Process for the preparation of prostaglandin analogs for the supply of nitric oxide

Abstract

The present invention relates to a process for the preparation of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I). According to the present invention, the compound (I) can be efficiently prepared in high purity by coupling bimatoprost in boronate protected form with 6-(nitrooxy)hexanoyl chloride and removing the boronate protecting group. The present 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 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa)) in an amount equal to or lower than 0.2%.

Description

Technical Field

[0001] This invention relates to a method suitable for large-scale preparation of 6-(nitrooxy)hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I), which allows for the acquisition of said product with high chemical purity. The invention also describes the preparation of high-purity 6-(nitrooxy)hexanoic acid (VIIIa) as a key synthetic intermediate. Background of the Invention

[0003] The 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I)

[0004]

[0005] It is a prostaglandin analogue that has been proven to be an effective IOP reducer (Impagnatiello F, Toris CB, Batugo M, Prasanna G, Borghi V, Bastia E, Ongini E, Krauss AHP; Invest Ophthalmol Vis Sci. 2015; 56:6558-64).

[0006] In the first phase 3 clinical trial that began in June 2020, the effects of two ophthalmic solutions containing compound (I) as the active pharmaceutical ingredient (0.065% and 0.1%) on reducing intraocular pressure (IOP) were evaluated in patients with open-angle glaucoma or ocular hypertension.

[0007] In recent years, various regulatory agencies have emphasized purity requirements and impurity identification for active pharmaceutical ingredients (APIs). Currently, any impurity is considered an organic substance, other than the drug substance itself, that can affect the efficacy and safety of a drug product. Therefore, the identification and quantification of impurities, especially those carrying mutagenic structural warnings, has become a mandatory regulatory requirement. Furthermore, because active pharmaceutical ingredients are intended for pharmaceutical use, the reagents, solvents, catalysts, etc., used in the synthesis of active ingredients are limited to those acceptable to the pharmaceutical industry.

[0008] 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I) is an oil whose large-scale purification is difficult because compound (I) cannot be crystallized; therefore, the presence of impurities is a key issue for large-scale production.

[0009] As is known from the prior art methods for preparing compound (I), the main impurity is: bimatoprost 15-(6-chlorohexanoyl) ester of formula (V) when a nitration step is performed at the end of the synthesis as disclosed in WO 2009 / 136281.

[0010]

[0011] Alternatively, it can be a byproduct of the nitration step in the preparation of intermediate 6-(nitrooxy)hexanoic acid (VIIIa), leading to the formation of bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (VII).

[0012]

[0013] Therefore, controlling reagent purity and reaction conditions is an important requirement for obtaining compound (I) with pharmaceutically acceptable purity.

[0014] WO 2009 / 136281 discloses a method for preparing compound (I). WO 2009 / 136281 discloses the synthesis of compound (I) and summarizes the preparation of bimatoprost 15-alkyl nitrate.

[0015] WO 2009 / 136281 discloses the synthesis of the following compound of formula (I) (Example B-1): by reacting the borate-protected form of bimatoprost (II) with 6-bromohexanoyl chloride to obtain the borate-protected form of bimatoprost 15-(6-bromohexanoyl) ester of formula (III), which is then deprotected by converting it to a nitric acid derivative in acetonitrile with silver nitrate and purified by reversed-phase chromatography to obtain the compound of formula (I).

[0016]

[0017] The main drawbacks of the above synthesis are the use of more than an equimolar amount of 6-bromohexanoyl chloride in the esterification reaction, which presents a potentially mutagenic structural warning, and the use of silver nitrate in the final step, which generates a large amount of silver salt in the wastewater. Another major drawback of this method is the formation of impurities and byproducts, such as bimatoprost 15-(6-bromohexanoyl) ester of formula (IV) and bimatoprost 15-(6-chlorohexanoyl) ester of formula (V), which are difficult to remove even after multiple purifications, because they have similar polarity, similar lipophilicity and / or solubility in chromatography as compound (I).

[0018]

[0019] Furthermore, as required by the regulatory agency, compound (V) was predicted to be positive for in vitro mutagenicity in bacteria using both statistical and expert rule-based methods. Compound (IV) also contained halogenated chains that were identified as potentially genotoxic. The removal of these impurities required repeated purification, which further reduced the yield and increased the cost of preparing compound (I) on a commercial scale.

[0020] According to the procedure disclosed in WO 2009 / 136281, bimatoprost 15-(6-bromohexanoyl) ester (IX) is an impurity derived from the incomplete reaction of compound (III) with silver nitrate after the removal of borate protection.

[0021] Bimatoprost 15-(6-chlorohexanoyl) ester (V) is a byproduct formed during esterification via a halogen exchange reaction between the bromine atom of the borate-protected form of bimatoprost 15-(6-bromohexanoyl) ester (III) and the free chloride anion of 4-dimethylaminopyridine hydrochloride. The borate-protected form of bimatoprost 15-(6-chlorohexanoyl) ester (VI) is also present.

[0022]

[0023] It does not react with silver nitrate and forms compound (V) after the protecting group is removed.

