoprost boronate is added to methyl tert-butyl ether, and the resulting solution is cooled to a temperature of approximately 0 °C to 5 °C. 4-Dimethylaminopyridine (approximately 2.1–2.3 equivalents) is added, and 6-(nitrooxy)hexanoyl chloride (compound (IV)) (1.5 equivalents) dissolved in methyl tert-butyl ether is added dropwise, maintaining the temperature of the mixture at approximately 0 °C to 5 °C. After the addition, the mixture is stirred at a temperature of approximately 0 °C to 5 °C for up to 4 hours and then overnight at 15 °C to 20 °C. The end of the reaction is monitored by HPLC analysis. (lS,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)7-oxohept-2-en-1 -yl]-2,4-dioxa-3-borabicyclo[3.2.1 ] oct-6-i 1} -1 -(2-phenylethyl)-prop-2-en-1 yl-6-(nitrooxy) hexanoate (compound (III)) is isolated by standard end-preparation methods (an example of preparation is described in Example 1); Step 6) Compound (III) is dissolved in methanol and the resulting solution is stirred for approximately 18 hours at a temperature of 17 °C to 25 °C; the conversion of compound (III) to compound (I) is monitored by *H NMR. In case the reaction stops, the mixture is evaporated and redissolved in fresh methanol until complete conversion; the reaction mixture is then concentrated under vacuum at a temperature below 40 °C and the crude compound (I) is isolated by standard preparation methods; Step 7) The crude product obtained (I) is purified by column chromatography using a silica gel column and a mixed solvent of dichloromethane and methanol to form compound (I) with an overall yield of more than 60% bimatoprost and a purity of 12 IF-2019-42693428-APN-ANP#INPI Page 12 of 21 of over 99%. The process of the invention provides a compound of formula (I) in high yield and purity while reducing the amount of by-products, in particular the amount of (S,E)-l-((lR,2R,3S,5R)-2-((Z)-7-(ethylamino)-7-oxohept-2-enyl)-3,5-dihydroxycyclopentyl)-5-phenylpent-l-en-3-yl 6-chlorohexanoate (compound (X)) which has a structural alert of potential mutagenicity). The above advantages make the invention process a cost-effective process, easily transferable to an industrial scale. Examples All the synthesis steps described below were performed under a nitrogen atmosphere. Example 1 Synthesis of (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-lyl]-3,5-dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6-(nitrooxy)hexanoic acid (I) (Batch I) Synthesis of 6-(nitrooxy)hexanoyl (IV) chloride Step 1: Synthesis of potassium salt of 6-hydroxyhexanoic acid (compound (VI)) A solution of potassium hydroxide (131.9 g, 0.98 eq.) in methanol (1250 ml, 5 vol.) was prepared under cooling at a temperature of 15–20 °C. 25.0 g of 2-caprolactone (1 eq.) and methanol (625 ml, 2.5 vol.) were introduced into a three-liter, three-necked round-bottom flask. The mixture was stirred until dissolved. A potassium hydroxide and methanol solution was added at a temperature of 5–20 °C within 0.5 hours. The mixture was stirred for 4.5 hours at a temperature of 15–20 °C. The reaction mixture was concentrated under vacuum (at a temperature of 40 °C or lower) to form the crude potassium salt of 6-hydroxyhexanoic acid (489.55 g). The crude salt was resuspended in methyl tert-butyl ether (1250 ml, 5 vol.) for 4 hours at a 13 IF-2019-42693428-APN-ANP#INPI Page 13 of 21. At a temperature of 20 °C to 25 °C, the product was filtered through a 3-pore filter, washed with methyl tert-butyl ether (2 x 250 ml, 2 x 1 vol.) and dried under vacuum (at a temperature equal to or less than 40 °C) to form the potassium salt of 6-hydroxyhexanoic acid (353.04 g) with a yield of 95.7%. Melting point 208 °C. The product was analyzed by 'H-NMR and HC1 assay. Step 2: Synthesis of 6-(nitrooxy)hexanoic (VII) acid This reaction is carried out on a 100 g scale to control the reaction temperature and reduce the time required to add the nitration mixture. The reaction is repeated to obtain the required amount of 6-(nitrooxy)hexanoic acid. Fuming HNO3 (4.6 eq.) was added to concentrated H2SO4 (3.1 eq.) at a temperature