Industrial process for 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 and high pure product
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- NICOX SA
- Filing Date
- 2023-07-06
- Publication Date
- 2026-07-10
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Figure 2024012261000001 
Figure 2024012261000002 
Figure 2024012261000003
Abstract
Description
[Technical field]
[0001] FIELD OF THEINVENTION The present invention relates to a process suitable for industrial scale production for the preparation of highly 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, which is substantially free of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost. The present invention relates to an ophthalmic pharmaceutical formulation containing said highly purified compounds. [Background technology]
[0002] 2. Background of the Invention 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, hereinafter also referred to as compound (I), has the following formula (I): [ka] has.
[0003] The chemical name of compound (I) is also (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.
[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 proven 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 efficacy as an intraocular pressure (IOP) lowering agent in patients with open-angle glaucoma or ocular hypertension has been evaluated.
[0005] Studying and monitoring the presence of impurities in active pharmaceutical ingredient(s) (API(s)) is a central topic in pharmaceutical manufacturing to ensure an adequate quality level in the manufactured product. The limits of each impurity in the manufactured API are the subject of specific guidelines published by international organizations. In the last years, there has been an increasing importance given to the quantification of impurities with potential genotoxicity, i.e. impurities that can cause DNA damage with gene mutations. Specifically, the limits of impurities with one or more alert functions are established according to specific guidelines.
[0006] 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 has been reported; the synthesis method is disclosed in the original compound patent WO2009 / 136281 and in the preparation method patent applications WO2019 / 162149 and WO2021 / 023693. The compound (I) prepared according to the methods disclosed in WO2019 / 162149 and WO2021 / 023693 has the potentially genotoxic compound of formula (II): [ka] 15-(6-chlorohexanoyl) ester of bimatoprost, and Formula (III): [ka] It contains two main impurities, which are 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost.
[0007] Another known chemical impurity contained in compound (I) is represented by formula (VII): [ka] the 5,6-trans-compound (I) (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, and Formula (VIII): [ka] The 15-epi-compound (I) is (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 and isomeric impurities such as
[0008] Pharmaceutical grade compound (I) is oily and cannot be purified by crystallization. Moreover, compound (I) and impurities 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost of formula (III) have similar polarity, so the presence of these impurities is a significant problem for the large-scale production of pharmaceutical grade compound (I). Therefore, the removal of these impurities requires several purification steps, which reduces the yield of the process and increases the cost of preparing compound (I) on a commercial scale.
[0009] WO 2019 / 162149 discloses an industrial process for the preparation of compound (I), comprising the steps of: [ka] The method includes protecting the C9-OH and C11-OH of bimatoprost with butyl-boronic acid to form bimatoprost butyl-boronate of formula (IV), followed by esterification of the C15-OH by reacting bimatoprost butyl-boronate (IV) with 6-(nitrooxy)hexanoyl chloride, and removing the butyl-boronate protecting group. The crude compound (I) is purified by flash chromatography. The 6-(nitrooxy)hexanoyl chloride is synthesized from 6-(nitrooxy)hexanoic acid, which is prepared by ring-opening reaction of ε-caprolactone, followed by nitration of 6-hydroxyhexanoic acid alkali 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 15-(6-chlorohexanoyl) ester of bimatoprost (II) and 0.40% (HPLC area %) of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester (III).
[0010] The 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) is a by-product formed during the esterification step by 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]octan-6-yl}-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate with free chlorine anion; at the end of the process, removal of the boronate ester protecting group results in the 15-(6-chlorohexanoyl) bimatoprost ester.
[0011] Bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of formula (III) is derived from the reaction of bimatoprost butyl-boronate of formula (IV) with 6-[6-nitrooxyhexanoyl]oxy}hexanoyl chloride, an impurity of the intermediate 6-(nitrooxy)hexanoyl chloride, and after esterification, removal of the butyl-boronate protection results in bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of formula (III).
[0012] WO 2021 / 023693 disclosed an improvement to the process disclosed in WO 2019 / 162149 that allows to reduce the formation of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III), in which 6-(nitrooxy)hexanoic acid is purified by reversed-phase chromatography at the end of the nitration step of 6-hydroxyhexanoic acid alkaline salt.
