Industrial process and high purity product for preparation of 6-(nitrooxy)-hexanoic acid (1s, 2e)-3-[(1r, 2r, 3s, 5r)-2-[(2z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3, 5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propylen-1-yl ester

Abstract

The invention relates to a method for preparing 6-(nitrooxy)-hexanoic acid (1S, 2E)-3-[(1R, 2R, 3S, 5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3, 5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propylene-1-yl ester, and particularly relates to a method for preparing 6-(nitrooxy)-hexanoic acid (1S, 2E)-3-[(1R, 2R, 3S, 5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3, 5 According to the present invention, a pharmaceutical grade compound (I) can be efficiently prepared by a one-pot reaction preparation step comprising esterification of 15-OH bimatoprost by coupling bimatoprost phenyl-borate with 6-(nitrooxy) hexanoic acid, removal of borate protecting groups, and then an efficient purification step. The invention also relates to high purity compounds (I) substantially free of the impurity bimatoprost 15-(6-chlorohexanoyl) ester and to ophthalmic pharmaceutical formulations containing the high purity compounds. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a method for preparing high-purity 6-(nitrooxy)-hexanoic acid (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 suitable for industrial-scale production, high-purity 6-(nitrooxy)-hexanoic acid (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] 6-(Nitrooxy)-hexanoic acid (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, hereinafter also referred to as compound (I), has the following formula (I):

[0003]

[0004] The chemical name of compound (I) can also be: (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.

[0005] 6-(Nitrooxy)-hexanoic acid (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 analog that has been shown to be an effective 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 has been evaluated for its efficacy as an intraocular pressure (IOP) lowering drug in patients with open-angle glaucoma or ocular hypertension.

[0006] Studying and monitoring the presence of impurities in active pharmaceutical ingredients (API) is a central theme in pharmaceutical production to ensure an appropriate level of quality in the manufactured product. The limits for each impurity in the API produced are the subject of specific guidelines published by international agencies. In the past few years, the quantitative analysis of potential genotoxic impurities, i.e. those that may cause DNA damage involving gene mutations, has become increasingly important. In particular, limits for impurities with one or more alert functions have been established according to specific criteria.

[0007] The preparation method of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propene-1-yl ester has been reported; its synthesis method is disclosed in the original compound patent with patent number WO2009 / 136281, as well as preparation patent applications WO2019 / 162149 and WO2021 / 023693. Compound (I) prepared according to the methods disclosed in WO2019 / 162149 and WO2021 / 023693 contains two major impurities, namely, bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) (which is a potentially genotoxic compound) and bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of formula (III)

[0008]

[0009] Other known chemical impurities contained in compound (I) are isomeric impurities, such as (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate, i.e., 5,6-trans-compound (I) of formula (VII)

[0010]

[0011] and (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate, i.e., 15-epi-compound (I) of formula (VIII)

[0012]

[0013] The presence of these impurities is a critical issue for the large-scale production of pharmaceutical grade compound (I) because it is an oil and cannot be purified by crystallization, and compound (I) and the impurities bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) and 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) have similar polarities. Therefore, removing these impurities requires multiple purifications, which reduces the yield of the process and increases the cost of preparing compound (I) on an industrial scale.

[0014] WO 2019 / 162149 discloses an industrial method for preparing compound (I). The method comprises: protecting the C9-OH and C11-OH of bimatoprost with butyl boronic acid to form bimatoprost butyl borate of formula (IV),

[0015]

[0016] C15-OH is then esterified by the reaction of bimatoprost butyl borate (IV) with 6-(nitrooxy) hexanoyl chloride, and the butyl borate protecting group is removed. The crude compound (I) is purified by flash chromatography. 6-(Nitrooxy) hexanoyl chloride is synthesized from 6-(nitrooxy) hexanoic acid, which is prepared by the ring-opening reaction of ε-caprolactone and the subsequent nitration of 6-hydroxyhexanoic acid alkali metal salt with a mixture of HNO3 and H2SO4 in dichloromethane. The intermediates 6-(nitrooxy) hexanoic acid and 6-(nitrooxy) hexanoyl chloride are used without purification. The resulting compound (I) contains 0.15% to 0.24% (HPLC area %) of bimatoprost 15-(6-chlorohexanoyl) ester (II) and 0.40% (HPLC area %) of 6-{[6-(nitrooxy) hexanoyl]oxy} hexanoic acid bimatoprost ester (III).

[0017] Bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) is a byproduct formed during the esterification step by the exchange reaction of the nitrooxy group of (1E,3S)-1-{(1S,5R,6R,7R)-3-butyl-7-[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate with the free chloride anion; at the end of the process, the boronate protecting group is removed to produce bimatoprost 15-(6-chlorohexanoyl) ester.