of 0 °C to 5 °C over 14 min, followed by the addition of CH2Cl2 (20 vol.) at a temperature of 0 °C to 5 °C over 12 min. The potassium salt of 6-hydroxyhexanoic acid (101.29 g, 1 eq.) was added in portions over 28 min at a temperature below 10 °C. The mixture was stirred for 2.2 hours at a temperature of 0 °C to 10 °C, and the reaction was monitored by *H-NMR, showing a conversion of 99.9%. The mixture was cooled to a temperature of 0 °C to 5 °C and a saturated aqueous solution of sodium chloride (286.71 g in 910 ml, 10 vol.) was carefully added at a temperature of 10 °C or lower over 17 min. After filtration of the insolubles, the organic layer was decanted, dried in sodium sulfate, and concentrated under vacuum (at a temperature of 40 °C or lower) to form 6-(nitrooxy)hexanoic acid with a yield of 87.7% and an HPLC purity of 97.0%. Step 3: Synthesis of 6-(nitrooxy)hexanoyl (IV) chloride 6-(Nitrooxy)hexanoic acid (230 g, 1 eq.) was dissolved in dichloromethane (150 mL, 5 vol.). The resulting solution was filtered through glass fibers, washed with dichloromethane (1 x 50 mL, 0.65 vol.), and analyzed by Karl Fischer titration (water content = 0.016%). The filtrate was cooled to 0–5 °C under nitrogen. IF-2019-42693428-APN-ANP#INPI Page 14 of 21 Subsequently, N,N-dimethylformamide (1.35 mL, 0.0059 vol.) and oxalyl chloride (108.5 mL, 1 eq.) were added at a temperature of 0 °C to 5 °C for 34 minutes. The reaction mixture was stirred at a temperature of 0 °C to 5 °C for 3.5 hours and then for 14 hours at a temperature of 15 °C to 20 °C. TLC monitoring showed the reaction to completion. The medium was concentrated under vacuum (at a temperature of 40 °C or lower) and evaporated with dichloromethane (4 x 1 L, 4 x 4.35 vol.) to form 6-(nitrooxy)hexanoyl chloride (240.01 g) in a 97.7% yield. Step 4: Prepare Methyl tert-butyl ether (2800 mL, 14 vol.) was loaded into a flask. Bimatoprost (200 g, 1 eq.) was added, and the apparatus was rinsed with methyl tert-butyl ether (200 mL, 1 vol.). Butylboronic acid (58.94 g, 1.13 eq.) was added to the resulting suspension in one portion, and the apparatus was rinsed with methyl tert-butyl ether (200 mL, 1 vol.). The mixture was heated at 40 °C for 1 hour. The reaction was monitored by ¹H NMR until conversion >97%. The reaction mixture was cooled to a temperature of 20–25 °C, clarified over a glass filter, and washed with methyl tert-butyl ether (200 mL, 1 vol.). The filtered product was transferred to a 4-liter three-necked round-bottom flask, the apparatus was rinsed with methyl tert-butyl ether (100 mL, 0.5 vol.), and the medium was heated to approximately 40 °C under vacuum for azeotropic distillation. The methyl tert-butyl ether rinse and azeotropic distillation continued until the water content of (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (compound (II)) was equal to or less than 0.25%. The compound of formula (II) was obtained in quantitative yield (281.22 g). Step 5: Preparation of (lS,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7oxohept-2-en-1 -yl]-2,4-dioxa-3-borabicyclo[3.2.1 ]oct-6-yl} -1 -(2-phenyl)-1-5-1-phenyl IF-2019-42693428-APN-ANP#INPI Page 15 of 21 6-(nitrooxy)hexanoate (III) Methyl tert-butyl ether (3196 mL, 11.6 vol.) was loaded into a 4 L three-necked round-bottom flask under nitrogen. (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (compound (II)) (276.48 g crude, 1 eq.) was added, and the apparatus was rinsed with methyl tert-butyl ether (398 mL, 1.44 vol.). The resulting solution was analyzed by Karl Fischer (water content = 0.072%) and cooled to a temperature of 0 °C to 5 °C. 4-Dimethylaminopyridine (138.5 g, 2.27 eq.) was added in one portion. A solution of 6-(nitrooxy)hexanoyl chloride (172.6 g, 1.5 eq.) in methyl tert-butyl ether (508 mL, 1.84 vol.) was added dropwise at 0–5 °C over 1 hour. The dropping funnel was rinsed with methyl tert-butyl ether (32 mL, 0.12 vol.). After stirring for 24 minutes at 0–5 °C, HPLC monitoring showed a conversion of 97.8%. The mixture was stirred at 15–20 °C for 14.5 hours.HPLC monitoring showed a conversion