[0013] Crude compound (I) prepared according to the method of WO 2021 / 023693 has an HPLC purity of 73% (HPLC area %) and contains, before final chromatographic purification, approximately 0.1% (HPLC area %) of 15-(6-chlorohexanoyl) bimatoprost ester of formula (II) and less than 0.05% (HPLC area %) (HPLC threshold) of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester (III).
[0014] The synthesis of compound (I) (Example B-1) disclosed in WO 2009 / 136281 includes the protection of 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 bimatoprost in butyl-boronate protected form, which is converted to its nitrate derivative with 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 structural alerts to possible mutagenicity; the use of silver nitrate, which generates large amounts of silver salts in wastewater; the possible formation of the impurity 15-(6-bromohexanoyl) ester of bimatoprost, derived from 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), derived from incomplete nitration of the 15-(6-chlorohexanoyl) ester of bimatoprost butyl-boronate. 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 15-(6-bromohexanoyl) ester of bimatoprost butyl-boronate and a free chlorine anion, which is formed during the esterification process and is promoted by the presence of a base in the reaction medium.
[0015] WO 2009 / 136281 does not mention the impurity profile of the final product, and experiments carried out by the inventors showed that compound (I) prepared according to the method disclosed in WO 2009 / 136281 contains about 8.34% (HPLC area %) of the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II).
[0016] WO 2009 / 136281 also discloses an alternative method for the preparation of 15-acyl alkylnitrate bimatoprost derivatives (Examples N-1 and O-1). The synthesis involves reacting boronate-protected bimatoprost with nitrate-alkyl carboxylic acid chloride in the presence of 4-dimethylaminopyridine (DMAP) supported on resin (PS-DMAP), removing boronate protecting group, and purifying by silica gel column chromatography. This alternative method avoids the use of 6-bromohexanoyl chloride and removing the silver salt, but the main drawback of the method is the use of 4-dimethylaminopyridine supported on resin, which makes the method unsuitable and expensive for commercial scale-up, and the addition of nitrate-alkyl carboxylic acid chloride in large excess.
[0017] WO 2009 / 136281 also discloses another method (Example Q1) for the preparation of 15-acylalkynitrate bimatoprost derivatives, in which the compounds are 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 drawback of this synthesis is the removal of unreacted nitrato-alkyl-(p-nitrophenyl)-carboxylates and the by-product p-nitrophenol by chromatographic methods.
[0019] Thus, a need exists for 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 that is suitable for the manufacture of ophthalmic pharmaceutical formulations.
[0020] The present invention solves the above problems and provides an industrially viable process for the preparation of highly 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 of formula (I). Summary of the Invention
[0021] Description of the Invention The present invention provides a process 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) (compound (I)) suitable for industrial scale production, which allows to prepare compound (I) in high yield and high purity.
[0022] The method of the present invention comprises an efficient one-pot reaction preparation process of crude compound (I) from bimatoprost and 6-(nitrooxy)hexanoic acid, followed by a highly efficient purification process of crude compound (I), which comprises firstly normal phase gravity silica gel column chromatography to remove almost all impurities derived from the preparation process, and then silica gel filtration chromatography with a working area to remove higher boiling point solvents.The method is applicable to large-scale preparation of compound (I), for example up to 650 grams. An important advantage of the process of the present invention is that it 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 process of the present invention does not contain 15-(6-chlorohexanoyl) ester of bimatoprost (II), and the total impurity amount is (≦) 0.20% (HPLC area %) or less. The high overall chemical yield of about 70% and efficient purification steps of the process make it a cost-saving method easily applicable for industrial scale preparation of pharmaceutical grade 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).
[0023] Another object of the present invention is 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), substantially free of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and which contains a total impurity amount of less than or equal to (≦) 0.20% (HPLC area %), preferably, the total impurity amount is less than or equal to (≦) 0.15% (HPLC area %), and most preferably, the total impurity amount is less than or equal to (≦) 0.10% (HPLC area %).
