INDUSTRIAL PROCESS 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 AND HIGH PURE PRODUCT

Abstract

The present invention relates to a process 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

##STR00001##

In accordance with the present invention, a pharmaceutical grade Compound (I) can be efficiently prepared by a one-pot reactions preparation step that includes the esterification of the 15-OH bimatoprost by coupling bimatoprost phenyl-boronate with 6-(nitrooxy) hexanoic acid and the removal of the boronate ester protecting group, followed by an efficient purification step.

The invention refers also to high purity Compound (I) substantially free of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost and to ophthalmic pharmaceutical formulations containing the high purity compound.

Claims

1. A process 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) ##STR00011## comprising the following steps: 1) reacting bimatoprost with phenylboronic acid in toluene at the refluxing temperature and removing water by azeotropic distillation to obtain bimatoprost phenyl-boronate of formula (V) ##STR00012## 2) cooling down the solution to 25 C. and adding N,N-diisopropyl carbodiimide, a catalytic amount of dimethylaminopyridine and 6-(nitrooxy) hexanoic acid 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) ##STR00013## 3) removing the phenylboronate protecting group under basic conditions; 4) separating 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 a normal phase gravity silica gel column chromatography using an eluent mixture containing diisopropyl ether, acetone and water in 40:15:0.5 volume ratio; 7) collecting the fractions of appropriate purity and evaporating the solvent to provide pure compound of formula (I); 8) dissolving the pure compound of formula (I) of step 7) in distilled methylene chloride and methanol, purifying the solution by gravity chromatography using an eluent mixture containing distilled methylene chloride and methanol in 30:1 volume ratio and collecting the fractions of appropriate purity and evaporating the solvent to obtain pure compound of formula (I); 9) dissolving the pure compound of formula (I) of step 8) in a ethanol, treating the solution with activated charcoal, thereafter removing the activated charcoal 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; said process being 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 the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and contains not more than () 0.20% (HPLC area %) of total impurities.

2. The process according to claim 1 wherein, in the reaction step 1), the bimatoprost:phenyl boronic acid molar ratio is 1:1.1.

3. The process according to claim 1 wherein, in the reaction step 2), N,N-diisopropyl carbodiimide 2.0 equiv., dimethylaminopyridine 0.2 equiv. and 6-(nitrooxy) hexanoic acid 1.8 to 2.2 equiv. are added.

4. The process according to claim 1, wherein in the reaction step 3) the phenylboronate protecting group is removed using a NaOH solution.

5. The process according to claim 4 wherein the phenylboronate protecting group is removed by quenching the reaction mixture obtained in step 2) with methanol, then adding a mixture of methylene chloride and 6.3 equiv. of NaOH as solution of NaOH 0.5M.

6. The process according to claim 1, wherein, in reaction step 4) the organic phase is washed first with an aqueous solution of sodium hydrogen sulfate and twice with an aqueous solution of NaCl 15% w/w.

7. The compound 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 of formula (I) as a drug substance containing an amount of total impurities of not more than () 0.20% (HPLC area %) and no amount of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) ##STR00014##

8. (canceled)

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

10. The compound of formula (I) according to claim 9 wherein the amount of total impurities is not more than () 0.10% (HPLC area %).

11. The compound of formula (I) according to claim 7, wherein said compound of formula (I) contains not more than () 0.05% w/w of the impurity 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) quantified by HPLC ##STR00015## and not more than () 0.05% w/w of the impurity (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) quantified by HPLC ##STR00016## and not more than () 0.05% of the impurity (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) quantified by HPLC ##STR00017##

12. The compound of formula (I) according to claim 7 wherein said compound of formula (I) contains less than (<) 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) quantified by HPLC ##STR00018## and not more than () 0.05% w/w (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) quantified by HPLC ##STR00019## and less than (<) 0.05% (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) quantified by HPLC ##STR00020##

13. An ophthalmic pharmaceutical composition comprising the compound of formula (I) according to claim 7 and at least a pharmaceutical acceptable excipient.

Description

DESCRIPTION OF THE INVENTION

[0023] 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 and that allows preparing high purity Compound (I) in high yield.

