PROCESS FOR THE PREPARATION OF A NITRIC OXIDE DONATING PROSTAGLANDIN ANALOGUE

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 of formula (I).

##STR00001##

In accordance with the present invention, the compound (I) can be efficiently prepared with high purity by coupling bimatoprost in a boronate protected form with 6-(nitrooxy)hexanoyl chloride and removing the boronate protecting group.

The invention also relates to a process for the preparation of 6-(nitrooxy) hexanoic acid having a HPLC purity equal to or greater than 99% and containing an amount of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa) equal to or lower than 0.2%.

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): ##STR00021## comprising the following steps: Step A) preparation of the 6-(nitrooxy)hexanoyl chloride (VIIIb) by: 1a) reacting the ?-caprolactone ##STR00022## with an inorganic base selected from KOH, NaOH and LiOH in a solvent selected from methanol, ethanol or isopropanol, at a temperature between 20? C. and the reflux temperature of the solvent to obtain 6-hydroxyhexanoic acid salt of formula (X) ##STR00023## wherein M is K, Na or Li; 2a) purifying the 6-hydroxyhexanoic acid salt of formula (X) obtained in step 1a) which comprises: i. adding methyl tert-butyl ether to the reaction mixture of step 1a) in a ratio methyl tert-butyl ether/volume of the reaction mixture 2:1; ii. filtering the solid; iii. slurring the solid with a mixture of methyl tert-butyl ether, methanol in a ratio of 3:1 to 5:1 and water in an amount of 0.3-1 mole of water for mole of ?-caprolactone; iv. isolating the 6-hydroxyhexanoic acid salt of formula (X) as a solid; 3a) reacting the 6-hydroxyhexanoic acid salt with a mixture of fuming HNO.sub.3 and concentrated H.sub.2SO.sub.4 in dichloromethane, at a temperature ranging from 0? C. to 10? C.; 4a) adding water to the nitration mixture of step 3a) while keeping the temperature between 0? C. and 5? C.; 5a) separating the organic phase, drying on sodium sulfate said organic phase and distilling off the solvent to obtain crude 6-(nitrooxy)hexanoic acid (VIIIa) ##STR00024## 6a) reacting the crude 6-(nitrooxy)hexanoic acid (VIIIa) with a chlorinating agent in dichloromethane to obtain the crude 6-(nitrooxy)hexanoyl chloride (VIIIb); Step B) 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 (I) comprising the following steps: 1b) reacting bimatoprost with butyl boronic acid in methyl tert-butyl ether at temperature about 40? C., to obtain compound (II): ##STR00025## 2b) reacting compound (II) with the crude 6-(nitrooxy)hexanoyl chloride (VIIIb) of step 6a) in the presence of 4-dimethylaminopyridine in free form in an aprotic organic solvent, to obtain the compound (XI) ##STR00026## 3b) removing the boronate protective group to obtain the crude compound of formula (I); 4b) purifying the crude compound (I) by column chromatography.

2. The process according to claim 1, wherein in step 1a) the inorganic base is KOH and the solvent is methanol and the reaction is carried out at the reflux temperature of methanol

3. The process according to claim 2, wherein in step 3a) the 6-hydroxyhexanoic acid potassium salt is added to a mixture of fuming HNO.sub.3 and concentrated H.sub.2SO.sub.4 in dichloromethane at a temperature from about 0? C. to 10? C. and the reaction mixture is vigorously stirred for 60?15 min at a temperature from 0? C. to 5? C.

4. The process according to claim 1, wherein in step 6a) the chlorinating agent is oxalyl chloride.

5. The process according to claim 1, wherein in step 2b) the aprotic solvent is methyl tert-butyl ether at a temperature ranging from 0? C. to 20?3? C.

6. The process according to claim 1, wherein in step 2b) the molar ratio of compound (II) to 4-dimethylaminopyridine is from 1:2.0 to 1:2.4.

7. The process according to claim 1, wherein in step 3b) the solvent used in the reaction is methanol and the reaction is carried out at room temperature.

8. The process according to claim 1, wherein in step 4b) the crude compound (I) is purified using high performance silica gel chromatography and methylene chloride/methanol as mobile phase.