[0024] WO 2009 / 136281 also discloses an alternative method for preparing 15-acylalkyl bimatoprost nitrate derivatives (Examples N-1 and O-1). This synthesis involves reacting the borate-protected form of bimatoprost (II) with a nitrate-alkylcarboxylic acid chloride in the presence of 4-dimethylaminopyridine (DMAP) supported on a resin (PS-DMAP), followed by removal of the borate protecting group and purification using silica gel chromatography.

[0025] The above method avoids the use of 6-bromohexanoyl chloride and the removal of silver salt from the final product. However, another major drawback of this method is the use of 4-dimethylaminopyridine supported on the resin, which makes the method unsuitable for commercial scale-up and expensive. Moreover, the nitrate-alkylcarboxylic acid chloride is added in two consecutive steps and in a large excess relative to the compound of formula (II); indeed, the alkylcarboxylic acid chloride is added in an amount of about 2 to 4 equivalents.

[0026] WO 2009 / 136281 also discloses another method for preparing 15-acylalkyl bimatoprost derivatives (Example Q1). In this method, the compound is obtained by esterifying the borate-protected form of bimatoprost (II) with an excess of nitrate-alkyl-(p-nitrophenyl)-carboxylic acid ester in the presence of 4-dimethylaminopyridine.

[0027] The main drawback of this method is that it uses chromatography to remove unreacted nitrate-alkyl-(p-nitrophenyl)-carboxylic acid esters and the byproduct p-nitrophenol, which is formed in equimolar amounts with the compound of formula (I).

[0028] WO 2016 / 155906 discloses a 15-nitrooxy derivative of fluoroprostaglandin, reporting the synthesis of a 15-nitrooxy-hexyl ester of fluoroprostaglandin isopropyl ester. This compound is prepared by reacting the borate-protected form of fluoroprostaglandin isopropyl ester with (4-nitrophenyl)-6-nitrooxyhexanoate in the presence of excess 4-dimethylaminopyridine.

[0029] As reported above, the main drawback of this method is the removal of unreacted nitrate-alkyl-(p-nitrophenyl)-carboxylic acid esters and, in particular, p-nitrophenol byproducts by chromatography.

[0030] WO 2019 / 162149 discloses a method for preparing 6-(nitrooxy)hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I). Compound (I) is prepared by coupling the borate-protected form of bimaprost with 6-(nitrooxy)hexanoyl chloride and removing the borate-protecting group. The crude compound (I) is purified by column chromatography.

[0031] 6-(nitrooxy)hexanoyl chloride is synthesized from 6-(nitrooxy)hexanoic acid, which is prepared by the ring-opening reaction of ε-caprolactone followed by nitration of the alkali metal salt of 6-hydroxyhexanoic acid with a mixture of HNO3 and H2SO4 in dichloromethane. Unpurified 6-(nitrooxy)hexanoic acid is used to prepare the corresponding acyl chloride, and crude 6-(nitrooxy)hexanoyl chloride is also used unpurified.

[0032] Experiments conducted by the inventors showed that compound (I) prepared using the method of WO 2019 / 162149 contained about 0.1% to 0.4% of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate impurity of formula (VII).

[0033]

[0034] Compounds (I) and (VII) have similar polarity, so the separation of these two compounds requires cumbersome column purification to remove impurity (VII), thus reducing the yield of the final product.

[0035] Therefore, there is a need to develop an industrially feasible method for preparing high-purity 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-heptene-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I) in high yield.

[0036] An impurity of formula (VII) was observed to form in the coupling step of the method in WO 2019 / 162149 because the crude 6-(nitrooxy)hexanoyl chloride of formula (VIIIb) contains the dimer impurity 6-[6-nitrooxyhexanoyl]oxy)hexanoyl chloride of formula (IXb).

[0037]

[0038] It also reacts with the borate protected form (II) of bimatoprost and, upon removal of the borate protection, forms compound (I) and bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (VII).

[0039] The 6-[6-nitrooxyhexanoyl]oxy}hexanoyl chloride of formula (IXb) is a byproduct derived from the crude mixture of the nitration step; indeed, nitration of the alkali metal salt of 6-hydroxyhexanoic acid with a mixture of nitric acid and sulfuric acid results in the formation of 6-(nitrooxy)hexanoic acid (VIIIa) and the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa).

[0040]

[0041] Experiments showed that the nitration mixture contained more than 80% of compound (VIIIa) and about 5% to about 9% of compound (IXa). Both compounds (VIIIa) and (IXa) reacted with a chlorinating agent to produce a mixture containing 6-(nitrooxy)hexanoyl chloride (VIIIb) and the dimer impurity 6-[6-nitrooxyhexanoyl]oxy}hexanoyl chloride (IXb). This crude mixture was reacted with compound (II) without further purification according to method WO2019 / 162149.

[0042] To control the formation of impurity (VII), it was unexpectedly found that adding water instead of a saturated aqueous solution of NaCl (brines) as described in WO 2019 / 162149 during the nitration of the alkali metal salt of 6-hydroxyhexanoic acid produced a crude nitration mixture containing a reduced amount of the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa). Specifically, this change in the post-treatment of the nitration step produced a crude nitration mixture containing 99.2% (a / a%) of 6-(nitrooxy)hexanoic acid (VIIIa) and 0.15% (a / a%) of the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa). Compounds (VIIIa) and (IXa) were identified and quantified by appropriate UHPLC analysis.