[0024] The process of the invention includes an efficient one-pot reactions preparation step of the crude Compound (I) from Bimatoprost and 6-(nitrooxy) hexanoic acid followed by a highly efficient purification step of the crude Compound (I) that includes, first, a normal phase gravity silica gel column chromatography to remove almost all the impurities deriving from the preparation step followed by a silica gel filtration chromatography that has the scope to remove the higher boiling solvents. The process is applicable to a large scale preparation of Compound (I), for example up to 650 grams. An important advantage of the process of the invention is that this process is suitable 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), indeed the compound obtained by the process of the invention does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost (II) and the amount of total impurities is not more than () 0.20% (HPLC area %). The high overall chemical yield of the process of about 70% and the high effective purification step make this process a cost-saving method easily applicable to an 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).

[0025] Another object of the 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 an amount of total impurities not more than () 0.20% (HPLC area %), preferably the amount of total impurities is not more than () 0.15% (HPLC area %), most preferably the amount of total impurities is not more than () 0.10% (HPLC area %).

[0026] The above definitions does not contain 15-(6-chlorohexanoyl) ester of bimatoprost (II) and substantially free of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) means that the amount of said compound is below the Limit of Detection (LoD) of the HPLC method that is disclosed hereunder.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to a process 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)

##STR00007## [0028] said process comprising the following steps: [0029] 1) reacting bimatoprost with phenylboronic acid in toluene at the refluxing temperature and removing water by azeotropic distillation to obtain bimatoprost phenyl-boronate of formula (V)

##STR00008## [0030] 2) cooling down the solution to 25 C. and adding N,N-diisopropyl carbodiimide, a catalytic amount of dimethylaminopyridine and 6-(nitrooxy) hexanoic acid 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)

##STR00009## [0031] 3) removing the phenylboronate protecting group under basic conditions; [0032] 4) separating the organic phase, washing the organic phase and evaporating the solvent; [0033] 5) stirring the raw product in dichloromethane, filtering the mixture and evaporating the solvent to obtain the crude compound of formula (I); [0034] 6) purifying the crude compound of formula (I) by applying a normal phase gravity silica gel column chromatography using an eluent mixture containing diisopropyl ether, acetone and water in 40:15:0.5 volume ratio; [0035] 7) collecting the fractions of appropriate purity and evaporating the solvent to obtain pure compound of formula (I); [0036] 8) dissolving the pure compound of formula (I) of step 7) in distilled methylene chloride and methanol, purifying the solution by gravity chromatography using an eluent mixture containing distilled methylene chloride and methanol in 30:1 volume ratio; collecting the fractions of appropriate purity and evaporating the solvent; [0037] 9) dissolving the pure compound of formula (I) of step 8) in a suitable solvent, treating the obtained solution with activated charcoal and thereafter 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; [0038] wherein the process is characterized in that the obtained compound of formula (I) does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and has an amount of total impurities not more than () 0.20% (HPLC area %); preferably the amount of the total impurities is not more than () 0.1% (HPLC area %).

[0039] In the reaction step 1), preferably the bimatoprost:phenyl boronic acid molar ratio is 1:1.1,

[0040] In the reaction step 2) preferably N,N-diisopropyl carbodiimide 2.0 equiv., dimethylaminopyridine 0.2 equiv. and 6-(nitrooxy) hexanoic acid 1.8 to 2.2 equiv. are added.

[0041] In the reaction step 3) the phenylboronate protecting group is preferably removed using a NaOH solution. In particular, the phenylboronate protecting group is removed by quenching the reaction mixture obtained in step 2) with methanol, then adding a mixture of methylene chloride and 6.3 equiv. of NaOH as solution of NaOH 0.5M.

[0042] Preferably in reaction step 4) the organic phase is washed first with an aqueous solution of sodium hydrogen sulfate and twice with an aqueous solution of NaCl 15% w/w.

[0043] Preferably the process of the invention gives the compound of formula (I) containing not more than () 0.10% (HPLC area %) of total impurities.