9. (canceled)

10. A process for the synthesis of the 6-(nitrooxy)hexanoic acid comprising: 1a) reacting the ?-caprolactone ##STR00027## with KOH in a methanol at the reflux temperature of the solvent; 2a)-i. adding methyl tert-butyl ether to the reaction mixture of step 1a) in a ratio methyl tert-butyl ether/volume of the reaction mixture 2:1; 2a)-ii. filtering the solid 6-hydroxyhexanoic acid potassium salt; 2a)-iii. slurring the solid with a mixture of methyl tert-butyl ether, methanol in a ratio of 3:1 to 5:1 and water in an amount of 0.3-1 mole of water for mole of ??caprolactone; 2a)-iv. isolating the 6-hydroxyhexanoic acid potassium salt as a solid; 3a) adding the 6-hydroxyhexanoic acid potassium salt of step 2a) in a mixture of fuming HNO.sub.3 and concentrated H.sub.2SO.sub.4 in dichloromethane, at a temperature ranging from 0? C. to 10? C. and vigorously stirring the reaction mixture at a temperature from 0? C. to 5? C.; 4a) adding water to the nitration mixture of step 3a) while keeping the temperature between 0? C. and 5? C.; 5a) separating the organic phase, drying on sodium sulfate said organic phase and distilling off the solvent to obtain 6-(nitrooxy)hexanoic acid characterized by a chemical purity equal to or greater than 99% and a content of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid equal to or less than 0.2%.

11. 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 characterized by a chemical purity?99%, and a content of the dimeric impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost (compound (VII)) below 0.05%.

Description

DESCRIPTION OF THE INVENTION

[0033] 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):

##STR00014##

comprising the following steps:
Step A) preparation of the 6-(nitrooxy)hexanoyl chloride (VIIIb) by: [0034] 1a) reacting the ?-caprolactone

##STR00015## [0035] with an inorganic base selected from KOH, NaOH and LiOH in a solvent selected from methanol, ethanol or isopropanol, at a temperature between 20? C. and the reflux temperature of the solvent to obtain 6-hydroxyhexanoic acid salt of formula (X)

##STR00016## [0036] wherein M is K, Na or Li; [0037] 2a) purifying the 6-hydroxyhexanoic acid salt of formula (X) obtained in step 1a) which comprises: [0038] i. adding methyl tert-butyl ether to the reaction mixture of step 1a) in a ratio methyl tert-butyl ether/volume of the reaction mixture 2:1; [0039] ii. filtering the solid; [0040] iii. slurring the solid with a mixture of methyl tert-butyl ether, methanol in a ratio of 3:1 to 5:1 and water in an amount of 0.3-1 mole of water for mole of ?-caprolactone; [0041] iv. isolating the 6-hydroxyhexanoic acid salt of formula (X) as a solid; [0042] 3a) reacting the 6-hydroxyhexanoic acid salt with a mixture of fuming HNO.sub.3 and concentrated H.sub.2SO.sub.4 in dichloromethane, at a temperature ranging from 0? C. to 10? C.; [0043] 4a) work-up of the reaction of step 3a) by adding water to the nitration mixture of step 3a) while keeping the temperature between 0? C. and 5? C.; [0044] 5a) separating the organic phase, drying on sodium sulfate said organic phase and distilling off the solvent to obtain crude 6-(nitrooxy)hexanoic acid (VIIIa)

##STR00017## [0045] 6a) reacting the crude 6-(nitrooxy)hexanoic acid (VIIIa) with a chlorinating agent in dichloromethane to obtain the crude 6-(nitrooxy)hexanoyl chloride (VIIIb);
Step B) 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 (I) by: [0046] 1b) reacting bimatoprost with butyl boronic acid in methyl tert-butyl ether at temperature about 40? C., to obtain compound (II):

##STR00018## [0047] 2b) reacting compound (II) with the crude 6-(nitrooxy)hexanoyl chloride (VIIIb) of step 6a) in the presence of 4-dimethylaminopyridine in free form in an aprotic organic solvent, to obtain the compound (XI)

##STR00019## [0048] 3b) removing the boronate protective group to obtain the crude compound of formula (I); [0049] 4b) purifying the crude compound (I) by column chromatography;
wherein said process is characterized in that the crude nitration mixture of step 5a) contains an amount equal to or greater than 99% of 6-(nitrooxy)hexanoic acid (VIIIa) and an amount equal to or less than 0.2% of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa).

