Industrial process and high-purity product for the preparation of (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)prop-2-en-1-yl 6-(nitrooxy)hexanoate

Abstract

The present invention relates to a method for preparing the compound (I) of hexanoic acid, 6-(nitrooxy)-, (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. According to the present invention, the pharmaceutical-grade compound (I) can be efficiently prepared by an esterification of 15-OH bimatoprost by coupling bimatoprost phenyl-boronate with 6-(nitrooxy)hexanoic acid and a one-pot reaction preparation process including removal of the boronate ester protecting group, and a subsequent efficient purification process. The present invention also refers to a high-purity compound (I) substantially free of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost, and an ophthalmic pharmaceutical preparation containing the high-purity compound.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a method suitable for industrial-scale production for preparing hexanoic acid, 6-(nitrooxy)-, (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, a high-purity hexanoic acid, 6-(nitrooxy)-, (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 substantially free of the impurity bimatoprost 15-(6-chlorohexanoyl) ester, and an ophthalmic pharmaceutical preparation containing the high-purity compound.

Background Art

[0002] Background of the Invention Hereinafter, hexanoic acid, 6-(nitrooxy)-, (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, also referred to as compound (I), has the following formula (I):

Chemical formula

[0003] The chemical name of the compound (I) is also (1S,2E)-3-{(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl]-3,5-dihydroxycyclopentyl}-1-(2-phenylethyl)prop-2-en-1-yl-6-(nitrooxy)hexanoate.

[0004] Hexanoic acid, 6-(nitrooxy)-, (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 is a prostaglandin analogue that has been proven to be effective as an IOP-lowering agent (Impagnatiello F, Toris CB, Batugo M, Prasanna G, Borghi V, Bastia E, Ongini E, Krauss AHP; Invest Ophthalmol Vis Sci. 2015; 56:6558-64), and its effectiveness as an IOP-lowering drug in patients with open-angle glaucoma or ocular hypertension has been evaluated.

[0005] Studying and monitoring the presence of impurities in the active pharmaceutical ingredient(s) (API(s)) is a central topic in pharmaceutical manufacturing to ensure an appropriate quality level in the manufactured product. The limits for each impurity in the manufactured API are the subject of specific guidelines published by international agencies. In recent years, the importance of quantifying impurities that may have genotoxicity, i.e., impurities that may cause DNA damage with gene mutations, has been increasing. Specifically, the limits for impurities with one or more alert functions are defined according to specific guidelines.

[0006] The preparation method of hexanoic acid, 6-(nitrooxy)-, (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 has already been reported; the said synthesis method is disclosed by the original compound patent International Publication No. WO2009 / 136281, and the preparation method patent application International Publication Nos. 2019 / 162149 and 2021 / 023693. The said compound (I) prepared according to the methods disclosed in International Publication No. 2019 / 162149 and International Publication No. 2021 / 023693 is a compound that may have genotoxicity, formula (II):

Chem.

Chem.

[0007] Other known chemical impurities contained in compound (I) are of formula (VII):

Chem.

Chem.

[0008] The pharmaceutical-grade compound (I) is an oil and cannot be purified by crystallization. Furthermore, the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III), which are impurities of compound (I), have similar polarities. Therefore, the presence of these impurities is an important problem for the large-scale production of pharmaceutical-grade compound (I). Therefore, the removal of these impurities requires several purifications that reduce the yield of the above method and increase the cost of preparing compound (I) on a commercial scale.

[0009] International Publication No. WO 2019 / 162149 discloses an industrial method for the preparation of compound (I). The method is of formula (IV):

Chemical formula

[0010] The 15-(6-chlorohexanoyl) ester of bimatoprost of the formula (II) is a by-product formed during the esterification step by an exchange reaction of the nitrooxy group of 6-(nitrooxy)hexanoate of (1E,3S)-1-{(1S,5R,6R,7R)-3-butyl-7-[(2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl]-2,4-dioxo-3-borabicyclo[3.2.1]octan-6-yl}-5-phenylpent-1-en-3-yl with the free chlorine anion; at the end of the method, removal of the boronate ester protecting group gives 15-(6-chlorohexanoyl)bimatoprost ester.

[0011] The bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III) is derived from the reaction of bimatoprost butyl-boronate of formula (IV) with 6-[6-nitrooxyhexanoyl]oxy}hexanoyl chloride, which is an impurity of the intermediate 6-(nitrooxy)hexanoyl chloride. After esterification, the removal of the butyl-boronate protection results in the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III).

[0012] International Publication No. 2021 / 023693 discloses an improvement of the method disclosed in International Publication No. 2019 / 162149, which enables the reduction of the formation of the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III). In this method, 6-(nitrooxy)hexanoic acid is purified by reverse-phase chromatography at the end of the nitration step of the alkali salt of 6-hydroxyhexanoic acid.