[0018] The 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) is derived from the reaction of the bimatoprost butyl borate of formula (IV) with 6-{[6-nitrooxyhexanoyl]oxy}hexanoyl chloride (which is an impurity of the intermediate 6-(nitrooxy)hexanoyl chloride); after esterification, the butyl borate protection is removed to produce the 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III).

[0019] WO 2021 / 023693 discloses an improvement to the method disclosed in WO 2019 / 162149, which is capable of reducing the formation of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III). In this method, at the end of the nitration step of the alkali metal salt of 6-hydroxyhexanoic acid, 6-(nitrooxy)hexanoic acid is purified by reverse phase chromatography.

[0020] The crude compound (I) prepared according to the method of WO 2021 / 023693 has an HPLC purity of 73% (HPLC area %) and contains approximately 0.1% (HPLC area %) of bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) and less than 0.05% (HPLC area %) (HPLC threshold) of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester (III) before the final chromatographic purification.

[0021] The synthesis of compound (I) disclosed in WO 2009 / 136281 (Example B-1) includes: protecting the C9-OH and C11-OH groups of bimatoprost with butyl boronic acid to form bimatoprost butyl borate of formula (IV), esterifying C15-OH with 6-bromohexanoyl chloride to obtain bimatoprost 15-(6-bromohexanoyl) ester in butyl borate protected form, converting it into a nitrate derivative by silver nitrate in acetonitrile, removing the butyl borate group, and purifying the product by reverse phase chromatography. The main disadvantages of the above synthesis are: the use of 6-bromohexanoyl chloride in excess of an equimolar amount, which has a warning structure of potential mutagenicity; the use of silver nitrate, which produces a large amount of silver salts in wastewater, may form bimatoprost 15-(6-bromohexanoyl) ester impurities produced by incomplete nitration of the intermediate bimatoprost butyl-boronate 15-(6-bromohexanoyl) ester, and the formation of bimatoprost 15-(6-chlorohexanoyl) ester byproduct of formula (II) produced by incomplete nitration of bimatoprost butyl-boronate 15-(6-chlorohexanoyl) ester. Bimatoprost butyl-boronate 15-(6-chlorohexanoyl) ester is a byproduct of the esterification reaction, which is formed by a halogen exchange reaction between the bromine atom of bimatoprost butyl-boronate 15-(6-bromohexanoyl) ester and free chloride anions, which are formed in the esterification step and whose formation is promoted by the presence of a base in the reaction medium.

[0022] WO 2009 / 136281 does not mention the impurity distribution of the final product. Experiments conducted by the inventors show that compound (I) prepared according to the method disclosed in WO 2009 / 136281 contains about 8.34% (HPLC area %) of bimatoprost 15-(6-chlorohexanoyl) ester of formula (II).

[0023] WO 2009 / 136281 also discloses an alternative method for preparing 15-acylalkyl nitrate bimatoprost derivatives (Examples N-1 and O-1). The synthesis includes reacting bimatoprost in a boronate protected form with a nitrate-alkyl carboxylic acid chloride in the presence of a resin-loaded 4-dimethylaminopyridine (DMAP) (PS-DMAP), removing the boronate protecting group, and purifying by silica gel column chromatography. Although this alternative method avoids the use of 6-bromohexanoyl chloride and removing silver salts, the main disadvantage of the method is the use of a resin-loaded 4-dimethylaminopyridine, which makes the method unsuitable for commercial scale-up and expensive, and in addition, a large excess of nitrate-alkyl carboxylic acid chloride is added.

[0024] WO2009 / 136281 also discloses another method for preparing 15-acylalkyl nitrate bimatoprost derivatives (Example Q1). In this method, the compound is obtained by esterifying bimatoprost butyl-boronate with an excess of nitrate-alkyl-(p-nitrophenyl)-carboxylate in the presence of 4-dimethylaminopyridine.

[0025] The major disadvantage of this synthesis is the removal of unreacted nitrate-alkyl-(p-nitrophenyl)-carboxylate and the by-product p-nitrophenol by chromatography.

[0026] Therefore, there is a need for high-purity 6-(nitrooxy)-hexanoic acid (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 preparation of ophthalmic pharmaceutical preparations.

[0027] The present invention solves the above problems and provides an industrially feasible method for preparing high-purity 6-(nitrooxy)-hexanoic acid (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). DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides a method for synthesizing 6-(nitrooxy)-hexanoic acid (1S, 2E)-3-[(1R, 2R, 3S, 5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propene-1-yl ester (compound (I)) of formula (I) suitable for industrial-scale production. The method can prepare high-purity compound (I) with high yield.