[0044] Another embodiment of the invention relates to a process for the synthesis of 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 formula (I) comprising the following steps: [0045] 1) reacting bimatoprost (1 equiv.) with phenyl boronic acid (1.1 equiv.) in toluene to protect the 9,11-hydroxy groups of bimatoprost; warming the reaction mixture to the reflux temperature of toluene to remove by water azeotropic distillation and stirring the reaction mixture at reflux temperature until formation of bimatoprost phenyl-boronate of formula (V); [0046] 2) cooling down the reaction mixture to 25 C. and adding N,N-diisopropyl carbodiimide (2.0 equiv.), a catalytic amount of dimethylaminopyridine (0.2 equiv.) and 6-(nitrooxy) hexanoic acid (1.8 to 2.2 equiv.) 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); [0047] 3) at the end of the esterification, quenching the reaction mixture with methanol, then adding methylene chloride and a solution of NaOH 0.5 M, thereafter, stirring the resulting mixture to complete the removal of the phenylboronate protecting group, wherein preferably 6.3 equiv. of NaOH are used; [0048] 4) separating the organic phase and washing the organic phase first with an aqueous solution of sodium hydrogen sulfate and twice with an aqueous solution of NaCl 15% w/w thereafter evaporating the solvent at a temperature below 45 C. [0049] 5) adding dichloromethane to the raw mixture and stirring the mixture at 10 C. for 30 minutes, removing N,N-diisopropyl urea by filtration and evaporating the solvent of the filtered solution to obtain the crude compound of formula (I); [0050] 6) purifying the crude compound of formula (I) by applying normal phase gravity gel column chromatography using an eluent mixture containing silica diisopropylether/acetone/water in 40:15:0.5 volume ratio; [0051] 7) collecting the fractions of appropriate purity and evaporating the solvent to obtain pure compound of formula (I); [0052] 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 methylene chloride/distilled methanol in 30:1 volume ratio; collecting the fractions of appropriate purity and evaporating the solvent to obtain pure compound of formula (I) as an oil; [0053] 9) dissolving the pure compound of formula (I) obtained in step 8) in ethanol, treating the obtained solution with activated charcoal, thereafter removing the activated charcoal by filtration and removing ethanol by evaporation under vacuum leading to the final pure compound of formula (I), wherein the process is characterized in that the obtained compound of formula (I) does not contain the 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and has an amount of total impurities not more than () 0.20% (HPLC area %); preferably the amount of the total impurities is not more than () 0.10% (HPLC area %).

[0054] The above equiv. (equivalent) means the molar equivalent of each reagent and it is calculated with respect to the moles of bimatoprost.

[0055] The above reported process is illustrated in the following Scheme.

##STR00010##

[0056] The process of the invention has several advantages, it allows eliminating the formation of 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and reducing the amount of 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III) to below the limit of quantification that is 0.05% w/w. Moreover, the purity assessment of Compound (I) performed by HPLC showed that also the amount of each of impurities 5,6-trans-Compound (I) of formula (VII) and 15-epi-Compound (I) of formula (VIII) is not more than () 0.05% w/w.

[0057] Another advantage of the invention process is that the synthesis of the crude compound of formula (I) can be performed in a one-pot reactions preparation in which the above reported steps 1) to 3) are conducted without isolating or purifying the resulting intermediates.

[0058] 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) does not contain of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) and which contains an amount of total impurities not more than () 0.20% (HPLC area %), preferably the amount of total impurities is not more than () 0.15% (HPLC area %), most preferably the amount of total impurities is not more than () 0.10% (HPLC area %).

[0059] An embodiment of the 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 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: [0060] not more than () 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III); [0061] not more than () 0.05% w/w (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 [0062] not more than () 0.05% w/w (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 wherein the amount of total impurities is not more than () 0.20% (HPLC area %).

[0063] Another embodiment of the 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-dihydroxy cyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: [0064] less than (<) 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester) of formula (III); [0065] not more than () 0.05% w/w (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 [0066] less than (<) 0.05% w/w (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

[0067] wherein the amount of total impurities is not more than () 0.20% (HPLC area %).

[0068] Another embodiment of the 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-dihydroxy cyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: [0069] less than (<) 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester) of formula (III); [0070] not more than () 0.05% w/w (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 [0071] less than (<) 0.05% w/w (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

[0072] wherein the amount of total impurities is not more than () 0.15% (HPLC area %).

[0073] Another embodiment of the 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-dihydroxy cyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: [0074] less than (<) 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester) of formula (III); [0075] not more than () 0.05% w/w (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 [0076] less than (<) 0.05% w/w (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

[0077] wherein the amount of total impurities is not more than () 0.10% (HPLC area %).