[0013] The crude compound (I) prepared according to the method of International Publication No. 2021 / 023693 has an HPLC purity of 73% (HPLC area %), and before the final chromatographic purification, it contains about 0.1% (HPLC area %) of the 15-(6-chlorohexanoyl)bimatoprost ester of formula (II) and less than 0.05% (HPLC area %) (HPLC threshold) of the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (III).

[0014] The synthesis of compound (I) (Example B-1) disclosed in International Publication No. WO 2009 / 136281 involves protection of the C9-OH and C11-OH groups of bimatoprost with butyl-boronic acid to form bimatoprost butyl-boronate of formula (IV), esterification of C15-OH with 6-bromohexanoyl chloride to give the 15-(6-bromohexanoyl) ester of the butyl-boronate-protected form of bimatoprost which is converted to a nitrate derivative by silver nitrate in acetonitrile, removal of the butyl-boronate group, and purification of the product by reverse-phase chromatography. The main drawbacks of the above synthesis are the use of more than equimolar amounts of 6-bromohexanoyl chloride which has a structural alert to the potential of mutagenicity; the use of silver nitrate which generates a large amount of silver salts in the wastewater; the possibility of formation of 15-(6-bromohexanoyl) ester impurities of bimatoprost due to incomplete nitration of the intermediate 15-(6-bromohexanoyl) ester of bimatoprost butyl-boronate; and the formation of the by-product 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) due to incomplete nitration of the 15-(6-chlorohexanoyl) ester of bimatoprost butyl-boronate. The 15-(6-chlorohexanoyl) ester of bimatoprost butyl-boronate is a by-product of the esterification reaction formed by a halogen exchange reaction between the bromine atom of the 15-(6-bromohexanoyl) ester of bimatoprost butyl-boronate and free chloride anions, which is formed during the esterification process and is promoted by the presence of a base in the reaction medium.

[0015] International Publication No. WO 2009 / 136281 does not mention the impurity profile of the final product, and experiments conducted by the inventors have shown that compound (I) prepared according to the method disclosed in International Publication No. WO 2009 / 136281 contains about 8.34% (HPLC area %) of the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II).

[0016] International Publication No. WO 2009 / 136281 also discloses an alternative method for the preparation of 15-acylalkynitrate bimatoprost derivatives (Examples N-1 and O-1). The synthesis involves reacting bimatoprost in its boronate-protected form with a nitrate-alkyl carboxylic acid chloride in the presence of 4-dimethylaminopyridine (DMAP) supported on a resin (PS-DMAP), followed by removal of the boronate protecting group and purification by silica gel column chromatography. This alternative method avoids the use of 6-bromohexanoyl chloride and the removal of the silver salt. However, the main drawback of this method is the use of 4-dimethylaminopyridine supported on a resin, which renders the method unsuitable and expensive for commercial scale-up, and furthermore, a large excess of nitrate-alkyl carboxylic acid chloride is added.

[0017] International Publication No. WO 2009 / 136281 also discloses another method (Example Q1) for the preparation of 15-acylalkynitrate bimatoprost derivatives. In this method, the compound is obtained by esterification of bimatoprost butyl-boronate with an excess of nitrate-alkyl-(p-nitrophenyl)-carboxylate in the presence of 4-dimethylaminopyridine.

[0018] The main drawbacks of this synthesis are the removal of unreacted nitrate-alkyl-(p-nitrophenyl)-carboxylate by chromatography and the removal of the by-product p-nitrophenol.

[0019] Therefore, there is a need for high-purity hexanoic acid, 6-(nitrooxy)-, (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, which is suitable for the manufacture of ophthalmic pharmaceutical formulations.

[0020] The present invention solves the above problems and provides an industrially feasible method for the preparation of high-purity hexanoic acid, 6-(nitrooxy)-, (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 formula (I).

Summary of the Invention

[0021] Description of the Invention The present invention is a method for the synthesis of hexanoic acid, 6-(nitrooxy)-, (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 (Compound (I)) suitable for industrial-scale production, which enables the preparation of Compound (I) in high yield and high purity.

[0022] The method of the present invention includes an efficient one-pot reaction preparation step of crude compound (I) from bimatoprost and 6-(nitrooxy)hexanoic acid, and subsequently, first, normal-phase gravity silica gel column chromatography for removing almost all impurities derived from the preparation step, and subsequently, silica gel filtration chromatography having an action range for removing higher-boiling solvents, including a highly efficient purification step of crude compound (I). The method is applicable to the large-scale preparation of compound (I) up to, for example, 650 grams. An important advantage of the method of the present invention is that this method is suitable for the preparation of hexanoic acid, 6-(nitrooxy)-, (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 formula (I) with high purity. In fact, the compound obtained by the method of the present invention does not contain the 15-(6-chlorohexanoyl) ester (II) of bimatoprost, and the total amount of impurities is (≦) 0.20% (HPLC area%). The high overall chemical yield of about 70% and the highly effective purification step of the method make this method an easy-to-apply cost-reduction method for the industrial-scale preparation of hexanoic acid, 6-(nitrooxy)-, (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 formula (I) for pharmaceutical grade.