[0024] The above definitions "free of 15-(6-chlorohexanoyl) ester of bimatoprost (II)" and "substantially free of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost 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 compound of formula (I): [ka] 5, wherein the 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 The method comprises the steps of: 1) Bimatoprost is reacted with phenylboronic acid in toluene at reflux temperature and water is removed by azeotropic distillation to give the compound of formula (V): [ka] obtaining bimatoprost phenyl-boronate; 2) The solution is cooled to 25° C., and N,N′-diisopropylcarbodiimide, a catalytic amount of dimethylaminopyridine, and 6-(nitrooxy)hexanoic acid are added to obtain a compound of formula (VI): [ka] obtaining (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]octan-6-yl}-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate; 3) removing the phenyl-boronate protecting group under basic conditions; 4) separating the organic phase, washing the organic phase and evaporating the solvent; 5) stirring the raw product in dichloromethane, filtering the mixture and evaporating the solvent 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 and water in a volume ratio of 40:15:0.5; 7) collecting fractions of suitable purity and evaporating the solvent to obtain the pure compound of formula (I); 8) dissolving the pure compound of formula (I) of step 7) in distilled methylene chloride and methanol and 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 suitable purity and evaporating the solvent; 9) dissolving the pure compound of formula (I) of step 8) in a suitable solvent and treating the resulting solution with activated charcoal, followed by separating the activated charcoal from the solution by filtration and removing the solvent by evaporation under vacuum to yield 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. Including, The method is characterized in that the compound of formula (I) obtained does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and has a total impurity content of (≦) 0.20% (HPLC area%) or less, preferably, the total impurity content is (≦) 0.1% (HPLC area%) or less.
[0026] In the 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 a NaOH solution. Specifically, the phenylboronate protecting group is removed by quenching the reaction mixture obtained in step 2) with methanol, followed by adding a mixture of methylene chloride and 6.3 equivalents of NaOH as a solution of 0.5M NaOH.
[0028] Preferably, in reaction step 4), the organic phase is first washed with an aqueous solution of sodium hydrogen sulfate and then washed twice with an aqueous solution of 15% w / w NaCl.
[0029] Preferably, the process of the present invention provides a compound of formula (I) containing no more than (≦) 0.10% (HPLC area %) total impurities.
[0030] Another embodiment of the present invention is a process comprising the steps of: 1) reacting bimatoprost (1 equivalent) with phenylboronic acid (1.1 equivalents) in toluene to protect the 9,11-hydroxy group of bimatoprost; warming the reaction mixture to the reflux temperature of toluene, removing water by azeotropic distillation, and stirring the reaction mixture at 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-oxohept-2-en-1-yl]-3-phenyl-2,4-dioxa-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, the reaction mixture is quenched with methanol, followed by the addition of methylene chloride and 0.5M NaOH solution, followed by 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 hydrogen sulfate and then twice with an aqueous solution of 15% w / w NaCl, followed by evaporating the solvent at a temperature below 45° C. 5) adding dichloromethane to the raw 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 suitable purity and evaporating the solvent to obtain the pure compound of formula (I); 8) dissolving the pure compound of formula (I) obtained in step 7) and purifying said 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; collecting fractions of suitable purity and evaporating the solvent to obtain the pure compound of formula (I) as an oil; 9) dissolving the pure compound of formula (I) obtained in step 8) in ethanol and treating the resulting solution with activated charcoal, followed by removal of the activated charcoal by filtration and removal of the ethanol by evaporation under vacuum to yield the final pure compound of formula (I). Including, The compound of formula (I) obtained does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and has a total impurity content of (≦) 0.20% (HPLC area%) or less, preferably, the total impurity content is (≦) 0.10% (HPLC area%) or less. The present invention relates to a process 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 "equiv." (equivalent) means the molar equivalent of each reagent and is calculated relative to the moles of bimatoprost.
[0032] The method reported above is shown in the following scheme: [ka] As exemplified in.