[0030] The method of the present invention comprises an efficient one-pot reaction preparation step for preparing a crude compound (I) from bimatoprost and 6-(nitrooxy)hexanoic acid, followed by an efficient purification step for the crude compound (I), which first comprises normal phase gravity silica gel column chromatography for removing almost all impurities originating from the preparation step, followed by silica gel filtration chromatography that can remove higher boiling point solvents. The method is suitable for large-scale preparation of compound (I), for example up to 650 grams. An important advantage of the process of the present invention is that the process is suitable for preparing high-purity 6-(nitrooxy)-hexanoic acid (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). In fact, the compound obtained by the process of the present invention does not contain bimatoprost 15-(6-chlorohexanoyl) ester (II) and the total impurity amount does not exceed (≤) 0.20% (HPLC area %). The high total chemical yield of about 70% and the efficient purification steps of the method make the method a cost-saving method that is easily applicable to the industrial-scale preparation of pharmaceutical-grade 6-(nitrooxy)-hexanoic acid (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] Another object of the present invention is 6-(Nitrooxy)-hexanoic acid (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 is substantially free of the impurity bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), and which contains a total impurity amount of no more than (≤) 0.20% (HPLC area %), preferably a total impurity amount of no more than (≤) 0.15% (HPLC area %), and most preferably a total impurity amount of no more than (≤) 0.10% (HPLC area %).

[0032] The above definitions "free of bimatoprost 15-(6-chlorohexanoyl) ester (II)" and "substantially free of the impurity bimatoprost 15-(6-chlorohexanoyl) ester of formula (II)" mean that the amount of the compound is below the detection limit (LoD) of the HPLC method disclosed below. DETAILED DESCRIPTION

[0033] The present invention relates to a method for preparing 6-(nitrooxy)-hexanoic acid (1S, 2E)-3-[(1R, 2R, 3S, 5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propene-1-yl ester of formula (I)

[0034]

[0035] The method comprises the following steps:

[0036] 1) reacting bimatoprost with phenylboronic acid in toluene at reflux temperature and removing water by azeotropic distillation to obtain bimatoprost phenylboronic acid ester of formula (V)

[0037]

[0038] 2) The solution was cooled to 25° C., and N,N′-diisopropylcarbodiimide, a catalytic amount of dimethylaminopyridine and 6-(nitrooxy)hexanoic acid were added to obtain 6-(nitrooxy)hexanoic acid (1E,3S)-1-{(1S,5R,6R,7R)-7-[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-3-phenyl-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-5-phenylpent-1-en-3-yl ester of formula (VI)

[0039]

[0040] 3) removing the phenylboronic acid ester protecting group under alkaline conditions;

[0041] 4) separating the organic phase, washing the organic phase, and evaporating the solvent;

[0042] 5) stirring the crude product in dichloromethane, filtering the mixture and evaporating the solvent to obtain a crude compound of formula (I);

[0043] 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 and water in a volume ratio of 40:15:0.5;

[0044] 7) collecting fractions of appropriate purity and evaporating the solvent to obtain a pure compound of formula (I);

[0045] 8) dissolving the pure compound of formula (I) obtained in step 7) in distilled dichloromethane and methanol, and purifying the solution by gravity chromatography using an eluent mixture containing distilled dichloromethane and methanol in a volume ratio of 30:1; collecting fractions of appropriate purity and evaporating the solvent;

[0046] 9) dissolving the pure compound of formula (I) obtained in step 8) in a suitable solvent, treating the resulting solution with activated carbon, then separating the activated carbon from the solution by filtration, and removing the solvent by vacuum evaporation to obtain pure 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propene-1-yl ester;

[0047] The method is characterized in that the obtained compound of formula (I) does not contain bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) and contains a total impurity amount of no more than (≤) 0.20% (HPLC area %); preferably, the total impurity amount does not exceed (≤) 0.1% (HPLC area %).

[0048] In the reaction step 1), the molar ratio of bimatoprost to phenylboronic acid is preferably 1:1.1.

[0049] In the 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.

[0050] In reaction step 3), the phenylboronic acid ester protecting group is preferably removed using a NaOH solution. In particular, the phenylboronic acid ester protecting group is removed by quenching the reaction mixture obtained in step 2) with methanol and then adding a mixture of dichloromethane and 6.3 equivalents of NaOH in the form of a 0.5M NaOH solution.

[0051] 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 NaCl aqueous solution.

[0052] Preferably, the process of the present invention yields a compound of formula (I) containing no more than (≤) 0.10% (HPLC area %) of total impurities.

[0053] Another embodiment of the present invention relates to a method for synthesizing 6-(nitrooxy)-hexanoic acid (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), comprising the following steps:

[0054] 1) reacting bimatoprost (1 equivalent) with phenylboronic acid (1.1 equivalent) in toluene to protect the 9,11-hydroxyl group of bimatoprost; heating the reaction mixture to the reflux temperature of toluene to remove water by azeotropic distillation, and stirring the reaction mixture at the reflux temperature until the bimatoprost phenyl-boronate of formula (V) is formed;

[0055] 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-oxohept-2-en-1-yl]-3-phenyl-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate of formula (VI);

[0056] 3) at the end of the esterification reaction, the reaction mixture is quenched with methanol, and then dichloromethane and 0.5M NaOH solution are added, after which the resulting mixture is stirred to complete the removal of the phenylboronic ester protecting group, wherein 6.3 equivalents of NaOH are preferably used;

[0057] 4) The organic phase was separated, washed twice with an aqueous sodium bisulfate solution, washed twice with a 15% w / w NaCl aqueous solution, and then the solvent was evaporated at a temperature below 45°C.