[0023] Another object of the present invention is hexanoic acid of formula (I), 6-(nitrooxy)-, (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, which is substantially free of the 15-(6-chlorohexanoyl) ester of bimatoprost, which is an impurity, and which contains a total impurity amount of (≦) 0.20% (HPLC area %), preferably the total impurity amount is (≦) 0.15% (HPLC area %), and most preferably the total impurity amount is (≦) 0.10% (HPLC area %).

[0024] "Free of the 15-(6-chlorohexanoyl) ester of bimatoprost (II) as defined above" and "substantially free of the 15-(6-chlorohexanoyl) ester of bimatoprost, which is an impurity, of formula (II)" mean that the amount of the compound is below the limit of detection (LoD) of the HPLC method disclosed herein.

[0025] Detailed Description of the Invention The present invention relates to a method for the preparation of hexanoic acid of formula (I):

Chemical formula

Chemical formula

Chemical formula

[0026] In reaction step 1), preferably, the molar ratio of bimatoprost:phenylboronic acid is 1:1.1; In reaction step 2), preferably, 2.0 equivalents of N,N'-diisopropylcarbodiimide, 0.2 equivalents of dimethylaminopyridine, and 1.8 to 2.2 equivalents of 6-(nitrooxy)hexanoic acid are added.

[0027] In reaction step 3), preferably, the phenylboronate protecting group is removed using an NaOH solution. Specifically, the phenylboronate protecting group is removed by quenching the reaction mixture obtained in step 2) with methanol and then adding a mixture of methylene chloride and 6.3 equivalents of NaOH as a 0.5 M NaOH solution.

[0028] Preferably, in reaction step 4), the organic phase is first washed with an aqueous sodium bisulfate solution and then washed twice with a 15% w / w aqueous NaCl solution.

[0029] Preferably, the method of the present invention gives a compound of formula (I) containing (≦) 0.10% (HPLC area %) or less of total impurities.

[0030] Another embodiment of the present invention is the following steps: 1) Reacting bimatoprost (1 equivalent) with phenylboronic acid (1.1 equivalents) in toluene to protect the 9,11-hydroxy groups of bimatoprost; heating the reaction mixture to the reflux temperature of toluene, removing water by azeotropic distillation, and stirring the reaction mixture at the reflux temperature until the formation of bimatoprost phenyl-boronate of formula (V); 2) Cooling the reaction mixture to 25 °C and adding N,N'-diisopropylcarbodiimide (2.0 equivalents), a catalytic amount of dimethylaminopyridine (0.2 equivalents) and 6-(nitrooxy)hexanoic acid (1.8 to 2.2 equivalents) to obtain (1E,3S)-1-{(1S,5R,6R,7R)-7-[(2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl]-3-phenyl-2,4-dioxo-3-borabicyclo[3.2.1]octan-6-yl}-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate of formula (VI); 3) At the end of the esterification, quenching the reaction mixture with methanol, then adding methylene chloride and 0.5 M NaOH solution, and then stirring the resulting mixture to complete the removal of the phenylboronate protecting group (wherein preferably 6.3 equivalents of NaOH are used); 4) Separating the organic phase and washing the organic phase first with an aqueous solution of sodium bisulfate and then twice with an aqueous solution of 15% w / w NaCl, and then evaporating the solvent at a temperature below 45 °C 5) Adding dichloromethane to the untreated mixture and stirring the mixture at 10 °C for 30 minutes, removing N,N'-diisopropylurea by filtration, and evaporating the solvent of the filtered solution to obtain the crude compound of formula (I); 6) Purifying the crude compound of formula (I) by applying normal phase gravity silica gel column chromatography using an eluent mixture containing diisopropyl ether / acetone / water in a volume ratio of 40:15:0.5; 7) Collecting fractions of appropriate purity and evaporating the solvent to obtain the pure compound of formula (I); 8) Dissolve the pure compound of formula (I) obtained in step 7), and purify the solution by gravity chromatography on a column packed with silica using an eluent mixture containing distilled methylene chloride / distilled methanol in a volume ratio of 30:1; collect fractions of appropriate purity, and evaporate the solvent to obtain the pure compound of formula (I) as an oil; 9) Dissolve the pure compound of formula (I) obtained in step 8) in ethanol, treat the resulting solution with activated carbon, then remove the activated carbon by filtration, and remove ethanol by evaporation under vacuum to yield the final pure compound of formula (I) comprising wherein the obtained compound of formula (I) does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and has a total impurity amount of (≦) 0.20% (HPLC area %), preferably, the total impurity amount is (≦) 0.10% (HPLC area %) or less A method for the synthesis of hexanoic acid, 6-(nitrooxy)-, (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 formula (I).