[0078] Another embodiment of the invention provides an ophthalmic pharmaceutical composition comprising hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), and which contains an amount of total impurities not more than () 0.20% (HPLC area %) and at least a pharmaceutically acceptable excipient, preferably the amount of total impurities is not more than () 0.15% (HPLC area %), most preferably the amount of total impurities is not more than () 0.10% (HPLC area %).

[0079] Another embodiment of the invention provides ophthalmic pharmaceutical compositions comprising hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: not more than () 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III), not more than () 0.05% w/w (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)), not more than () 0.05% w/w 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 of formula (VIII) (15-epi-Compound (I)), wherein the amount of total impurities is not more than () 0.20% (HPLC area %), and at least a pharmaceutical acceptable excipient.

[0080] Another embodiment of the invention provides an ophthalmic pharmaceutical composition comprising hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain (15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: less than (<) 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III), not more than () 0.05% w/w (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)), less than (<) 0.05% w/w (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 wherein the amount of total impurities is not more than () 0.20% (HPLC area %) and at least a pharmaceutical acceptable excipient.

[0081] Another embodiment of the invention provides ophthalmic pharmaceutical compositions comprising hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: less than (<) 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III), not more than () 0.05% w/w (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)), less than (<) 0.05% w/w (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 wherein the amount of total impurities is not more than () 0.15% (HPLC area %) and at least a pharmaceutical acceptable excipient.

[0082] Another embodiment of the invention provides ophthalmic pharmaceutical compositions comprising hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) that does not contain 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), wherein said compound of formula (I) contains: less than (<) 0.05% w/w 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III), not more than () 0.05% w/w (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)), less than (<) 0.05% w/w (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 wherein the amount of total impurities is not more than () 0.10% (HPLC area %) and at least a pharmaceutical acceptable excipient.

[0083] The purity and the impurity profile of Compound (I) were assessed by HPLC methods disclosed hereunder (Method 1 and Method 2).

[0084] The above reported definitions does not contain 15-(6-chlorohexanoyl) ester of bimatoprost or substantially free of 15-(6-chlorohexanoyl) ester of bimatoprost mean that the amount of 15-(6-chlorohexanoyl) ester of bimatoprost is below the Limit of Detection (LoD) of the HPLC method disclosed hereunder.

[0085] The above reported definition the amount of 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester is less than 0.05% w/w means that the amount of 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester is below the Limit of Quantification (LoQ) of the HPLC method disclosed hereunder.

[0086] The above reported definition the amounts 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 hereunder.

[0087] The above reported definition the amounts of 15-epi-Compound (I) a less than 0.05% w/w means that the amount of 15-epi-Compound (I) is blow the Limit of Quantification of the HPLC method disclosed hereunder.

Example 1

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))

[0088] The starting material bimatoprost is commercially available.

1a) Preparation of 6-(nitrooxy) hexanoic acid

Synthesis of methyl 6-hydroxyhexanoate

[0089] Dry methanol (300 mL) was charged in a 1 L three-necked flask equipped with thermometer, dropping funnel, reflux condenser and magnetic stirrer followed by conc. sulfuric acid (2.8 mL, 0.052 mole 0.06 eq). The mixture was heated to reflux temperature (T=63 C.). To the mixture was added dropwise a solution of -caprolactone (100 g, 0.876 mole, 1 eq) in dry methanol (200 mL). The reaction was stirred for 2 hours at reflux temperature. TLC was performed to monitor residual -caprolactone. Reaction mixture was then cooled down to 0-5 C. Conc. NaHCO.sub.3 solution (100 mL) was slowly added keeping the temperature below 10 C. The volatile solvent was removed on rotary evaporator under vacuum at temperature of 40 C. To the residue were added water and methyl tert-butyl ether (MTBE) (300 mL). The phases were stirred for 5 minutes, then separated. The aqueous phase was extracted with MTBE (2200 mL). The combined organic phase was washed with brine (2100 mL). The organic phase was dried over Na.sub.2SO.sub.4, then concentrated on rotary evaporator under vacuum at temperature of 40 C. to obtain methyl 6-hydroxyhexanoate as a colorless oil (103.58 g, Y: 80.9%). GC purity: 96.2%.