[0058] 5) adding dichloromethane to the crude mixture and stirring the mixture at 10° C. for 30 minutes, filtering to remove N,N′-diisopropylurea and evaporating the solvent of the filtered solution to obtain a crude compound of formula (I);

[0059] 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;

[0060] 7) collecting fractions of appropriate purity and evaporating the solvent to obtain a pure compound of formula (I);

[0061] 8) dissolving the pure compound of formula (I) obtained in step 7) and purifying the solution by gravity chromatography on a column packed with silica using an eluent mixture containing distilled dichloromethane / distilled methanol in a volume ratio of 30:1; collecting fractions of appropriate purity and evaporating the solvent to obtain the pure compound of formula (I) in the form of an oil;

[0062] 9) dissolving the pure compound of formula (I) obtained in step 8) in ethanol, treating the obtained solution with activated carbon, then removing the activated carbon by filtration and removing the ethanol by vacuum evaporation to obtain the final pure compound of formula (I), wherein the method is characterized in that the obtained compound of formula (I) does not contain bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) and contains a total impurity amount of not more than (≤) 0.20% (HPLC area %); preferably, the total impurity amount does not exceed (≤) 0.10% (HPLC area %).

[0063] The above-mentioned "equivalent" refers to the molar equivalent of each reagent, and it is calculated relative to the molar number of bimatoprost.

[0064] The above method is illustrated in the following process.

[0065]

[0066] The process of the present invention has several advantages, it eliminates the formation of bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) and reduces the amount of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of formula (III) to a quantification limit of less than 0.05% w / w. In addition, the purity evaluation of compound (I) by HPLC shows that the amount of impurities 5,6-trans compound (I) of formula (VII) and 15-epi-compound (I) of formula (VIII) is also no more than (≤) 0.05% w / w.

[0067] Another advantage of the process 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, wherein the above steps 1) to 3) are carried out without isolating or purifying the obtained intermediates.

[0068] The present invention provides 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propene-1-yl ester of formula (I), which does not contain the impurity bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), and the total impurity amount contained is not more than (≤) 0.20% (HPLC area %), preferably, the total impurity amount is not more than (≤) 0.15% (HPLC area %), and most preferably, the total impurity amount is not more than (≤) 0.10% (HPLC area %).

[0069] One embodiment of the present invention provides 6-(nitrooxy)-hexanoic acid (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 bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), wherein the compound of formula (I) comprises:

[0070] - not more than (≤) 0.05% w / w of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of formula (III);

[0071] - not more than (≤) 0.05% w / w of (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-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

[0072] - not more than (≤) 0.05% w / w of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate (15-epi-compound (I)) of formula (VIII); and

[0073] The total impurity content does not exceed (≤) 0.20% (HPLC area %).

[0074] Another embodiment of the present invention provides 6-(nitrooxy)hexanoic acid (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 bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), wherein the compound of formula (I) comprises:

[0075] - less than (<) 0.05% w / w of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of formula (III);

[0076] - not more than (≤) 0.05% w / w of (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-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

[0077] - less than (<) 0.05% w / w of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate of formula (VIII) (15-epi-compound (I)); and

[0078] The total impurity content does not exceed (≤) 0.20% (HPLC area %).

[0079] Another embodiment of the present invention provides 6-(nitrooxy)hexanoic acid (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 bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), wherein the compound of formula (I) comprises:

[0080] - less than (<) 0.05% w / w of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of formula (III);

[0081] - not more than (≤) 0.05% w / w of (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-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

[0082] - less than (<) 0.05% w / w of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate of formula (VIII) (15-epi-compound (I)); and

[0083] The total impurity content does not exceed (≤) 0.15% (HPLC area %).

[0084] Another embodiment of the present invention provides 6-(nitrooxy)hexanoic acid (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 bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), wherein the compound of formula (I) comprises:

[0085] - less than (<) 0.05% w / w of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of formula (III);

[0086] - not more than (≤) 0.05% w / w of (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-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

[0087] - less than (<) 0.05% w / w of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate of formula (VIII) (15-epi-compound (I)); and

[0088] The total impurity content does not exceed (≤) 0.10% (HPLC area %).