[0031] The above "equivalent (equiv.)" (equivalent) means the molar equivalent of each reagent and is calculated relative to the mole of bimatoprost.

[0032] The method reported above is as follows:

Chemical Structure

[0033] The method of the present invention has several advantages, eliminating the formation of the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and enabling the amount of the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III) to be reduced to less than the quantification limit of 0.05% w / w. Further, the evaluation of the purity of compound (I) performed by HPLC also showed that the amounts of each of the 5,6-trans-compound (I) of formula (VII) and the 15-epi-compound (I) of formula (VIII), which are impurities, are also (≦) 0.05% w / w or less.

[0034] Another advantage of the method of the present invention is that the synthesis of the crude compound of formula (I) can be carried out in a one-pot reaction preparation in which steps 1) to 3) reported above are performed without isolating or purifying the obtained intermediate.

[0035] The present invention provides a hexanoic acid of formula (I), 6-(nitrooxy)-, (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 that does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) as an impurity, and it contains a total impurity amount of (≦) 0.20% (HPLC area %), preferably the total impurity amount is (≦) 0.15% (HPLC area %) or less, and most preferably the total impurity amount is (≦) 0.10% (HPLC area %) or less.

[0036] One embodiment of the present invention provides a hexanoic acid of formula (I), 6-(nitrooxy)-, (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 that does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein the compound of formula (I) is as follows: · 6-{[6-(Nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) at (≤) 0.05% w / w or less; · (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(Ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl 6-(nitrooxy)hexanoate (5,6-trans-compound (I)) of formula (VII) at (≤) 0.05% w / w or less; and · (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(Ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl-6-(nitrooxy)hexanoate (15-epi-compound (I)) of formula (VIII) at (≤) 0.05% or less contain; and where the total amount of impurities is (≤) 0.20% (HPLC area%).

[0037] Another embodiment of the present invention provides a hexanoic acid, 6-(nitrooxy)-, (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 formula (I) that does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), where the compound of formula (I) is as follows: · 6-{[6-(Nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) at (<) 0.05% w / w or less; · (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(Ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl 6-(nitrooxy)hexanoate (5,6-trans-compound (I)) of formula (VII) at (≤) 0.05% w / w or less; and · less than (<) 0.05% w / w of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl 6-(nitrooxy)hexanoate (15-epi-compound (I)) of formula (VIII) containing; and wherein the total amount of impurities is (≦) 0.20% (HPLC area %) or less.

[0038] Another embodiment of the present invention provides a hexanoic acid, 6-(nitrooxy)-, (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 formula (I) that does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein the compound of formula (I) is as follows: · less than (<) 0.05% w / w of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of formula (III); · (≦) 0.05% w / w or less of (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl 6-(nitrooxy)hexanoate (5,6-trans-compound (I)) of formula (VII); and · less than (<) 0.05% w / w of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl 6-(nitrooxy)hexanoate (15-epi-compound (I)) of formula (VIII) containing; and wherein the total amount of impurities is (≦) 0.15% (HPLC area %) or less.

[0039] Another embodiment of the present invention provides a hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein the compound of formula (I) is as follows: · less than (<) 0.05% w / w of the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III); · less than or equal to (≦) 0.05% w / w of the (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VII) (5,6-trans-compound (I)); and · less than (<) 0.05% w / w of the (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VIII) (15-epi-compound (I)) contains; and wherein the total amount of impurities is less than or equal to (≦) 0.10% (HPLC area%).

[0040] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition containing hexanoic acid, 6-(nitrooxy)-, (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 formula (I) which does not contain the (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and contains a total amount of impurities of (≦) 0.20% (HPLC area %), preferably the total amount of impurities is (≦) 0.15% (HPLC area %), and most preferably the total amount of impurities is (≦) 0.10% (HPLC area %), and at least one pharmaceutically acceptable excipient.

[0041] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising hexanoic acid, 6-(nitrooxy)-, (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 formula (I) that does not contain the (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and at least one pharmaceutically acceptable excipient, wherein the compound of formula (I) is (≤) 0.05% w / w or less of the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III); (≤) 0.05% w / w or less of the (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl 6-(nitrooxy)hexanoate (5,6-trans-compound (I)) of formula (VII); (≤) 0.05% w / w or less of the (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl-6-(nitrooxy)hexanoate (15-epi-compound (I)) of formula (VIII), wherein the total amount of impurities is (≤) 0.20% (HPLC area %).

[0042] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) which does not contain the (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and at least one pharmaceutically acceptable excipient, wherein the compound of formula (I) has less than (<) 0.05% w / w of the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III); at most (≦) 0.05% w / w of the (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VII) (5,6-trans-compound (I)); less than (<) 0.05% w / w of the (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VIII) (containing the 15-epi-compound (I)), and wherein the total amount of impurities is at most (maximum) 0.20% (HPLC area %).