[0050] 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 (I) prepared according to the process of the invention has a chemical purity?99% and contains less than 0.05% of the impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost (VII). Said compound contains about 0.11% of the impurity 15-(6-chlorohexanoyl) ester of bimatoprost (V). The compounds were identified and quantified by HPLC analysis, the amount of the compounds were expressed as area percentage (a/a %); 0.05% is the HPLC detection limit.

[0051] In step 1a) the ring opening reaction of ?-caprolactone is preferably carried out in presence of potassium hydroxide in methanol at temperature between 20? C. and the reflux temperature. During the workup of the reaction mixture of step 1a), the purification of 6-hydroxyhexanoic acid potassium salt is obtained by slurring the solid of step 2a)-ii. in a mixture containing methyl tert-butyl ether, methanol in a ratio of 3:1 to 5:1 and water 0.3-1 mole of water for mole of ccaprolactone (step 2a)-iii.). 6-hydroxyhexanoic acid potassium salt is obtained as a solid.

[0052] The crude nitration mixture of step 5a) contains an amount equal to or greater than 99% of 6-(nitrooxy)hexanoic acid (VIIIa) and an amount of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa) equal to or less than 0.2%. The chemical yield of the 6-(nitrooxy)hexanoic acid (VIIIa) is 80-85%. The chemical purity was assessed by analytical UHPLC method.

[0053] The preferred chlorinating agent used in step 6a) is oxalyl chloride.

[0054] The step 2b) is carried out at a temperature ranging from 0? C. to 25? C. and it is preferably carried out in aprotic organic solvent selected from methyl tert-butyl ether, N,N-dimethylformamide or dichloromethane; most preferably, the organic solvent is methyl tert-butyl ether. 4-Dimethylaminopyridine (DMAP) in free form means that DMAP is not bound to a resin. The molar ratio of compound (II) to 6-(nitrooxy)hexanoyl chloride (VIIIb) preferably ranges from 1:1.4 to 1:1.6.

[0055] The molar ratio of compound (II) to 4-dimethylaminopyridine is preferably from 1:2.0 to 1:2.4.

[0056] In step 3b), the removal of the boronate protecting group is preferably carried out using methanol at a temperature from 17? C. to 25? C.

[0057] A preferred process for preparing the compound of formula (I) comprises the following steps, which are depicted in Scheme 1:

Step A

[0058] 1a) reacting the ?-caprolactone with potassium hydroxide in methanol at a temperature between 20? C. and reflux temperature of methanol, preferably at reflux temperature of methanol, and
2a) purifying the 6-hydroxyhexanoic acid potassium salt obtained in step 1a) which comprises: [0059] i. adding methyl tert-butyl ether (MTBE) to the reaction mixture of step 1a) in a ratio of 2:1 MTBE/methanol; [0060] ii. filtering the solid; [0061] iii. slurring the solid in a mixture of methyl tert-butyl ether, methanol in a ratio of 3:1 to 5:1 and water in an amount of 0.3-1 mole of water for mole of ccaprolactone; [0062] iv. isolating the 6-hydroxyhexanoic acid potassium salt as a solid.
3a) adding the 6-hydroxyhexanoic acid potassium salt (formula (X), M=K) to a mixture of fuming HNO.sub.3 and concentrated H.sub.2SO.sub.4 in dichloromethane at a temperature from about 0? C. to 10? C. and vigorously stirring the reaction mixture at a temperature from 0? C. to 5? C. and monitoring the progress of the reaction (up to 99.9% of conversion);
4a) carefully adding water to the nitration mixture by and keeping the temperature at 0? to 5? C.
5a) separating the organic phase, drying on sodium sulfate said organic phase and distilling off the solvent to yield crude 6-(nitrooxy)hexanoic acid (VIIIa) containing an amount of 6-(nitrooxy)hexanoic acid (VIIIa) equal to or greater than 99% of and an amount of the by-product 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa)) equal to less than 0.2%.
6a) reacting the crude 6-(nitrooxy)hexanoic acid with oxalyl chloride to obtain 6-(nitrooxy)hexanoyl chloride (VIIIb) that is used without further purification in Step B of the process;