Synthesis of methyl 6-nitrooxyhexanoate

[0090] Conc. sulfuric acid (45.6 mL 0.85 mol, 3.1 eq) which was cooled to 0-5 C. was added to a 500 mL three-necked flask equipped with thermometer, dropping funnel and magnetic stirrer. Then fuming nitric acid (47.2 mL, 1.12 mol, 4.1 eq) was added dropwise to it while the temperature was kept between 0-5 C. To the cooled mixture was added slowly dry dichloromethane (100 mL, 2.5 V) then stirred between 0-5 C. for 30 min. At this temperature methyl 6-hydroxyhexanoate (40 g, 0.27 mol, 1 eq.) in dry (dichloromethane) DCM (100 mL) was added for 1 hour. After 30 min stirring at 0-5 C. the reaction mixture was monitored by TLC which showed the completed reaction. The reaction mixture was quenched by dropping it to cooled water (200 mL) (T<10 C., 2 hours). Then the phases were separated. The organic phase was dried over Na.sub.2SO.sub.4, then concentrated on rotary evaporator under vacuum at temperature of 40 C. to obtain methyl 6-nitrooxyhexanoate (49.46 g). as a yellow oil in a 94.5% yield.

Synthesis of crude 6-(nitrooxy) hexanoic acid

[0091] 1.5 M NaOH aqueous solution (208 mL) was charged into a 1 L three-necked flask equipped with thermometer, dropping funnel and magnetic stirrer. Methyl 6-nitrooxyhexanoate (49.64 g, 0.26 mol, 1 eq) dissolved in methanol (248 mL, 5V) was added dropwise at room temperature (Tmax=35 C.). After the addition, the mixture was stirred for 2 hours at room temperature. Controlled by TLC the reaction mixture showed the reaction was completed. The pH of the reaction mixture was set to 2.5 using 183 mL 2M HCl aqueous solution. The resulted mixture was extracted with MTBE (100 mL) then the water phase was extracted MTBE (550 mL). The combined organic phase was washed with brine (230 mL), then concentrated on rotary evaporator under vacuum at temperature of 40 C. to obtain crude 6-(nitrooxy) hexanoic acid as a yellow oil (44.2 g). Assay is assessed by UPLC method.

Purification of 6-(nitrooxy) hexanoic acid

[0092] The crude 6-(nitrooxy) hexanoic acid was purified by column chromatography according to the following procedures:

[0093] Eluent was prepared by mixing toluene and methanol in a volumetric ratio of 40:1. Silica was stirred with eluent in a slurry dispenser and the column was loaded and prepared. The crude material (1161 g, assay of 91.8% by UPLC) was dissolved in toluene and the solution was loaded on the top of the column. The elution was performed at a flow rate of 155 L/h and fractions of 10 L were collected and analyzed by TLC. Purest fractions were combined and concentrated under vacuum at temperature below 55 C.5 C. The purified 6-(nitrooxy) hexanoic acid (952 g, 5.37 mole) was isolated as an oil in a 82% yield having a 98.7% purity by UPLC and containing a not detectable amount of 6-[6-nitrooxyhexanoyl]oxy}hexanoic acid.

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))

[0094] Toluene (28 Kg, 49 vol) was charged in a reactor followed by Bimatoprost (663 g, 1.595 mol, 1 equiv) and phenylboronic acid (214 g, 1.755 mole, 1.1 equiv). The reaction is heated at 110 C.5 C. and vapor temperature was maintained at 90 C. for 4 hours to ensure azeotropic distillation of water for a total volume of solvent of around 12 L. Reaction mixture was then cooled down to 25 C.5 C.