[0089] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising 6-(nitrooxy)-hexanoic acid (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) and at least one pharmaceutically acceptable excipient, wherein the compound of formula (I) does not contain bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) and contains no more than (≤) 0.20% (HPLC area %) of total impurities, preferably, the total impurities do not exceed (≤) 0.15% (HPLC area %), and most preferably, the total impurities do not exceed (≤) 0.10% (HPLC area %).

[0090] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising 6-(nitrooxy)-hexanoic acid (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) and at least one pharmaceutically acceptable excipient, wherein the compound of formula (I) does not contain bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), wherein the compound of formula (I) contains no more than (≤) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III), no more than (≤) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (VII), and no more than (≤) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III). -(nitrooxy)hexanoic acid (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (5,6-trans-compound (I)) and not more than (≤) 0.05% w / w of 6-(nitrooxy)hexanoic acid (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (15-epi-compound (I)), wherein the total impurity amount does not exceed (≤) 0.20% (HPLC area %).

[0091] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising 6-(nitrooxy)-hexanoic acid (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), wherein the compound of formula (I) does not contain bimatoprost 15-(6-chlorohexanoyl) ester 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), and no more than (≤) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (VII). -(nitrooxy)hexanoic acid (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (5,6-trans-compound (I)), less than (<) 0.05% w / w of 6-(nitrooxy)hexanoic acid (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (15-epi-compound (I)) of formula (VIII), wherein the total impurity amount does not exceed (≤) 0.20% (HPLC area %).

[0092] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising 6-(nitrooxy)-hexanoic acid (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), wherein the compound of formula (I) does not contain bimatoprost 15-(6-chlorohexanoyl) ester 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), and no more than (≤) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (VII). -(nitrooxy)hexanoic acid (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (5,6-trans-compound (I)), less than (<) 0.05% w / w of 6-(nitrooxy)hexanoic acid (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (15-epi-compound (I)) of formula (VIII), wherein the total impurity amount does not exceed (≤) 0.15% (HPLC area %).

[0093] Another embodiment of the present invention provides an ophthalmic pharmaceutical composition comprising 6-(nitrooxy)-hexanoic acid (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), wherein the compound of formula (I) does not contain bimatoprost 15-(6-chlorohexanoyl) ester 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), and no more than (≤) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (VII). -(nitrooxy)hexanoic acid (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (5,6-trans-compound (I)), less than (<) 0.05% w / w of 6-(nitrooxy)hexanoic acid (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester (15-epi-compound (I)) of formula (VIII), wherein the total impurity amount does not exceed (≤) 0.10% (HPLC area %).

[0094] The purity and impurity profile of Compound (I) were evaluated by the HPLC methods disclosed below (Method 1 and Method 2).

[0095] The above definition "free of bimatoprost 15-(6-chlorohexanoyl) ester" or "substantially free of bimatoprost 15-(6-chlorohexanoyl) ester" means that the amount of bimatoprost 15-(6-chlorohexanoyl) ester is below the detection limit (LoD) of the HPLC method disclosed below.

[0096] The above definition "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 quantitation (LoQ) of the HPLC method disclosed below.

[0097] The above definition "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 less than the quantification limit of the HPLC method disclosed below.

[0098] The above definition "the amount of 15-epi-Compound (I) is less than 0.05% w / w" means that the amount of 15-epi-Compound (I) is below the quantification limit of the HPLC method disclosed below.

[0099] Example 1

[0100] Synthesis of 6-(Nitrooxy)-hexanoic acid (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))

[0101] The starting material bimatoprost is commercially available.

[0102] 1a) Preparation of 6-(nitrooxy)hexanoic acid

[0103] Synthesis of 6-Hydroxyhexanoic Acid Methyl Ester

[0104] Anhydrous methanol (300 mL) was added to a 1L three-necked flask equipped with a thermometer, a dropping funnel, a reflux condenser and a magnetic stirrer, followed by concentrated sulfuric acid (2.8 mL, 0.052 mol, 0.06 eq). The mixture was heated to reflux temperature (T = 63 ° C). A solution of ε-caprolactone (100 g, 0.876 mol, 1 eq) in anhydrous methanol (200 mL) was added dropwise to the mixture. The reaction was stirred at reflux temperature for 2 hours. TLC was performed to monitor the residual ε-caprolactone. The reaction mixture was then cooled to 0-5 ° C. Concentrated NaHO3 solution (100 mL) was slowly added, keeping the temperature below 10 ° C. The volatile solvent was removed in vacuo on a rotary evaporator at 40 ° C. Water and methyl tert-butyl ether (MTBE) (300 mL) were added to the residue. The mixture was stirred for 5 minutes and then phase separated. The aqueous phase was extracted with MTBE (2×200 mL). The combined organic phases were washed with brine (2 x 100 mL). The organic phase was dried over Na2SO4 and then concentrated in vacuo on a rotary evaporator at 40°C to give methyl 6-hydroxyhexanoate (103.58 g, yield: 80.9%) as a colorless oil. GC purity: 96.2%.