Step B

[0063] 1b) reacting bimatoprost with butyl boronic acid (1.1-1.8 eq) in methyl tert-butyl ether (MTBE) at temperature about 40? C., then removing water by azeotropic distillation to obtain bimatoprost boronate (II);
2b) reacting bimatoprost boronate (II) with the crude 6-(nitrooxy)hexanoyl chloride (VIIIb) obtained in step 6a) (1.4-1.6 equivalent), in methyl tert-butyl ether in the presence of 4-dimethylaminopyridine (2.0-2.4 equivalent) at a temperature ranging from 0? C. to 20?3? C., to obtain (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (XI);
3b) reacting (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy) hexanoate (XI) with methanol at room temperature to remove the protective group and 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 (I).
4b) purifying the crude compound (I) by high performance silica gel chromatography to obtain 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 (I) using methylene chloride/methanol as mobile phase

[0064] The HPLC quantitative analysis of the isolated compound (I) after chromatography purification had a chemical purity greater than 99% containing an amount of 15-(6-chlorohexanoyl) ester of bimatoprost (V) about 0.11% and an amount of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost (VII) below the HPLC detection limit of 0.05%.

##STR00020##

[0065] Another object of the invention is a pharmaceutical formulation containing hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) and at least a pharmaceutically acceptable excipient, wherein 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 a chemical purity of 99%, and contains an amount of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (V)) about 0.11% and an amount of the dimeric impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost (compound (VII)) below 0.05%.

[0066] In the present application the quantification of the compounds expressed as percentage % is determined by analysis HPLC (a/a %).

EXAMPLES

[0067] All synthesis steps described below were conducted under nitrogen atmosphere.

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))

Synthesis of 6-(nitrooxy)hexanoyl chloride (Compound (VIIIb))

Steps 1a) and 2a): Synthesis of 6-Hydroxyhexanoic Acid Potassium Salt (Compound (X))

[0068] A solution of potassium hydroxide (141.12 g corresponding to 122.8 g as pure material) in methanol (1250 ml) was prepared under cooling at 15? C. to 20? C. and was added at 5? C. to 37? C. within 0.25 hour to a solution of 250.0 g of ?-caprolactone in methanol (675 ml). The mixture was stirred 18 hours at 20? C. to 63? C. Then the solvent was concentrated to a volume of approximatively 675 mL and methyl tert-butyl ether (1250 ml) was added at around 57? C. The reaction was stirred for 48 hours at RT and the solid was isolated by filtration to give 380.9 g of crude 6-hydroxyhexanoic acid potassium salt. The crude was re-slurred in MTBE (1475 ml) and Methanol (375 ml) and water (19.7 ml) at 22?3? C. for 8 hours and filtered. The solid was re slurred in methyl tert-butyl ether (500 ml) and filtered again and then dried under vacuum at 50? C. for 12 hours to give 354.7 g of 6-hydroxyhexanoic acid potassium salt (93.2% yield) with a purity of 98%. Melting point 208? C.

Steps 3a) to 5a): Synthesis of 6-(nitrooxy)hexanoic acid (Compound (VIIIa))

[0069] Fuming HNO.sub.3 (485.5 g, 4.6 equivalents) was added to concentrated H.sub.2SO.sub.4 (553.0 g, 3.1 equivalent) at a temperature from 0? C. to 5? C. in 30 min, then Dichloromethane (5820 ml) was added at a temperature from 0? C. to 5? C. in 23 min. 6-Hydroxyhexanoic acid potassium salt (306.09 g, 1 equivalent) was added portion-wise in 35 min at temperature below 5? C. The mixture was stirred for 2 hours at 0? C. to 5? C. and the reaction was monitored by .sup.1H-NMR showing 99.9% conversion. Water (2.9 L) was added maintaining temperature between 0? C. to 5? C. within 20 min. The organic layer was decanted, dried over sodium sulfate and concentrated under vacuum to give 318.4 g of 6-(nitrooxy)hexanoic acid (94.8% yield) having a 99.2% HPLC purity and a content of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa)) of 0.15%.