of 99.8%. The reaction mixture was cooled to a temperature of 0 °C to 5 °C and deionized water (1351 mL, 4.89 vol.) was added over 20 minutes at a maximum temperature of 15 °C. The mixture was stirred for 5 minutes and decanted. The aqueous layer was analyzed and then discarded. A 1N aqueous hydrochloric acid solution was prepared by mixing deionized water (493 mL, 1.78 vol.) and 11.6 N hydrochloric acid (46.6 mL, 1.08 eq.). The organic layer was washed with the hydrochloric acid solution. The aqueous layer was analyzed and then discarded. The organic layer was washed first with deionized water (1351 mL, 4.88 vol.) and then with a saturated sodium chloride solution (3 x 1177 mL, 3 x 4.25 vol.) previously prepared by mixing deionized water (3530 mL, 12.8 vol.) and sodium chloride (1175 g, 425% w / w). The aqueous layers (pH = 4 after the last wash) were analyzed and discarded. The organic layer was dried in sodium sulfate (240 g, 86.8% w / w), washed with 16 IF-2019-42693428-APN-ANP#INPI Page 16 of 21 methyl tert butyl ether (481 mL, 1.74 vol.) was concentrated under vacuum to form (1 S,2E)-3{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-l-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-l-(2-phenylethyl)-prop-2-en-l-yl 6-(nitrooxy)hexanoate (compound (III)) in quantitative yield (327 g). Step 6: Synthesis of (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2hepten-l-yl]-3,5-dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6(nitrooxy)-hexanoic acid (I) (crude compound) (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]2,4-dioxa-3-borabicyclo[3.2.1]oct-6-y 1}-1-(2-phenylethyl)-prop-2-en-1-yl o-(nitrooxy)hexanoate (compound III) (325.4 g crude, leq.) in methanol (2873 mL, 8.83 vol.). The resulting solution was loaded into the flask, and the equipment was rinsed with methanol (918 mL, 2.82 vol.). The mixture was stirred at a temperature of 17 °C to 24 °C for 2.40 hours, monitoring the reaction by ¹H-NMR. The mixture was stirred for an additional 15 hours at 16–20 °C. As ¹H-NMR monitoring showed no change, the methanol was extracted under vacuum at 35–40 °C. Methanol (918 mL, 2.82 vol.) was added to the residue, and the mixture was stirred at 20–25 °C for 4 hours. ¹H-NMR monitoring showed a 94.6% conversion. The reaction mixture was concentrated under vacuum at a temperature below 40 °C. The residue was dissolved in methyl tert-butyl ether (3530 mL, 10.85 vol.).The resulting solution was washed with deionized water (1770 mL, 5.44 vol.). The aqueous layer (pH = 7) was discarded. A sodium chloride solution (1045 g, 321% w / w) was prepared in deionized water (3134 mL, 9.63 vol.). The organic layer was washed with the sodium chloride solution (2 x 1567 mL, 2 x 4.82 vol.). The aqueous layers were discarded. The organic layer was dried in sodium sulfate (320 g, 98% w / w), washed with methyl tert-butyl ether (640 mL, 1.97 vol.), and concentrated under vacuum at a temperature below 40 °C to obtain the crude compound (I) (292.77 g) in quantitative yield. IF-2019-42693428-APN-ANP#INPI Page 17 of 21 Step 7: Purification of (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2hepten-l-yl]-3,5-dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6(nitrooxy)hexanoic acid (I) The mixture obtained in step 6) (crude compound (I)) was divided into 4 portions and purified using a silica gel column (750 g x 4, 10.34 vol.) and dichloromethane / methanol as the eluent with a gradient from 100:0 to 95:5 in a Combiflash. The fractions were monitored by TLC and analyzed by HPLC (% area). The HPLC fractions (% area) > 98% were blended and concentrated under vacuum at 50 °C or less to obtain 188.83 g of Compound (I), which was further loaded into the 4 L three-necked round-bottom flask and dissolved in absolute ethanol (1510 mL, 8 vol.). The apparatus was rinsed with absolute ethanol (378 mL, 2 vol.). Activated carbon (19 g, 10% w / w) was added and the mixture was stirred at a temperature of 20 °C to 25 °C for 0.5 hours. The carbon was filtered and washed with absolute ethanol (189 mL, 1 vol.).The filtered product was