[0095] Dimethylaminopyridine (DMAP) (39 g, 0.319 mole, 0.2 equiv) and N,N-diisopropyl carbodiimide (DIC) (403 g, 3,190 mole, 2.0 equiv) were added to the cooled reaction mixture, followed by a solution of 6-(nitrooxy) hexanoic acid (565 g, 3.190 mole, 2.0 equiv) in toluene (0.5 L, 0.75 vol). The reaction was stirred for 1 to 3 hours at 25 C.5 C. TLC was performed to monitor residual bimatoprost phenyl-boronate (Compound (V)

[0096] The reaction mixture was quenched with methanol (1.3 L, 2 vol) and was stirred 5 to 10 min The reaction mixture was then transferred via vacuum into an extractor containing 0.5M sodium hydroxide solution (20 L) and dichloromethane (10 L, 15 vol). Reactor was washed with several portions of methylene chloride that were added to the reaction mixture. The reaction mixture was stirred for 2 to 3 hours, then left to settle for 60 min and the organic phase was collected in a mobile tank. Dichloromethane (10 L, 15 vol) was charged into the extractor, the mixture was stirred for 5 to 10 min and left to settle for 15 min. The organic layer was collected. The organic layers were charged again in the extractor and a 1M NaHSO.sub.4 solution (20 L) was transferred via vacuum into the extractor. The mixture is stirred for 5 to 10 min, left to settle for 15 min and the lower organic phase was collected in a mobile tank. The aqueous layer was removed. The organic phase was charged again in the extractor and a 15% NaCl solution (20 L) was added and the mixture was stirred for 5 to 10 min and left to settle for 15 min. The organic layer was collected and the aqueous phase was removed. The organic layer was charged again in the extractor and a 15% NaCl w/w solution (20 L) was added. The mixture was stirred for 5 to 10 min and left to settle for 15 min. The organic layer was collected evaporated under vacuum at a temperature of 45 C.5

[0097] The crude product was dissolved in dichloromethane (7 L, 10.5 vol) and the mixture was transferred into a reactor. The mixture was cooled to 5-10 C. and kept for 30-60 min at this temperature to allow N,N-diisopropyl urea precipitation. The mixture is filtered through the filter in a mobile tank. The reactor is washed with dichloromethane (2 L, 3 vol), solution is filtered and collected in the mobile tank. Then the solution in the mobile tank is transferred to a flask and the solvent is evaporated under vacuum at temperature of 45 C.5 C. to obtain the 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).

1c) Purification of the 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))

First Chromatography

[0098] The eluent mixture 1 was prepared by mixing diisopropylether/acetone/water in 40:15:0.5 volume ratio.

[0099] Silica gel (30 Kg, 45 vol) was suspended in the eluent mixture 1 (66.3 Kg, 100 vol), the suspension was stirred for 3 min and loaded in the column.

[0100] The crude product was dissolved in the eluent mixture (1 L, 1.5 vol) and charged on the top of the column. The elution was performed and fractions of 15 L were collected and analyzed by TLC. Fractions were combined according to purity and analyzed by HPLC and UPLC.

[0101] Selected fractions with desired purity were collected and the solvent was evaporated using a rotary evaporator at temperature of 45 C.5 C. until dryness. The residue was dissolved in dichloromethane (5 L, 7.5 vol) and the solvent was evaporated again until dryness.

[0102] The Compound (I) was stored at 5 C.3 C.

Silica Gel Filtration Chromatography

[0103] Eluent mixture 2 was prepared by mixing distilled dichloromethane and distilled methanol in 30:1 volume ratio.

[0104] Silica 75 S (6.6 Kg, 10 vol) was added to eluent mixture 2 (13 Kg, 19.6 vol) and the suspension was stirred and poured to fill the column. Compound (I) was dissolved in dichloromethane (1 L, 1.5 vol) and the solution was loaded on the top of the column.

[0105] The elution was performed and fractions were controlled by TLC and the selected fractions with desired purity were collected; the combined fractions were analyzed by HPLC and UPLC.

[0106] Fractions were evaporated in a rotary evaporator under vacuum at temperature of 45 C.5 C. The pure Compound (I) isolated as an oil was stored at 5 C.3 C.

Treatment with Activated Charcoal (Clarification)

[0107] The pure Compound (I) was dissolved in ethanol (6 L, 9 vol) and activated charcoal (60 g, around 9% w/w) was added. The mixture was stirred for 30 min at 20 C.5 C. and the activated charcoal is removed by filtration. The solvent was evaporated under vacuum at 45 C.5 C. until constant weight; 649.6 g (1.13 mole) pure Compound (I) was isolated as an oil. The overall yield of the process was 70.8%.