Step 6a): Synthesis of 6-(nitrooxy)hexanoyl chloride (Compound (VIIIb))

[0070] 6-(nitrooxy)hexanoic acid (270.8 g, 1 equivalent) was dissolved in dichloromethane (1220 ml) and cooled to 0? C. to 5? C. under nitrogen. Then N,N-dimethylformamide (1.6 ml) and oxalyl chloride (198.2 g) were added at 0? C. to 5? C. within 30 minutes. The reaction mixture was stirred at 0? C. to 5? C. for 1 hour and then for 4 hours at 15? C. to 20? C. Then the reaction mixture was concentrated under vacuum at temperature equal to or below 40? C. and co-evaporated with dichloromethane to give 6-(nitrooxy)hexanoyl chloride (323.9 g, yield 99.6% yield, titration AgNO.sub.3=92.0%).

Step 1b): Synthesis of (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (Compound (II))

[0071] Bimatoprost (249.0 g, 1 equivalent) was suspended in Methyl tert-butyl ether (4 L) and Butyl boronic acid (69.0 g, 1.13 equivalents) was added. The mixture was heated to 40? C. for 1 hour. The reaction was monitored by .sup.1H NMR till conversion>97%.

[0072] The reaction mixture was cooled down to about 20? C., filtered and rinsed with methyl tert-butyl ether (250 ml). The mixture was concentrated at a temperature about 40? C. under vacuum by azeotropic distillation. Rinsing with methyl tert-butyl ether and azeotropic distillation were continued till the water content of (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (compound (II)) was equal to or below 0.25%.

[0073] (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide was isolated as a crude with quantitative yield (313.1 g corresponding to 298.6 g of pure material).

Step 2b): Synthesis of (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy) hexanoate (compound (XI))

[0074] (Z)-7-[(1S,5R,6R,7R)-3-Butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (compound (II)) (313.1 g, 1 equivalent) was dissolved in methyl tert-butyl ether (4.27 L) under nitrogen and cooled to 0? C. to 5? C. 4-Dimethylaminopyridine (162.7 g, 2.27 equivalents) was added in one portion. A solution of 6-(nitrooxy)hexanoyl chloride (compound (VIIIb)) (172.1 g, 1.5 equivalents) in methyl tert-butyl ether (600 mL), was added dropwise at 0? C. to 5? C. within 40 min. After stirring for 2 h and 30 min at 0? C. to 5? C., and at 15? to 20? C. for 16.5 hours, the reaction mixture was cooled at 0? C. to 5? C. and deionized water was added within 20 minutes at a maximum temperature of 10? C.

[0075] The mixture was stirred for 5 min. The aqueous layer was separated, and discarded. The organic layer was washed with a 1N hydrochloric acid solution then with deionized water and last with brine.

[0076] The organic layer was dried over sodium sulfate and concentrated under vacuum to afford (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy) hexanoate (compound (XI)) (402.9 g, 94.9% yield)

Steps 3b) and 4b): 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))

[0077] (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy) hexanoate (compound (XI)) (396 g crude, lequivalent) was dissolved in methanol (4.20 L) and the resulting solution was stirred at room temperature for 24 hours monitoring the reaction by .sup.1H-NMR. Then methanol was removed under vacuum at 35? C. to 40? C. The residue was dissolved in methanol, stirred for 14 hours and evaporated under vacuum at 35? C. to 40? C. The residue was dissolved in methyl tert-butyl ether and washed with deionized water and then brine. The organic layer was dried over sodium sulfate and concentrated under vacuum at a temperature below 40? C. to afford 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 (352.3 g, 96.5% yield).

[0078] The residue was divided in 3 equal amounts (around 107 g each) and purified by chromatography on column using high performance silica gel (SNAP Ultra column, 1500 g of silica) using dichloromethane/methanol as eluent with a gradient 100:0 to 95:5 v/v on a Biotage Isolera LS system. The fractions were monitored by TLC and UHPLC. Fractions were concentrated under vacuum at temperature equal to or below 50? C. to give compound (I) as an oil (242.5 g, 90.3% yield).