concentrated under industrial vacuum at 45–50°C for 2 hours and then under high vacuum at 45–50°C for 5 hours. Monitoring by 'H NMR on DMSO-d6 showed no residual solvents. The (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-l-yl]-3,5dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6-(nitrooxy)-hexanoic acid (I) (174.4 g) was obtained with an overall yield of 63% of compound (II). The purity by HPLC was 99.47%. Following the same procedure described in Example 1, two further batches (2 and 3) of the compound of formula (I) were prepared. Table 1 below reports the purity of the compound of formula (I) from the 3 batches and the overall yield of compound (II). Table 1 Batch Initial Quantity Final Quantity Purity Overall Yield IF-2019-42693428-APN-ANP#INPI Page 18 of 21 of compound (Π) of compound (Π) by HPLC with respect to compound (II) 1 (Example 1) 231.72 174.4 99.47 63% 2 225.8 148 99.4 54.9% 3 321.28 236 99.7 61.5% Example 2 (Comparative example) Synthesis of (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-lyl]-3,5-dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6-(nitrooxy)hexanoic acid (I) according to the procedure disclosed in Document WO 2009 / 136281 (Figure 3 - Scheme 3) Step A: Preparation of (Z)-7-[(lS,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5phenyl-pent-l-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (II) Butylboronic acid (1.129 eq.) was added to a solution of bimatoprost (1 g, 1 eq.) in dichloromethane (16 vol.). The mixture was heated at 40 °C for 1 hour, monitoring the reaction progress by ¹H-NMR. The solvent was extracted under reduced pressure for 2 hours. Dichloromethane (16 vol.) was added, and the mixture was heated at 40 °C for another hour. The solvent was extracted under pressure for 40 min. Dichloromethane (16 vol.) was added, and the mixture was heated at 40 °C for 16 hours. The solvent was evaporated, and the crude product was dried under high vacuum at 40 °C for 3 hours, yielding compound (II) in quantitative yield, which was used in the next step without further purification. MS: m / z = 438 [M+H]+ Step B: Synthesis of (S,E)-l-((lS,5R,6R,7S)-3-butyl-7-((Z)-7-(ethylamino)-7oxohept-2-en-l-yl)-2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl)-5-phenylpent-l-en-3-yl 6bromo hexanoate (VIII). 4-Dimethylaminopyridine (1.1 eq.) and 6-bromohexanoyl chloride (1.15 eq.) were added to a solution of compound (II) (0.8 g, 1 eq.) in dichloromethane (15.3 vol.) IF-2019-42693428-APN-ANP#INPI Page 19 of 21 cooled to 0-5 °C. The mixture was stirred for 0.5 hours at a temperature of 0 °C to 5 °C and 16 hours at a temperature of 20 °C to 25 °C. 4-Dimethylaminopyridine (0.25 eq.) and 6-bromohexanoyl chloride (0.25 eq.) were added, and the mixture was stirred for an additional 19 hours. The reaction was monitored by 1H-NMR until complete conversion. The mixture was diluted with dichloromethane (15.3 vol.), and the organic solution was washed with deionized water (6.25 vol.) and brine (6.25 vol.). The organic phase was dried in Na₂SO₄ and concentrated under vacuum to obtain compound (VIII) as a light yellow oil (calculated as a quantitative yield), which was used in the next step without further purification. Step C: Synthesis of (lS,2E)-3-[(lR,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2hepten-l-yl]-3,5-dihydroxycyclopentyl]-l-(2-phenylethyl)-2-propen-l-yl ester of 6-(nitrooxy)-hexanoic acid (I) Silver nitrate (3.72 eq.) was added to a solution of compound (VIII) (0.8 g, 1 eq.) in acetonitrile (9.4 vol.). The mixture was stirred for 18 hours at a temperature of 20 °C to 25 °C. Conversion was monitored by 'H-NMR in DMSO. Silver nitrate (0.5 eq.) was added and the mixture was stirred for an additional 20 hours until HPLC showed a conversion of 99.7%. The mixture was filtered through a Whatman filter. The filtrate was concentrated under vacuum. The residue was dissolved in ethyl acetate (30 vol.). The organic phase was washed with deionized water (5 vol.) and brine (5 vol.). After drying on Na₂SC₄, the layer was concentrated under vacuum. The residue was subjected