[0043] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising hexanoic acid of formula (I), 6-(nitrooxy)-, (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, which does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and at least one pharmaceutically acceptable excipient, wherein the compound of formula (I) contains less than (<) 0.05% w / w of the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III); at most (≦) 0.05% w / w of the (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VII) (5,6-trans-compound (I)); less than (<) 0.05% w / w of the (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VIII) ((15-epi-compound (I)), and wherein the total amount of impurities is at most (≦) 0.15% (HPLC area%).

[0044] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising hexanoic acid of formula (I), 6-(nitrooxy)-, (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 which does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and at least one pharmaceutically acceptable excipient, (wherein the compound of formula (I) contains less than (<) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III); at most (≦) 0.05% w / w of (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VII) (5,6-trans-compound (I)); less than (<) 0.05% w / w of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpenta-1-en-3-yl-6-(nitrooxy)hexanoate of formula (VIII) (15-epi-compound (I)), and wherein the total amount of impurities is at most (≦) 0.10% (HPLC area %).

[0045] The purity of compound (I) and the impurity profile were evaluated by the HPLC methods (Method 1 and Method 2) disclosed herein.

[0046] The definitions “not containing 15-(6-chlorohexanoyl) ester of bimatoprost” or “substantially free of 15-(6-chlorohexanoyl) ester of bimatoprost” reported above mean that the amount of 15-(6-chlorohexanoyl) ester of bimatoprost is below the limit of detection (LoD) of the HPLC method disclosed herein.

[0047] The definition reported above, "the amount of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate is less than 0.05% w / w", means that the amount of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate is below the limit of quantification (LoQ) of the HPLC method disclosed in this specification.

[0048] The definition reported above, "the amount of 5,6-trans-compound (I) is less than 0.05% w / w", means that the amount of 5,6-trans-compound (I) is below the limit of quantification of the HPLC method disclosed in this specification.

[0049] The definition reported above, "the amount of 15-epi-compound (I) less than 0.05% w / w", means that the amount of 15-epi-compound (I) is below the limit of quantification of the HPLC method disclosed in this specification.

Example 1

[0050] Synthesis of hexanoic acid, 6-(nitrooxy)-, (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 (Compound (I))

[0051] The starting material bimatoprost is commercially available.

[0052] 1a) Preparation of 6-(nitrooxy)hexanoic acid

[0053] Synthesis of methyl 6-hydroxyhexanoate 1 L three-necked flask equipped with a thermometer, dropping funnel, reflux condenser and magnetic stirrer was charged with dry methanol (300 mL), followed by concentrated sulfuric acid (2.8 mL, 0.052 mol, 0.06 eq). The mixture was heated to the reflux temperature (T = 63 °C). A solution of ε-caprolactone (100 g, 0.876 mol, 1 eq) in dry methanol (200 mL) was added dropwise to the mixture. The reaction mixture was stirred at the reflux temperature for 2 h. TLC was performed to monitor the residual ε-caprolactone. The reaction mixture was then cooled to 0 - 5 °C. While maintaining the temperature below 10 °C, concentrated NaHCO3 solution (100 mL) was slowly added. The volatile solvent was removed using a rotary evaporator at a temperature of 40 °C under vacuum. Water and methyl tert-butyl ether (MTBE) (300 mL) were added to the residue. The phases were stirred for 5 min and then separated. The aqueous phase was extracted with MTBE (2 × 200 mL). The combined organic phases were washed with brine (2 × 100 mL). The organic phase was dried over Na2SO4 and then concentrated using a rotary evaporator at a temperature of 40 °C under vacuum to obtain methyl 6-hydroxyhexanoate (103.58 g, yield: 80.9%) as a colorless oil. GC purity: 96.2%.

[0054] Synthesis of methyl 6-nitrooxyhexanoate To a 500 mL three-necked flask equipped with a thermometer, a dropping funnel, and a magnetic stirrer, concentrated sulfuric acid (45.6 mL, 0.85 mol, 3.1 eq) cooled to 0 - 5 °C was added. Next, while maintaining the temperature between 0 - 5 °C, fuming nitric acid (47.2 mL, 1.12 mol, 4.1 eq) was added dropwise thereto. To the cooled mixture, dry dichloromethane (100 mL, 2.5 vol) was slowly added, and then the mixture was stirred at 0 - 5 °C for 30 minutes. At this temperature, methyl-6-hydroxyhexanoate (40 g, 0.27 mol, 1 eq) in dry (dichloromethane) DCM (100 mL) was added over 1 hour. After stirring at 0 - 5 °C for 30 minutes, the reaction mixture was monitored by TLC, indicating the completion of the reaction. The reaction mixture was quenched by dropping it into cooled water (200 mL) (T < 10 °C, maximum 2 hours). Next, the phases were separated. The organic phase was dried over Na2SO4 and then concentrated under vacuum at 40 °C using a rotary evaporator to obtain methyl-6-nitrooxyhexanoate (49.46 g) as a yellow oil in a 94.5% yield.