[0033] The method of the present invention has several advantages, it allows to eliminate the formation of 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and reduce the amount of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) to less than the limit of quantification, which is 0.05% w / w. Furthermore, the evaluation of the purity of compound (I) carried out by HPLC also showed that the amount of each of the impurities 5,6-trans-compound (I) of formula (VII) and 15-epi-compound (I) of formula (VIII) is also less than (≦) 0.05% w / w.
[0034] 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 in which steps 1) to 3) reported above are carried out without isolation or purification of the resulting intermediates.
[0035] The present invention provides 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 impurity 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and which contains a total impurity amount of (≦) 0.20% (HPLC area %) or less, 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 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) has the following structure: (≦) 0.05% w / w or less of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III); (≦) 0.05% w / w or less 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 (≦)0.05% or less 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)) Contains; and Here, the total amount of impurities is (≦) 0.20% (HPLC area %) or less.
[0037] Another embodiment of the present invention provides 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 said compound of formula (I) is as follows: (<)0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III); (≦) 0.05% w / w or less 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 (<)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) Contains; and Here, the total amount of impurities is (≦) 0.20% (HPLC area %) or less.
[0038] Another embodiment of the present invention provides 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 (<) less than 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III); (≦) 0.05% w / w or less 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 (<) 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)) Contains; and Here, the total amount of impurities is (≦) 0.15% (HPLC area %) or less.
[0039] Another embodiment of the present invention provides 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 (<)0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III); (≦) 0.05% w / w or less 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 (<) 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)) Contains; and Here, the total amount of impurities is (≦) 0.10% (HPLC area %) or less.
[0040] 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) and at least one pharma- ceutically acceptable excipient, which does not contain the (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and contains a total impurity amount of (≦) 0.20% (HPLC area %) or less, 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.
[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) which does not contain (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and at least one pharma- ceutical acceptable excipient, wherein said compound of formula (I) is present in an amount of (≦) 0.05% w / w or less of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost of formula (III) esters; (≦) 0.05% w / w or less 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 (5,6-trans-compound (I)) of formula (VII); (≦) 0.05% w / w or less of containing (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), wherein the amount of total impurities is less than or equal to (≦) 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-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) which does not contain (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and at least one pharma- ceutical acceptable excipient, wherein said compound of formula (I) is present in an amount of less than (<) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost of formula (III) esters; (≦) 0.05% w / w or less 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 (5,6-trans-compound (I)) of formula (VII); (<) 0.05% w / w or less 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 (5,6-trans-compound (I)) of formula (VII); III), (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 (containing 15-epi-compound (I)), and wherein the amount of total impurities is less than or equal to (maximum) 0.20% (HPLC area %).
[0043] 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) and at least one pharma- ceutical acceptable excipient, which does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) is present in an amount of less than (<) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost of formula (III). esters; (≦) 0.05% w / w or less 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 (5,6-trans-compound (I)) of formula (VII); (<) 0.05% w / w or less 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 (5,6-trans-compound (I)) of formula (VII); III) (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))), and wherein the amount of total impurities is less than or equal to (≦) 0.15% (HPLC area %).
[0044] 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) and at least one pharma- ceutical acceptable excipient, which does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) is present in an amount of less than (<) 0.05% w / w of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost of formula (III). esters; (≦) 0.05% w / w or less 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 (5,6-trans-compound (I)) of formula (VII); (<) 0.05% w / w or less 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 (5,6-trans-compound (I)) of formula (VII); VIII) (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)), and wherein the amount of total impurities is less than or equal to (≦) 0.10% (HPLC area %).
[0045] The purity and impurity profile of Compound (I) was assessed by the HPLC methods disclosed herein (Method 1 and Method 2).
[0046] The definitions "free of the 15-(6-chlorohexanoyl) ester of bimatoprost" or "substantially free of the 15-(6-chlorohexanoyl) ester of bimatoprost" reported above mean that the amount of the 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}hexanoic acid ester is less than 0.05% w / w" means that the amount of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester is below the limit of quantitation (LoQ) of the HPLC method disclosed herein.
[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 herein.