[0079] 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)) was dissolved in ethanol and treated with charcoal for 30 min, filtered and the solvent was evaporated at a temperature below 50? C. under vacuum. Compound (I) was isolated as an oil (212.7 g, 92.4% yield). The purity 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)) was of 99.89% (assessed by UHPLC), the content of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (V)) was 0.11% and the content of the dimeric impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost (compound (VII)) was below 0.05%.

Example 2 (Comparative Example)

[0080] The synthesis reported below was performed according to the method disclosed in WO 2019/162149

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 (I)

Synthesis of 6-(nitrooxy)hexanoyl chloride (VIIIb)

Synthesis of 6-Hydroxyhexanoic Acid Potassium Salt (Compound (X))

[0081] A solution of potassium hydroxide 90% (89.7 g) in methanol (830 ml) was prepared under cooling at 10? C-20? C. 165.2 g of ?-caprolactone (1 eq.) and methanol (415 ml) were introduced in a 4 L three-necked round-bottomed flask. The mixture was stirred until dissolution. Then potassium hydroxide solution in methanol was added at 0?-30? C. within 25 min. The mixture was stirred for 20 hours at 15?-20? C. The reaction mixture was concentrated under vacuum (at a temperature equal to or below 40? C.) to give crude 6-hydroxyhexanoic acid potassium salt. The crude solid was suspended in methyl tert-butyl ether (830 ml) for 2 hours at 15?-20? C., filtered through a pore 3 filter, washed with methyl tert-butyl ether (2?165 ml) and dried under vacuum at 35? C. to give 6-hydroxyhexanoic acid potassium salt (228.1 g, 98.3% HCl assay) in 92.7% yield as a white powder.

Synthesis of 6-(nitrooxy)hexanoic acid (VIIIa)

[0082] The nitration reaction was performed on around 100 g scale in order to control the reaction temperature and to reduce the time of the addition of nitrating mixture. The reaction was performed twice. The nitration mixture was quenched by adding a saturated sodium chloride aqueous solution (brine). Results of the two separate nitration reactions are reported in Table 1.

[0083] Nitration 1: 6 L glass reactor under nitrogen was charged with concentrated sulfuric acid (200.9 g) and cooled to 0-5? C. Fuming HNO.sub.3 (187.9 g) was carefully added dropwise at a temperature from 0? C. to 10? C. in 20 min. Then Dichloromethane (2.23 L) was added and the reaction mixture was stirred for 45 min. 6-Hydroxyhexanoic acid potassium salt (110.1 g, 1 eq.) was added portion-wise in 30 min at temperature between ?5? C. to 5? C. The 5 mixture was stirred for 2.5 hours at temperature between ?5? C. to 5? C. and then overnight at 20? C., the reaction was monitored by .sup.1H-NMR showing 99.6% conversion. Saturated sodium chloride aqueous solution (315.3 gin 1.0 L) was added carefully at a temperature equal to or below 10? C. within 25 min. Precipitation of large amount of inorganic salts was observed. The reaction mixture was transferred to a separating funnel taking care of not transferring inorganic salts. The organic layer was decanted, dried over sodium sulfate and concentrated under vacuum (at a temperature equal to or below 40? C.) to give crude 6-(nitrooxy)hexanoic acid (105.4 g, 85.2%). HPLC purity=83.7% with an amount of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa) of 8.8%.

[0084] Nitration 2: The reaction was performed using 109.97 g of 6-hydroxyhexanoic acid potassium salt to obtain 101.6 g of crude 6-(nitrooxy)hexanoic acid (VIIIa) (82%) that had 89.7% HPLC purity and an amount of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa)) of 5.8%.

[0085] The two batches were combined and subjected to the following steps without further purification

Synthesis of 6-(nitrooxy)hexanoyl chloride (VIIIb)

[0086] 6-(Nitrooxy)hexanoic acid (193 g) was dissolved in dichloromethane (870 mL). The resulting cloudy solution was filtered on glass fibers washed with dichloromethane (130 mL) and the clear solution was analyzed by Karl Fischer (water content=0.018%). The solution was cooled to 0? C. to 5? C. under nitrogen. Subsequently, N,N-dimethylformamide (1.2 mL) and oxalyl chloride (141.4 g) were added at 0? C. to 5? C. within 30 minutes. The reaction mixture was stirred at 0? C. to 5? C. for 1 hour and 14 hours at 15? C. to 20? C. TLC monitoring showed the complete reaction. The mixture was concentrated under vacuum (temperature is equal to or below 40? C.) and co-evaporated with dichloromethane (4?870 ml) to give 6-(nitrooxy)hexanoyl chloride (212.4 g) with 91.9% yield.