[0108] The purity assessment of the isolated Compound (I) was performed by HPLC analysis using the two methods described below, the HPLC assay of Compound (I) was 99.91% (HPLC area %) assessed by HPLC/Method 1.

[0109] The Limit of Detection (LoD) and the Limit of Quantification (LoQ) for 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II), 6-{[6-(nitrooxy) hexanoyl]oxy}hexanoic acid bimatoprost ester of formula (III), 5,6-trans-Compound (I) of formula (VII) and 15-epi-Compound (I) of formula (VIII) are reported in Table 1.

[0110] The impurities profile of the isolated Compound (I) is reported in Table 2.

[0111] Table 3 reports the contents of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost (II) in the Compound (I) prepared according to the processes disclosed in the prior art.

[0112] Method 1: the HPLC analysis was performed using the following measurement conditions: [0113] Instrument: Waters HPLC System with PDA detector [0114] Stationary phase: Zorbax RX SIL, 2504.6 mm, 5 m [0115] Column temperature: 35 C. [0116] Mobile phase: hexane:Ethanol:acetonitrile:acetic acid=935:60:5:0.5 [0117] Run Time: 60 min [0118] Flow rate: 1.5 ml/min [0119] Injected Volume: 20 l [0120] Detection: UV, 210 nm [0121] Sample solvent: ethanol:hexane 2:8

[0122] Method 2: the HPLC analysis was performed to quantify the 15-epi-Compound (I) of formula (VIII) using the following measurement conditions: [0123] Instrument: Waters HPLC System with PDA detector [0124] Stationary phase: Chiralpak AD-H, 2504.6 mm, 5 m [0125] Column temperature: 25 C. [0126] Mobile phase: Hexane:Ethanol:TFA-850:150:1 [0127] Run Time: 30 min [0128] Flow rate: 1.0 mL/min [0129] Injected Volume: 10 L [0130] Detection: 210 nm [0131] Sample solvent: Hexane:Ethanol8:2

TABLE-US-00001 TABLE 1 Limit of Detection (LoD) and Limit of Quantification (LoQ) of the impurities LoD LoQ IMPURITY % w/w % w/w 15-(6-chlorohexanoyl) ester of bimatoprost of formula (II) 0.01 0.03 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid bimatoprost 0.02 0.05 ester of formula (III) (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7-(ethylamino)-7- 0.01 0.05 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)) (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7-(ethylamino)-7- 0.02 0.05 oxohept-2-en-1-yl)-3,5-dihydroxycyclopentyl)-5- phenylpent-1-en-3-yl 6-(nitrooxy) hexanoate (15-epi-Compound (I) of formula (VIII))

TABLE-US-00002 TABLE 2 HPLC analysis of the isolated Compound (I) of Example 1 Main and total impurities Method 1 Method 2 % RT RT IMPURITY (w/w) (min) (min) 15-(6-chlorohexanoyl) ester of bimatoprost of <LoD 15.4 formula (II) 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid <LoD 22.1 bimatoprost ester of formula (III) (1E,3S)-1-((1R,2R,3S,5R)-2-((2E)-7- <LoQ.sup. 23.6 (ethylamino)-7-oxohept-2-en-1-y1)-3,5- dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6- (nitrooxy) hexanoate (5,6-trans-Compound (I) of formula (VII)) (1E,3R)-1-((1R,2R,3S,5R)-2-((2Z)-7- <LoD 13.9 (ethylamino)-7-oxohept-2-en-1-y1)-3,5- dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6- (nitrooxy) hexanoate (15-epi-Compound (I) of formula (VIII)) Total impurities 0.07* NA NA RT = Retention time .sup.the measured amount of 5,6-trans-Compound (I) of formula (VII) was 0.04% w/w calculated as medium value of two determinations by HPLC analysis *HPLC area %

TABLE-US-00003 TABLE 3 Summary of the contents of the impurity 15-(6-chlorohexanoyl)ester of bimatoprost (II) in the Compound (I) prepared according to the processes disclosed in the prior art WO 2009/136281 WO 2019/162149 WO 2021/023693 15-(6-chlorohexanoy1) 8.34 0.15-0.24 0.11 ester of bimatoprost of (HPLC area %) (% w/w) (HPLC area %) formula (II)