[0049] The above reported definition "an 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 herein. 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 A 1 L three-neck 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 moles, 0.06 equiv). The mixture was heated to reflux temperature (T=63°C). To the mixture was added a solution of ε-caprolactone (100 g, 0.876 moles, 1 equiv) in dry methanol (200 mL). The reaction was stirred at reflux temperature for 2 h. TLC was performed to monitor residual ε-caprolactone. The reaction mixture was then cooled to 0-5°C. Concentrated NaHCO3 solution (100 mL) was added slowly, keeping the temperature below 10°C. Volatile solvents were removed on a rotary evaporator under vacuum at a temperature of 40°C. 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 phase was washed with brine (2×100 mL). The organic phase was dried over Na2SO4 and then concentrated on a rotary evaporator under vacuum at a temperature of 40° C. to give methyl 6-hydroxyhexanoate (103.58 g, yield: 80.9%) as a colorless oil. GC purity: 96.2%.
[0054] Synthesis of methyl 6-nitrooxyhexanoate A 500 mL three-neck flask equipped with a thermometer, dropping funnel and magnetic stirrer was charged with concentrated sulfuric acid (45.6 mL, 0.85 moles, 3.1 equiv.) cooled to 0-5 °C. Then, fuming nitric acid (47.2 mL, 1.12 moles, 4.1 equiv.) was added dropwise thereto, keeping the temperature between 0-5 °C. To the cooled mixture, dry dichloromethane (100 mL, 2.5 vol.) was slowly added and then stirred between 0-5 °C for 30 min. At this temperature, methyl-6-hydroxyhexanoate (40 g, 0.27 moles, 1 equiv.) in dry (dichloromethane) DCM (100 mL) was added in 1 h. After stirring for 30 min at 0-5 °C, the reaction mixture was monitored by TLC, which showed the completion of the reaction. The reaction mixture was quenched by adding it dropwise to chilled water (200 mL) (T<10 °C, maximum 2 h). The phases were then separated. The organic phase was dried over Na2SO4 and then concentrated on a rotary evaporator under vacuum at 40° C. to give methyl-6-nitrooxyhexanoate (49.46 g) as a yellow oil in 94.5% yield.
[0055] Synthesis of crude 6-(nitrooxy)hexanoic acid A 1 L three-neck flask equipped with a thermometer, dropping funnel, and magnetic stirrer was charged with 1.5 M aqueous NaOH (208 mL). Methyl-6-nitrooxyhexanoate (49.64 g, 0.26 mol, 1 equiv.) dissolved in methanol (248 mL, 5 vol.) was heated at room temperature (T 最大 = 35 °C). After the addition, the mixture was stirred at room temperature for 2 h. The reaction mixture was checked by TLC, which showed the reaction was complete. The pH of the reaction mixture was set to 2.5 using 183 mL of 2 M HCl aqueous solution. The resulting mixture was extracted with MTBE (100 mL), and then the aqueous phase was extracted with MTBE (5 x 50 mL). The combined organic phase was washed with brine (2 x 30 mL), and then concentrated on a rotary evaporator under vacuum at a temperature of 40 °C to obtain crude 6-(nitrooxy)hexanoic acid (44.2 g) as a yellow oil. The assay was evaluated by UPLC method.
[0056] Purification of 6-(nitrooxy)hexanoic acid The 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. The column was prepared by stirring the silica with the eluent in a slurry dispenser and loading it onto 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 performed at a flow rate of 15±5 L per hour, and 10 L fractions were collected and analyzed by TLC. The purest fractions were combined and concentrated under vacuum at a temperature below 55° C.±5° C. The purified 6-(nitrooxy)hexanoic acid (952 g, 5.37 moles) was isolated in 82% yield as an oil with a purity of 98.7% by UPLC and 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)) The reactor was charged with toluene (28 Kg, 49 vol), followed by bimatoprost (663 g, 1.595 moles, 1 equiv.) and phenylboronic acid (214 g, 1.755 moles, 1.1 equiv.). The reaction was heated to 110° C.±5° C. and the vapor temperature was maintained at 90° C. for 4 hours, ensuring azeotropic distillation with water for a total volume of approximately 12 L of solvent. The reaction mixture was then cooled to 25° C.±5° C.