Preparation of (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (Compound (II))

[0087] Methyl tert-butyl ether (3900 mL, 14 vol.) was charged in a flask. Bimatoprost (249.1 g, 1 eq.) was added and the equipment was rinsed with methyl tert-butyl ether (250 mL, 1 vol.). Butyl boronic acid (70.66 g, 69.2 g in pure material, 1.13 eq.) was added to the resulting suspension in one portion, the equipment was rinsed with methyl tert-butyl ether (250 mL, 1 vol.). The mixture was heated to 40? C. for 2 h. The reaction was monitored by .sup.1H NMR till conversion>97%.

[0088] The reaction mixture was cooled to 20? C. to 25? C., clarified on a glass filter and washed with methyl tert-butyl ether (250 mL, 1 vol.). The filtrate was concentrated under vacuum at a temperature around 40? C. by azeotropic distillation. Rinsing with methyl tert-butyl ether and azeotropic distillation were continued till the water content of (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (compound (II)) was 0.2%. Compound of formula (II) was analyzed by 1H NMR to determine residual MTBE.

[0089] (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (compound (II)) was obtained in quantitative yield (386.7 g of crude material, uncorrected).

Preparation of (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (XI)

[0090] Methyl tert-butyl ether (4100 mL, 11.6 vol.) was charged in a 4 L three-neck round-bottomed flask under nitrogen. (Z)-7-[(1S,5R,6R,7R)-3-Butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide (compound (II)) (378 g crude, 1 eq.) was added, equipment was rinsed with methyl tert-butyl ether (510 mL, 1.44 vol.). The resulting solution was cooled to 0? C. to 5? C.

[0091] 4-Dimethylaminopyridine (177.5 g, 2.27 eq.) was added in one portion. A solution of 6-(nitrooxy)hexanoyl chloride (204.4 g, corresponding to 188 g in pure, 1.5 eq.) in methyl tert-butyl ether (650 mL, 1.84 vol.) was added dropwise at 0? C. to 5? C. within 45 min. The dropping funnel was rinsed with methyl tert-butyl ether (40 mL, 0.12 vol.). After stirring for 2 hours at 0? C. to 5? C. The mixture was stirred at 15 to 20? C. for 17.5 h. HPLC monitoring showed 99.0% conversion. Deionized water (1730 mL, 4.89 vol.) was added within 9 min at a maximum temperature of 25? C.

[0092] The mixture was decanted. Aqueous layer was analyzed then discarded. Organic layer was washed with 1M hydrochloric acid aqueous solution. Aqueous layer was analyzed then discarded. Organic layer was washed first with deionized water (1351 mL, 5 vol) then with a saturated sodium chloride solution (3?1177 mL, 3?4.25 vol.).

[0093] Aqueous layers (pH=4 after the last washing) were analyzed and discarded. Organic layer was dried over sodium sulfate (240 g, 86.8% w/w), washed with methyl tert-butyl ether (616 mL, 2 vol.) and concentrated under vacuum to afford (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (compound (XI) in quantitative yield (417.4 g, corresponding to 322.1 g of pure product, yield 85.8% pure).

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 (I) (Crude Compound).