[0105] Synthesis of 6-Nitrooxyhexanoic acid methyl ester

[0106] The concentrated sulfuric acid (45.6mL, 0.85mol, 3.1 equivalents) cooled to 0-5°C was added to a 500mL three-necked flask equipped with a thermometer, a dropping funnel and a magnetic stirrer. Then fuming nitric acid (47.2mL, 1.12mol, 4.1 equivalents) was added dropwise while maintaining the temperature at 0-5°C. Anhydrous dichloromethane (100mL, 2.5V) was slowly added to the cooled mixture and then stirred at 0-5°C for 30 minutes. At this temperature, anhydrous dichloromethane (100mL) solution of 6-hydroxyhexanoic acid methyl ester (40g, 0.27mol, 1 equivalent) was added over 1 hour. After stirring at 0-5°C for 30 minutes, the reaction mixture was monitored by TLC to show that the reaction was complete. The reaction mixture was dropped into cold water (200mL) (T<10°C~2 hours) to terminate the reaction. Each phase was then separated. The organic phase was dried over Na2SO4 and then concentrated on a rotary evaporator at 40°C in vacuo to give methyl 6-nitrooxyhexanoate (49.46 g) as a yellow oil in a yield of 94.5%.

[0107] Synthesis of crude 6-(nitrooxy)hexanoic acid

[0108] In a 1L three-necked flask equipped with a thermometer, a dropping funnel and a magnetic stirrer, a 1.5M NaOH aqueous solution (208mL) was added. At room temperature, 6-nitrooxyhexanoic acid methyl ester (49.64g, 0.26mol, 1 equivalent) dissolved in methanol (248mL, 5V) was added dropwise (Tmax=35°C). After addition, the mixture was stirred at room temperature for 2 hours. The reaction mixture was monitored by TLC, indicating that the reaction was complete. The pH value of the reaction mixture was adjusted to 2.5 using 183mL 2M HCl aqueous solution. The mixture obtained was extracted with MTBE (100mL), and then the aqueous phase was extracted with MTBE (5×50mL). The combined organic phase was washed with brine (2×30mL), and then concentrated in vacuo on a rotary evaporator at a temperature of 40°C to obtain a yellow oily 6-(nitrooxy)hexanoic acid crude product (44.2g). Evaluated and analyzed by UPLC method.

[0109] Purification of 6-(Nitrooxy)hexanoic acid

[0110] Crude 6-(nitrooxy)hexanoic acid was purified by column chromatography according to the following procedure:

[0111] The eluent was prepared by mixing toluene and methanol in a volume ratio of 40: 1. The silica and eluent were stirred together in a slurry distributor and loaded and prepared on the column. The crude material (1161 g, 91.8% by UPLC analysis) was dissolved in toluene and the solution was loaded on the top of the column. The elution was carried out at a flow rate of 15 ± 5 L / h, and the fractions were collected in 10 L portions and analyzed by TLC. The purest fractions were combined and concentrated under vacuum at a temperature below 55 ° C ± 5 ° C. Purified 6-(nitrooxy)hexanoic acid (952 g, 5.37 mol) was isolated as an oil with a yield of 82% and a UPLC purity of 98.7%, containing an undetectable amount of 6-{[6-nitrooxyhexanoyl]oxy}hexanoic acid.

[0112] 1b) Synthesis of crude 6-(nitrooxy)-hexanoic acid (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))

[0113] Toluene (28 Kg, 49 volumes) was added to the reactor, followed by bimatoprost (663 g, 1.595 mol, 1 eq) and phenylboronic acid (214 g, 1.755 mol, 1.1 eq). The reaction was heated at 110°C ± 5°C and the vapor temperature was maintained at 90°C for 4 hours to ensure azeotropic distillation of water, with a total solvent volume of about 12 L. The reaction mixture was then cooled to 25°C ± 5°C.

[0114] 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 a solution of 6-(nitrooxy)hexanoic acid (565 g, 3.190 mol, 2.0 eq) in toluene (0.5 L, 0.75 vol). The reaction was stirred at 25°C ± 5°C for 1 to 3 hours. TLC was performed to monitor the residual bimatoprost phenyl-boronic acid ester (compound (V)).