[0058] Dimethylaminopyridine (DMAP) (39 g, 0.319 moles, 0.2 equiv.) and N,N'-diisopropylcarbodiimide (DIC) (403 g, 3,190 moles, 2.0 equiv.) were added to the cooled reaction mixture, followed by a solution of 6-(nitrooxy)hexanoic acid (565 g, 3.190 moles, 2.0 equiv.) 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-boronate (compound (V)).
[0059] The reaction mixture was quenched with methanol (1.3 L, 2 vol) and stirred for 5-10 min. 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 vol). The reactor was washed several times with methylene chloride, which was added to the reaction mixture. The reaction mixture was stirred for 2-3 h, then allowed to stand for 60 min, and the organic phase was collected in a transfer tank. Dichloromethane (10 L, 15 vol) was charged to the extractor, the mixture was stirred for 5-10 min, and allowed to stand for 15 min. The organic layer was collected. The organic layer was again charged to the extractor and 1 M NaHSO4 solution (20 L) was transferred via vacuum to the extractor. The mixture was stirred for 5-10 min, allowed to stand for 15 min, 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 and 15% NaCl solution (20 L) was added and the mixture was stirred for 5 to 10 minutes and allowed to stand 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 allowed to stand for 15 minutes. The organic layer was collected and evaporated under vacuum at a temperature of 45°C ± 5°C.
[0060] The crude product was dissolved in dichloromethane (7 L, 10.5 vol) and the mixture was transferred to a reactor. The mixture was cooled to 5-10° C. and held at this temperature for 30-60 min to precipitate N,N'-diisopropylurea. The mixture was filtered through a filter into a transfer tank. The reactor was washed with dichloromethane (2 L, 3 vol) and the solution was filtered and collected in the transfer tank. The solution in the transfer tank was then 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 vol) was suspended in Eluent Mixture 1 (66.3 Kg, 100 vol) and the suspension was stirred for 3 minutes and loaded onto the column.
[0064] The crude product was dissolved in the eluent mixture (1 L, 1.5 volumes) and loaded onto the top of the column. Elution was performed and 15 L fractions were collected and analyzed by TLC. Fractions were combined according to purity and analyzed by HPLC and UPLC.
[0065] Selected fractions having the 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 vol) and the solvent was evaporated to dryness again.
[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 performed, fractions were checked by TLC and selected fractions with desired purity were collected; combined fractions were analyzed by HPLC and UPLC.
[0070] The fractions were evaporated on a rotary evaporator under vacuum at a temperature of 45° C.±5° C. Pure compound (I), isolated as an oil, was stored at 5° C.±3° C.
[0071] Treatment with activated carbon (clarification) Pure compound (I) was dissolved in ethanol (6 L, 9 volumes) and activated charcoal (60 g, approximately 9% w / w) was added. The mixture was stirred at 20° C.±5° C. for 30 minutes and the activated charcoal was removed by filtration. The solvent was evaporated under vacuum at 45° C.±5° C. to 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%.
[0072] The purity of the isolated Compound (I) was assessed by HPLC analysis using the two methods described below, with the HPLC assay of Compound (I) being 99.91% (HPLC area %) as assessed by HPLC / Method 1.
[0073] The limits of detection (LoD) and limits of quantification (LoQ) of the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), the 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost 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 isolated compound (I) is reported in Table 2.
[0075] Table 3 reports the content of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost (II) in compound (I) prepared according to the methods disclosed in the prior art.