[0055] Synthesis of Crude 6-(Nitrooxy)hexanoic Acid To a 1 L three-necked flask equipped with a thermometer, a dropping funnel, and a magnetic stirrer, 1.5 M aqueous NaOH solution (208 mL) was charged. Methyl-6-nitrooxyhexanoate (49.64 g, 0.26 mol, 1 eq) dissolved in methanol (248 mL, 5 vol) was added dropwise at room temperature (T 最大 = 35 °C). After the addition, the mixture was stirred at room temperature for 2 hours. Examination by TLC showed that the reaction mixture indicated the completion of the reaction. The pH of the reaction mixture was set to 2.5 using 183 mL of 2 M aqueous HCl solution. The resulting mixture was extracted with MTBE (100 mL), and then the aqueous phase was extracted with MTBE (5 × 50 mL). The combined organic phases were washed with brine (2 × 30 mL) and then concentrated under vacuum at 40 °C using a rotary evaporator to obtain crude 6-(nitrooxy)hexanoic acid (44.2 g) as a yellow oil. The assay was evaluated by the UPLC method.

[0056] Purification of 6-(Nitrooxy)hexanoic Acid Crude 6-(nitrooxy)hexanoic acid was purified by column chromatography according to the following procedure: The eluent was prepared by mixing toluene and methanol in a volume ratio of 40:1. Silica was stirred with the eluent in a slurry dispenser and packed into a column to prepare the column. The crude material (1161 g, 91.8% assay by UPLC) was dissolved in toluene and the solution was loaded onto the top of the column. Elution was carried out at a flow rate of 15 ± 5 L per hour, 10 L fractions were collected and analyzed by TLC. The fractions with the highest purity were combined and concentrated under vacuum at a temperature less than 55 °C ± 5 °C. The purified 6-(nitrooxy)hexanoic acid (952 g, 5.37 mol) had a purity of 98.7% by UPLC and was isolated as an oil in an 82% yield containing no detectable amount of 6-[6-nitrooxyhexanoyl]oxy}hexanoic acid.

[0057] 1b) Synthesis of crude hexanoic acid, 6-(nitrooxy)-, (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 (Compound (I)) Toluene (28 Kg, 49 volumes) was charged into a reactor, followed by bimatoprost (663 g, 1.595 mol, 1 equivalent) and phenylboronic acid (214 g, 1.755 mol, 1.1 equivalents). The reaction mixture was heated at 110 °C ± 5 °C, the vapor temperature was maintained at 90 °C for 4 hours, and azeotropic distillation was ensured with water relative to a total solvent volume of about 12 L. Next, the reaction mixture was cooled to 25 °C ± 5 °C.

[0058] Dimethylaminopyridine (DMAP) (39 g, 0.319 mol, 0.2 eq) and N,N'-diisopropylcarbodiimide (DIC) (403 g, 3.190 mol, 2.0 eq) were added to the cooled reaction mixture, followed by addition of a solution of 6-(nitrooxy)hexanoic acid (565 g, 3.190 mol, 2.0 eq) in toluene (0.5 L, 0.75 volume). The reaction was stirred at 25 °C ± 5 °C for 1 to 3 hours. TLC was performed to monitor the bimatoprost phenyl-boronate (Compound (V)) of the residue.

[0059] The reaction mixture was quenched with methanol (1.3 L, 2 volumes) and stirred for 5 - 10 minutes. The reaction mixture was then transferred via vacuum to an extractor containing 0.5 M sodium hydroxide solution (20 L) and dichloromethane (10 L, 15 volumes). The reactor was washed several times with methylene chloride and this was added to the reaction mixture. The reaction mixture was stirred for 2 to 3 hours and then left standing for 60 minutes, and the organic phase was collected in a transfer tank. Dichloromethane (10 L, 15 volumes) was charged to the extractor, the mixture was stirred for 5 to 10 minutes and left standing for 15 minutes. The organic layer was collected. The organic layer was charged again to the extractor and 1 M NaHSO4 solution (20 L) was transferred to the extractor via vacuum. The mixture was stirred for 5 to 10 minutes and left standing for 15 minutes, and the lower organic phase was collected in a transfer tank. The aqueous layer was removed. The organic phase was charged again to the extractor, 15% NaCl solution (20 L) was added, the mixture was stirred for 5 to 10 minutes and left standing for 15 minutes. The organic layer was collected and the aqueous phase was removed. The organic layer was charged again to the extractor and 15% w / w NaCl solution (20 L) was added. The mixture was stirred for 5 to 10 minutes and left standing for 15 minutes. The organic layer was collected and evaporated at a temperature of 45 °C ± 5 °C under vacuum.