[0094] (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy) hexanoate (compound (XI)) (407.3 g crude, leq.) was dissolved in methanol (3550 mL, 8.9 vol.). The resulting solution was charged in the flask and equipment was rinsed with methanol (1140 mL, 2.8 vol.). The mixture was stirred at 15? C. to 25? C. for 15 h monitoring the reaction by .sup.1H-NMR. Methanol was then removed under vacuum at 35? C. to 40? C. Methanol (3550 mL, 8.8 vol.) was added and the solution was transferred into a reactor. Reaction was stirred at 20-22? C. for 16 h. IPC was performed by NMR and showed completion of the reaction. The reaction mixture was concentrated under vacuum at a temperature below 40? C. The residue was dissolved in methyl tert-butyl ether (4350 mL, 10.85 vol.). The resulting solution was washed with deionized water (2190 mL). The aqueous layer (pH=7) was discarded. Organic layer was washed with the sodium chloride solution (2?1930 mL, 2?4.8 vol.). Aqueous layers were discarded. Organic layer was dried over sodium sulfate (395 g, 1 eq w/w), washed with methyl tert-butyl ether (790 mL, 2 vol.) and concentrated under vacuum at a temperature below 40? C. to afford 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)) (367.1 g, 98.3% yield) having a HPLC purity of 80.4%.

[0095] The mixture was purified using silica gel column (750 g?6, 10.34 vol.) and dichloromethane/methanol as eluent with a gradient 100:0 to 95:5 on a Combiflash. The fractions were monitored by TLC and analyzed by HPLC (% area). Fractions were concentrated under vacuum at temperature equal to or below 50? C. to afford 206 g 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)) that were charged in a 4 L three-neck round-bottomed flask and dissolved in absolute ethanol (2100 mL, 10 vol.), activated charcoal (21 g, 10%/w) was added and the mixture was stirred at 20? C. to 25? C. for 0.5 h. The charcoal was filtered and washed with absolute ethanol (210 mL, 1 vol.). The filtrate was concentrated under industrial vacuum at 45? C. to 50? C. for 4 h then under high vacuum at 45? C. to 50? C. for 8 h. Monitoring by .sup.1H NMR in DMSO-d6 showed no residual solvents.

[0096] 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 (192.9 g.) was obtained with 62% overall yield from compound (II). HPLC purity was 99.13%.

TABLE-US-00001 TABLE 1 Quantitative analysis and chemical yield of 6-(nitrooxy)hexanoic acid prepared according to the process of the present invention (Example 1) and to the process disclosed in WO 2019/162149 (Example 2 - comparative) Effective yield of Final amount HPLC (VIIIa) Starting of Crude Purity of Amount of corrected amount of Compound Compound compound by HPLC compound X (VIIIa) (VIIIa) (IXa) data Example (g) (g) (a/a %) (a/a %) (%) Example 1 306.1 318.4 99.2 0.15 99.0 Example 2 110.9 105.4 83.7 8.80 76.4 (Nitration 1) Example 2 110.0 101.6 89.7 5.82 79.6 (Nitration 2)

TABLE-US-00002 TABLE 2 UHPLC quantitative analysis of compound (I) and its impurity profile prepared according to the process of the present invention (Example 1) and to the process disclosed in WO 2019/162149 (Example 2 - comparative) Content (a/a %) Example Compound (I) Impurity (VII) Impurity (V) 1 99.89 <0.05 0.11 2 99.13 0.40 0.12

[0097] Table 1 reports the results of the quantitative analysis and chemical yield of 6-(nitrooxy)hexanoic acid (VIIIa) prepared according to the nitration reactions of the present invention and of the process disclosed in WO 2019/162149. The results show that the addition of water, instead of a saturated solution of NaCl in water (brine) as described in WO 2019/162149, during the work-up of the nitration reaction of the 6-hy droxyhexanoic acid alkali salt led to a crude nitration mixture containing almost pure compound (VIIIa) (purity of 99.2% (a/a %) and containing a reduced amount of the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa) (0.15% a/a %). Compound (VIIIa) was also obtained in high chemical yield such as 99%. The use of the crude nitration mixture of step 5a) allows to obtain the final product 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 (I) that contains and amount of the impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost (VII) less than 0.05%. Indeed, the results of Table 2, which reports the quantitative analysis of compound (I) and of the main impurities prepared according to the process of the invention (Example 1) and to a method discloses in WO 2019/162149 (Example 2-comparative), show that the process of the invention provides compound (I) with a content of the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid ester of bimatoprost (compound (VII)) below the detection limit of 0.05% and a content of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (V)) of 0.11%. The process disclosed in the prior art leads to compound (I) having a lower chemical purity and a content of compound (VII) from 0.1% to 0.4%.

[0098] The results demonstrate that the process of the invention represents an improved process easily transferable to the industrial scale.