[0076] Method 1: HPLC analysis was carried out using the following measurement conditions: Instrumentation: Waters HPLC system equipped with a PDA detector. Stationary phase: Zorbax RXSIL, 250×4.6mm, 5μm Column temperature: 35℃ Mobile phase: hexane:ethanol:acetonitrile:acetic acid = 935:60:5:0.5 Run Time: 60 minutes Flow rate: 1.5mL / min Injection volume: 20μL Detection: UV, 210nm Sample solvent: ethanol:hexane 2:8
[0077] Method 2: HPLC analysis was carried out to quantify the 15-epi-compound (I) of formula (VIII) using the following measurement conditions: Instrumentation: Waters HPLC system equipped with a PDA detector. Stationary phase: Chiralpak AD-H, 250×4.6mm, 5μm Column temperature: 25℃ Mobile phase: hexane:ethanol:TFA=850:150:1 Run Time: 30 minutes Flow rate: 1.0mL / min Injection volume: 10μL Detection: 210nm Sample solvent: hexane:ethanol = 8:2 [Table 1] [Table 2] [Table 3]
Claims
1. The following steps: 1) At reflux temperature in toluene, bimatoprost is reacted with phenylboronic acid, and water is removed by azeotropic distillation to obtain formula (V): 【Chemistry 11】 The process of obtaining bimatoprost phenyl-boronate; 2) The solution is cooled to 25°C, and then N,N'-diisopropylcarbodiimide, a catalytic amount of dimethylaminopyridine and 6-(nitrooxy)hexanoic acid are added to form formula (VI): 【Chemistry 12】 The process of obtaining (1E,3S)-1-{(1S,5R,6R,7R)-7-[(2Z)-7-(ethylamino)-7-oxohepta-2-en-1-yl]-3-phenyl-2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl}-5-phenylpenta-1-en-3-yl 6-(nitrooxy)hexanoate; 3) A step of removing the phenyl-boronate protecting group under basic conditions; 4) Separating the organic phase and evaporating the solvent; 5) Stirring the untreated product in dichloromethane, filtering the mixture, and evaporating the solvent to obtain the crude compound of formula (I); 6) A 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) Collecting a fraction of appropriate purity and evaporating the solvent to provide a pure compound of formula (I); 8) Dissolve the pure compound of formula (I) from step 7) in distilled methylene chloride and methanol, purify the solution by gravity chromatography using an eluent mixture containing distilled methylene chloride and methanol in a volume ratio of 30:1, collect a fraction of appropriate purity, and evaporate the solvent to obtain the pure compound of formula (I); 9) The process includes dissolving the pure compound of formula (I) from step 8) in ethanol, treating the solution with activated carbon, 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, Equation (I): 【Chemistry 13】 A method 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 method characterized in that 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 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and contains (≤) 0.20% (HPLC area%) or less of total impurities.
2. The method according to claim 1, wherein in 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 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 method according to claim 1 or 2, wherein in reaction step 3), the phenylboronate protecting group is removed using a NaOH solution.
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 solution of 0.5 M NaOH.
6. The method according to claim 1 or 2, wherein in reaction step 4), the organic phase is first washed with an aqueous sodium bisulfate solution, and then washed twice with an aqueous 15% w / w NaCl solution.
7. (≦) Contains a total amount of impurities of 0.20% (HPLC area %) or less, and is an impurity of formula (II): 【Chemistry 14】 A compound of formula (I) that contains no 15-(6-chlorohexanoyl) ester of bimatoprost, or contains it in an amount below the HPLC detection limit.
8. A 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 is (≤) 0.15% (HPLC area%) or less.
10. The compound of formula (I) according to claim 9, wherein the total amount of impurities is (≤) 0.10% (HPLC area%) or less.
11. (≤) 0.05% w / w or less is an impurity, as shown in formula (III): 【Chemistry 15】 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester, and (≤) 0.05% w / w or less is an impurity, as shown in formula (VII): 【Chemistry 16】 、 (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, and (≤) 0.05% or less of an impurity, formula (VIII): 【Chemistry 17】 (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 A compound of formula (I) according to any one of claims 7 to 10, comprising the above.
12. Formula (III) for less than 0.05% w / w: [Chemistry 18] 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost ester, and Formula (VII) for concentrations less than or equal to 0.05% w / w: 【Chemistry 19】 (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, and Formula (VIII) for less than 0.05%: 【Chemistry 20】 (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 A compound of formula (I) according to claim 11, comprising the above.
13. An ophthalmic pharmaceutical composition comprising a compound of formula (I) according to any one of claims 7 to 10, and at least one pharmaceutically acceptable excipient.