[0115] The reaction mixture was quenched with methanol (1.3L, 2 volumes), stirred for 5-10 minutes, and then the reaction mixture was transferred to an extractor containing 0.5M sodium hydroxide solution (20L) and dichloromethane (10L, 15 volumes) by vacuum. The reactor was washed with several portions of dichloromethane and dichloromethane was added to the reaction mixture. The reaction mixture was stirred for 2 to 3 hours, then allowed to stand for 60 minutes, and the organic phase was collected in a mobile storage tank. Dichloromethane (10L, 15 volumes) was added to the extractor, the mixture was stirred for 5-10 minutes and allowed to stand for 15 minutes. The organic layer was collected. The organic layer was added to the extractor again, and 1M NaHO4 solution (20L) was transferred to the extractor by vacuum. The mixture was stirred for 5 to 10 minutes, allowed to stand for 15 minutes, and the lower organic phase was collected in a mobile storage tank. The water layer was removed. The organic phase was added to the extractor again, 15% NaCl solution (20L) was added, the mixture was stirred for 5-10 minutes, and allowed to stand for 15 minutes. Collect the organic layer and remove the aqueous phase. Add the organic layer to the extractor again and add 15% NaCl w / w solution (20 L). Stir the mixture for 5 to 10 minutes and let stand for 15 minutes. Collect the organic layer and evaporate in vacuo at a temperature of 45°C ± 5°C.

[0116] The crude product is dissolved in dichloromethane (7L, 10.5 volumes), and the mixture is transferred to a reactor. The mixture is cooled to 5-10 ° C and kept at this temperature for 30-60 minutes to precipitate N, N'-diisopropylurea. The mixture is filtered into a mobile storage tank through a filter. The reactor is washed with dichloromethane (2L, 3 volumes), the solution is filtered and collected in a mobile storage tank. The solution in the mobile storage tank is then transferred to a flask, and the solvent is evaporated in vacuo at a temperature of 45 ° C ± 5 ° C to obtain crude 6- (nitrooxy) -hexanoic acid (1S, 2E) -3- [(1R, 2R, 3S, 5R) -2- [(2Z) -7- (ethylamino) -7- oxo -2- heptene -1- base] -3,5- dihydroxycyclopentyl] -1- (2-phenylethyl) -2- propene -1- base ester (crude compound I).

[0117] 1c) Purification of crude 6-(nitrooxy)-hexanoic acid (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))

[0118] First Chromatography

[0119] Prepare eluent mixture 1 by mixing diisopropyl ether / acetone / water in a volume ratio of 40:15:0.5.

[0120] Silica gel (30 Kg, 45 vol) was suspended in Eluent Mixture 1 (66.3 Kg, 100 vol), the suspension was stirred for 3 minutes and loaded into the column.

[0121] The crude product was dissolved in an eluent mixture (1 L, 1.5 volumes) and loaded onto the top of the column. Elution was performed, and fractions were collected in 15 L portions and analyzed by TLC. Fractions were combined according to purity and analyzed by HPLC and UPLC.

[0122] Selected fractions of desired purity were collected and the solvent was evaporated to dryness using a rotary evaporator at a temperature of 45° C.±5° C. The residue was dissolved in dichloromethane (5 L, 7.5 volumes) and the solvent was evaporated to dryness again.

[0123] Compound (I) was stored at 5°C ± 3°C.

[0124] Silica gel filtration chromatography

[0125] Eluent mixture 2 was prepared by mixing distilled dichloromethane and distilled methanol in a volume ratio of 30:1.

[0126] Silica 75S (6.6 Kg, 10 volumes) was added to eluent mixture 2 (13 Kg, 19.6 volumes), stirred and the suspension was 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.

[0127] Elution was performed, fractions were controlled by TLC, selected fractions having the desired purity were collected; the combined fractions were analyzed by HPLC and UPLC.

[0128] The fractions were evaporated in vacuo in a rotary evaporator at 45° C.±5° C. The isolated pure compound (I) was stored at 5° C.±3° C. as an oil.

[0129] Treatment with activated carbon (clarification)

[0130] 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 under vacuum at 45°C ± 5°C until constant weight; 649.6 g (1.13 mol) of pure compound (I) was isolated as an oil. The overall yield of the process was 70.8%.

[0131] The purity of the isolated compound (I) was evaluated by HPLC analysis using the following two methods. The HPLC analysis purity of the compound (I) evaluated by HPLC / method 1 was 99.91% (HPLC area %).

[0132] The limits of detection (LoD) and quantification (LoQ) of bimatoprost 15-(6-chlorohexanoyl) ester of formula (II), bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of formula (III), 5,6-trans-compound (I) of formula (VII) and 15-epi-compound (I) of formula (VIII) are reported in Table 1.

[0133] The impurity profile of the isolated compound (I) is reported in Table 2.

[0134] Table 3 reports the content of the impurity bimatoprost 15-(6-chlorohexanoyl) ester (II) in compound (I) prepared according to the method disclosed in the prior art.