[0060] The crude product was dissolved in dichloromethane (7 L, 10.5 volumes), and the mixture was transferred to a reactor. The mixture was cooled to 5 - 10 °C and this temperature was maintained for 30 - 60 minutes to precipitate N,N'-diisopropylurea. The mixture was filtered through a filter into a mobile tank. The reactor was washed with dichloromethane (2 L, 3 volumes), the solution was filtered and collected in the mobile tank. Next, the solution in the mobile tank was transferred to a flask, and the solvent was evaporated under vacuum at a temperature of 45 °C ± 5 °C to obtain crude hexanoic acid, 6-(nitrooxy)-, (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 (crude compound (I)).

[0061] 1c) Purification of crude hexanoic acid, 6-(nitrooxy)-, (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 (compound (I))

[0062] First chromatography Eluent mixture 1 was prepared by mixing diisopropyl ether / acetone / water in a volume ratio of 40:15:0.5.

[0063] Silica gel (30 Kg, 45 volumes) was suspended in eluent mixture 1 (66.3 Kg, 100 volumes), the suspension was stirred for 3 minutes and loaded into a column.

[0064] The crude product was dissolved in eluent mixture (1 L, 1.5 volumes) and charged onto the top of the column. Elution was carried out and 15 L fractions were collected and analyzed by TLC. The fractions were combined according to purity and analyzed by HPLC and UPLC.

[0065] The selected fraction having the desired purity was collected, and the solvent was evaporated using a rotary evaporator until dried at a temperature of 45 °C ± 5 °C. The residue was dissolved in dichloromethane (5 L, 7.5 volumes), and the solvent was evaporated again until dried.

[0066] Compound (I) was stored at 5 °C ± 3 °C.

[0067] Silica gel filtration chromatography Eluent mixture 2 was prepared by mixing distilled dichloromethane and distilled methanol in a volume ratio of 30:1.

[0068] Silica 75S (6.6 Kg, 10 volumes) was added to eluent mixture 2 (13 Kg, 19.6 volumes), and the suspension was stirred and poured to fill the column. Compound (I) was dissolved in dichloromethane (1 L, 1.5 volumes), and the solution was loaded onto the top of the column.

[0069] Elution was carried out, the fractions were examined by TLC, and the selected fractions having the desired purity were collected; the combined fractions were analyzed by HPLC and UPLC.

[0070] The fractions were evaporated using a rotary evaporator at a temperature of 45 °C ± 5 °C under vacuum. The pure compound (I) isolated as an oil was stored at 5 °C ± 3 °C.

[0071] Treatment with activated carbon (clarification) The pure compound (I) was dissolved in ethanol (6 L, 9 volumes), and activated carbon (60 g, about 9% w / w) was added. The mixture was stirred at 20 °C ± 5 °C for 30 minutes, and the activated carbon was removed by filtration. The solvent was evaporated at 45 °C ± 5 °C under vacuum until a constant weight was obtained. 649.6 g (1.13 moles) of pure compound (I) was isolated as an oil. The overall yield of the said method was 70.8%.

[0072] The purity evaluation of the isolated compound (I) was performed by HPLC analysis using the two methods described below. The HPLC assay of compound (I) was evaluated by HPLC / Method 1 and was 99.91% (HPLC area %).

[0073] The detection limits (LoD) and quantification limits (LoQ) of the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), the bimatoprost ester of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of formula (III), the 5,6-trans-compound (I) of formula (VII), and the 15-epi-compound (I) of formula (VIII) are reported in Table 1.

[0074] The impurity profile of the isolated compound (I) is reported in Table 2.

[0075] Table 3 reports the content of the 15-(6-chlorohexanoyl) ester (II) of bimatoprost, an impurity in the compound (I) prepared according to the method disclosed in the prior art.

[0076] Method 1: The HPLC analysis was performed using the following measurement conditions. Instrument: Waters HPLC system equipped with a PDA detector Stationary phase: Zorbax RXSIL, 250×4.6 mm, 5 μm Column temperature: 35 °C Mobile phase: hexane:ethanol:acetonitrile:acetic acid = 935:60:5:0.5 Run time: 60 minutes Flow rate: 1.5 mL / min Injection volume: 20 μL Detection: UV, 210 nm Sample solvent: ethanol:hexane 2:8

[0077] Method 2: The HPLC analysis was performed using the following measurement conditions to quantify the 15-epi-compound (I) of formula (VIII). Instrument: Waters HPLC system equipped with a PDA detector Stationary phase: Chiralpak AD-H, 250×4.6 mm, 5 μm Column temperature: 25 °C Mobile phase: hexane:ethanol:TFA = 850:150:1 Running time: 30 minutes Flow rate: 1.0 mL / min Injection volume: 10 μL Detection: 210 nm Sample solvent: hexane:ethanol = 8:2