to silica gel column chromatography with dichloromethane / methanol (95:5) as the eluent. The fractions were monitored by TLC, and only the fractions exhibiting a spot were blended and concentrated under vacuum at a temperature of 400°C or lower, yielding compound (I) with an overall yield of 86% along with 4.27% bimatoprost. The reversed-phase HPLC (% area) showed that the purity of compound (I) was IF-2019-42693428-APN-ANP#INPI Page 20 of 21 of 77% and the content of 15-(6-chlorohexanoyl) bimatoprost ester (compound (X)) was 8.34%. Table a2 Example Compound (I) Overall yield of compound (II) (%) Purity of compound (I) (%) Impurities Yield of Impurity (%) 1 Batch 1 63% 99.47 Comp. (X) 0.16 Batch 2 54.9% 99.40 Comp. (X) 0.24 Batch 3 61.5% 99.76 Comp. (X) 0.15 2 86% 77 Comp. (X) 8.34 bimatoprost 4.27 Table 2 reports the yield of Compound (I) and the main impurities 5 formed during its preparation according to the process of the invention (Example 1) and with a method described in document WO 2009 / 136281 (Example 2). Compound (X) is the 15-(6-chlorohexanoyl) ester of bimatoprost which has a structural alert of potential mutagenicity. The results show that the process of the invention provides compound 10 (I) with a chemical purity greater than 99% with a content of compound (X) of 0.15% to 0.26%; the process disclosed in the prior art leads to compound (I) having a chemical purity of 77% and a compound (X) content of 8.34%, i.e., more than 30 times greater than the amount of compound (X) formed in the process of the invention. The results demonstrate that the process of the invention represents an improvement over the method described in the prior art. IF-2019-42693428-APN-ANP#INPI Page 21 of 21 Argentine Republic - National Executive Branch 2019 - Year of Exports Additional Signature Sheet Graphic Report Number: IF-2019-42693428-APN-ANP#INPI CITY OF BUENOS AIRES Thursday, May 9, 2019 Reference: 20190100442 The document was imported by the GEDO system with a total of 21 page(s). Digitally signed by GESTION DOCUMENTAL ELECTRONICA - GDE DN: cn=ELECTRONIC DOCUMENTARY MANAGEMENT - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564 Date: 2019.05.09 09:53:47 -03'00' Mariela Flavia Gonnet Administrative Advisor National Patent Administration National Institute of Industrial Property Digitally signed by GESTION DOCUMENTAL ELECTRONICA GDE DN: cn=GEST10N ELECTRONIC DOCUMENTARY - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564 Date: 2019.05.09 09:53:47 -03'00'

Claims

1. A process for the preparation of (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of 6-(nitrooxy)hexanoic acid of formula (I): (FORMULA 1) said process characterized in that it comprises the following steps: a) reacting the compound of formula (II): (FORMULA 2) with 6-(nitrooxy)hexanoyl chloride of formula (IV): (FORMULA 3) in an aprotic organic solvent in the presence of free 4-dimethylaminopyridine at a temperature ranging from 0°C to room temperature, to obtain the compound of formula (III): (FORMULA 4), b) removing the protecting group from boronate of the compound of formula (III) to obtain the compound of formula (I), wherein the elimination of the boronate protecting group is carried out by reaction with methanol at a temperature of 17ºC to 24ºC;wherein all steps are carried out under a nitrogen atmosphere, wherein in said process the 6-(nitrooxy)hexanoyl chloride of formula (IV) is prepared by a process comprising the following steps: i) reacting 2-caprolactone of formula (V): (FORMULA 5) with an inorganic base selected from KOH, NaOH and LiOH, carried out in a solvent selected from methanol, ethanol or isopropanol to obtain the salt of 6-hydroxyhexanoic acid of formula (VI), (FORMULA 6) where M is K, Na or Li; ii) nitrating the compound of formula (VI) with a mixture of HNO3 and H2SO3 in dichloromethane to obtain 6-(nitrooxy)hexanoic acid of formula (VII) (FORMULA 7); iii) converting 6-(nitrooxy)hexanoic acid of formula (VII) with a chlorination reagent in dichloromethane to 6-(nitrooxy)hexanoyl chloride of formula (IV). 8 Claims follow;