[0135] Method 1 : HPLC analysis was performed using the following assay conditions:

[0136] Instrument: Waters HPLC system with PDA detector Stationary phase: Zorbax RX SIL, 250 × 4.6 mm, 5 μm

[0137] Column temperature: 35°C

[0138] Mobile phase: hexane: ethanol: acetonitrile: acetic acid = 935:60:5:0.5

[0139] Running time: 60 minutes

[0140] Flow rate: 1.5ml / min

[0141] Injection volume: 20 μL

[0142] Detection: UV, 210nm

[0143] Sample solvent: ethanol: hexane 2:8

[0144] Method 2 : HPLC analysis was performed to quantify the 15-epi-compound (I) of formula (VIII) using the following measurement conditions:

[0145] Instrument: Waters HPLC system with PDA detector Stationary phase: Chiralpak AD-H, 250×4.6 mm, 5 μm

[0146] Column temperature: 25°C

[0147] Mobile phase: Hexane:Ethanol:TFA-850:150:1

[0148] Run time: 30 minutes

[0149] Flow rate: 1.0mL / min

[0150] Injection volume: 10 μL

[0151] Detection: 210nm

[0152] Sample solvent: hexane: ethanol - 8:2

[0153]

[0154]

Claims

1. A method for preparing 6-(nitrooxy)-hexanoic acid (1S, 2E)-3-[(1R, 2R, 3S, 5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propene-1-yl ester of formula (I) The following steps are involved: 1) reacting bimatoprost with phenylboronic acid in toluene at reflux temperature and removing water by azeotropic distillation to obtain bimatoprost phenylboronic acid ester of formula (V) 2) The solution was cooled to 25° C., and N,N′-diisopropylcarbodiimide, a catalytic amount of dimethylaminopyridine and 6-(nitrooxy)hexanoic acid were added to obtain 6-(nitrooxy)hexanoic acid (1E,3S)-1-{(1S,5R,6R,7R)-7-[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-3-phenyl-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-5-phenylpent-1-en-3-yl ester of formula (VI) 3) removing the phenylboronic acid ester protecting group under alkaline conditions; 4) separating the organic phase and evaporating the solvent; 5) stirring the crude product in dichloromethane, filtering the mixture and evaporating the solvent to obtain a 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 and water in a volume ratio of 40:15:0.5; 7) collecting fractions of appropriate purity and evaporating the solvent to obtain a pure compound of formula (I); 8) dissolving the pure compound of formula (I) obtained in step 7) in distilled dichloromethane and methanol, purifying the solution by gravity chromatography using an eluent mixture containing distilled dichloromethane 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) obtained in step 8) in ethanol, treating the solution with activated carbon, filtering to remove the activated carbon, and removing the solvent by vacuum evaporation to obtain pure 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propene-1-yl ester; The method is characterized in that the obtained 6-(nitrooxy)-hexanoic acid (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 bimatoprost 15-(6-chlorohexanoyl) ester of formula (II) and contains a total impurity amount of no more than (≤) 0.20% (HPLC area %).

2. The method according to claim 1, wherein: In the reaction step 1), the molar ratio of bimatoprost to phenylboronic acid is 1:1.

1.

3. The method according to claim 1 or 2, wherein in the reaction step 2), 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.

4. The process according to any one of the preceding claims, wherein in reaction step 3), the phenylboronic ester protecting group is removed using NaOH solution.

5. The process of claim 4, wherein the phenylboronic ester protecting group is removed by quenching the reaction mixture obtained in step 2) with methanol followed by addition of a mixture of dichloromethane and 6.3 equivalents of NaOH in the form of a 0.5 M NaOH solution.

6. A method according to any one of the preceding claims, wherein: In reaction step 4), the organic phase was first washed with an aqueous sodium hydrogen sulfate solution and twice with a 15% w / w NaCl aqueous solution.

7. A compound of formula (I) containing a total impurity content of no more than (≤) 0.20% (HPLC area %) and containing no impurity of formula (II) bimatoprost 15-(6-chlorohexanoyl) ester Or its content is below the detection limit of HPLC.

8. The compound of formula (I) according to claim 7, wherein the HPLC detection limit is 0.01% w / w.

9. The compound of formula (I) according to claim 7, wherein the total amount of impurities does not exceed (≤) 0.15% (HPLC area %).

10. The compound of formula (I) according to claim 9, wherein the total amount of impurities does not exceed (≤) 0.10% (HPLC area %).

11. The compound of formula (I) according to any one of claims 7 to 10, wherein the compound of formula (I) contains no more than (≤) 0.05% w / w of the impurity 6-{[6-(nitrooxy)hexanoyl]oxy}bimatoprost hexanoate of formula (III) and no more than (≤) 0.05% w / w of the impurity 6-(nitrooxy)hexanoic acid (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester of formula (VII) and no more than (≤) 0.05% of the impurity 6-(nitrooxy)hexanoic acid (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl ester of formula (VIII) 12. The compound of formula (I) according to any one of claims 7 to 11, 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) and not more than (≤) 0.05% w / w of (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate of formula (VII) and less than (<) 0.05% of (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate of formula (VIII) 13. An ophthalmic pharmaceutical composition comprising a compound of formula (I) according to any one of claims 7 to 12 and at least one pharmaceutically acceptable excipient.

Images