Table 1

Table 2

Table 3

Claims

1. The following steps: 1) Reacting bimatoprost with phenylboronic acid in toluene at reflux temperature and removing water by azeotropic distillation to obtain a bimatoprost phenyl-boronate of formula (V): 【Chemical 11】 The step of obtaining bimatoprost phenyl-boronate; 2) Cooling the solution to 25 °C and adding N,N'-diisopropylcarbodiimide, a catalytic amount of dimethylaminopyridine and 6-(nitrooxy)hexanoic acid to obtain a (1E,3S)-1-{[(1S,5R,6R,7R)-7-[(2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl]-3-phenyl-2,4-dioxo-3-borabicyclo[3.2.1]octan-6-yl]-5-phenylpent-1-en-3-yl} 6-(nitrooxy)hexanoate of formula (VI): 【Chemical Formula 12】 The step of obtaining (1E,3S)-1-{[(1S,5R,6R,7R)-7-[(2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl]-3-phenyl-2,4-dioxo-3-borabicyclo[3.2.1]octan-6-yl]-5-phenylpent-1-en-3-yl} 6-(nitrooxy)hexanoate; 3) The step of removing the phenyl-boronate protecting group under basic conditions; 4) The step of separating the organic phase and evaporating the solvent; 5) The step of stirring the untreated product in dichloromethane, filtering the mixture and evaporating the solvent to obtain a crude compound of formula (I); 6) The step of purifying the crude compound of formula (I) by applying normal phase gravity silica gel column chromatography using an eluent mixture containing diisopropyl ether, acetone and water in a volume ratio of 40:15:0.5; 7) The step of collecting fractions of appropriate purity and evaporating the solvent to provide a pure compound of formula (I); 8) Dissolving the pure compound of formula (I) from step 7) in distilled methylene chloride and methanol, purifying the solution by gravity chromatography using an eluent mixture containing distilled methylene chloride and methanol in a volume ratio of 30:1, collecting fractions of appropriate purity and evaporating the solvent to obtain a pure compound of formula (I); 9) Dissolving the pure compound of formula (I) from step 8) in ethanol, treating the solution with activated carbon, then removing the activated carbon by filtration and removing the solvent by evaporation under vacuum to produce pure hexanoic acid, 6-(nitrooxy)-, (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, Formula (I): 【Chemical 13】 A method for the preparation of hexanoic acid, 6-(nitrooxy)-, (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, wherein the obtained hexanoic acid, 6-(nitrooxy)-, (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 does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and contains total impurities of (≤) 0.20% (HPLC area %), a method characterized thereby. **Claim 2** The method according to claim 1, wherein in reaction step 1), the molar ratio of said bimatoprost:phenylboronic acid is 1:1.

1. **Claim 3** The method according to claim 1 or 2, wherein in reaction step 2), 2.0 equivalents of N,N'-diisopropylcarbodiimide, 0.2 equivalent of dimethylaminopyridine, and 1.8 to 2.2 equivalents of 6-(nitrooxy)hexanoic acid are added. **Claim 4** The method according to any one of the above claims, wherein in reaction step 3), the phenylboronate protecting group is removed using an NaOH solution. **Claim 5** The method according to claim 4, wherein the phenylboronate protecting group is removed by quenching the reaction mixture obtained in step 2) with methanol and then adding a mixture of methylene chloride and 6.3 equivalents of NaOH as a 0.5 M NaOH solution. **Claim 6** The method according to any one of the above claims, wherein in reaction step 4), the organic phase is first washed with an aqueous sodium bisulfate solution and then washed twice with a 15% w / w aqueous NaCl solution. **Claim 7** A compound of formula (I) containing a total impurity amount of (≤) 0.20% (HPLC area %) or less and not containing the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) which is an impurity, or containing an amount below the HPLC detection limit. 【Chemical Formula 14】 **Claim 8** The compound of formula (I) according to claim 7, wherein the HPLC detection limit is 0.01% w / w. **Claim 9** ​ The compound of formula (I) according to claim 7, wherein the total impurity content is (≤) 0.15% (HPLC area %).

10. The compound of formula (I) according to claim 9, wherein the total impurity content is (≤) 0.10% (HPLC area %).

11. The impurity of formula (III) which is (≤) 0.05% w / w: 【Chemical Formula 15】 bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate, and The impurity of formula (VII) which is (≤) 0.05% w / w: 【Chemical 16】 、 (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate, and The impurity of formula (VIII) which is (≤) 0.05%: 【Chemical 17】 (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate The compound of formula (I) according to any one of claims 7 to 10, containing

12. The compound of formula (III) which is (<) 0.05% w / w: 【Chemical Formula 18】 bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate, and The compound of formula (VII) which is (≤) 0.05% w / w: 【Chemical Formula 19】 (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate, and The compound of formula (VIII) which is (<) 0.05%: 【Chemical 20】 (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate The compound of formula (I) according to any one of claims 7 to 11, containing

13. An ophthalmic pharmaceutical composition comprising the compound of formula (I) according to any one of claims 7 to 12 and at least one pharmaceutically acceptable excipient.

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