[0043] It is believed that, compared to adding a commonly used saturated NaCl aqueous solution (brinest water), adding water at the end of the nitration reaction allows for rapid dissolution of the inorganic salt and better separation of the organic phase containing 6-(nitrooxy)hexanoic acid (VIIIa) from the acidic aqueous phase containing unreacted HNO3 / H2SO4. This improved post-treatment of the nitration reaction reduces / inhibits side reactions that lead to the formation of the byproduct 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa).

[0044] In addition, another advantage of this change to the nitration step is that it reduces the volume of organic solvent used for post-reaction treatment, which allows this step to be scaled up by at least three times.

[0045] Therefore, the present invention provides an improved method suitable for large-scale production of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I), which contains less than 0.05% of the impurity bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (VII) (measured by HPLC as a / a%).

[0046]

[0047] A key advantage of the method of this invention is that it very effectively reduces the level of impurity (VII) while simultaneously providing compound (I) in good yield. This method is a favorable alternative to other methods, although other methods can theoretically achieve the same degree of reduction in the level of impurity (VII), but require additional purification steps including the 6-(nitrooxy)hexanoic acid intermediate.

[0048] Because 6-(nitrooxy)hexanoic acid is a liquid and both 6-(nitrooxy)hexanoic acid (VIIIa) and 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa) are polar compounds, it is not easy to purify large quantities of 6-(nitrooxy)hexanoic acid (VIIIa) for industrial-scale preparation.

[0049] Furthermore, the present invention relates to a method for the industrial synthesis of 6-(nitrooxy)hexanoic acid (VIIIa).

[0050]

[0051] The substance has a purity of 99% or greater and a content of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa) of 0.2% or less.

[0052] Summary of the Invention

[0053] This invention relates to a method for preparing 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I):

[0054]

[0055] The method includes the following steps:

[0056] Step A) is as follows: 6-(nitrooxy)hexanoyl chloride (VIIIb) is prepared.

[0057] 1a) ε-caprolactone

[0058]

[0059] The 6-hydroxyhexanoate of formula (X) is obtained by reacting an inorganic base selected from KOH, NaOH, and LiOH in a solvent selected from methanol, ethanol, or isopropanol at a temperature from 20°C to the solvent reflux temperature.

[0060]

[0061] Where M is K, Na, or Li;

[0062] 2a) Purification of the 6-hydroxyhexanoate of formula (X) obtained in step 1a), comprising:

[0063] i. Add methyl tert-butyl ether to the reaction mixture of step 1a) at a volume ratio of 2:1 (methyl tert-butyl ether / reaction mixture);

[0064] ii. Filtering solids;

[0065] iii. Prepare a slurry from the solid using a mixture of methyl tert-butyl ether, methanol and water in a ratio of 3:1 to 5:1, wherein the amount of water is 0.3-1 mol per mole of ε-caprolactone;

[0066] iv. 6-hydroxyhexanoate solid of formula (X);

[0067] 3a) React 6-hydroxyhexanoate with a mixture of fuming HNO3 and concentrated H2SO4 in dichloromethane at a temperature ranging from 0°C to 10°C;

[0068] 4a) Post-treatment of the reactants in step 3a) by adding water to the nitration mixture in step 3a) while maintaining the temperature between 0°C and 5°C;

[0069] 5a) Separate the organic phase, dry the organic phase with sodium sulfate, and distill off the solvent to obtain crude 6-(nitrooxy)hexanoic acid (VIIIa).

[0070]

[0071] 6a) Crude 6-(nitrooxy)hexanoic acid (VIIIa) is reacted with a chlorinating agent in dichloromethane to obtain crude 6-(nitrooxy)hexanoic acid.

[0072] (VIIIb) hexanoyl chloride;

[0073] Step B) prepares 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I) as follows:

[0074] 1b) Reaction of bimatoprost with butylboronic acid in methyl tert-butyl ether at a temperature of approximately 40°C yields...

[0075] Compound (II) is obtained:

[0076]

[0077] 2b) Compound (II) is reacted with crude 6-(nitrooxy)hexanoyl chloride (VIIIb) from step 6a) in an aprotic organic solvent in the presence of free 4-dimethylaminopyridine to obtain compound (XI).

[0078]

[0079] 3b) Remove the borate ester protecting group to obtain the crude compound of formula (I);

[0080] 4b) Purification of crude compound (I) by column chromatography;

[0081] The method is characterized in that the crude nitration mixture in step 5a) contains 99% or more of 6-(nitrooxy)hexanoic acid (VIIIa) and 0.2% or less of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa).

[0082] The 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I) prepared according to the method of the present invention has a chemical purity of ≥99% and contains less than 0.05% of the impurity bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (VII). The compound contains about 0.11% of the impurity bimaprost 15-(6-chlorohexanoyl) ester (V). The compound was identified and quantified by HPLC analysis, and the amount of the compound is expressed as area percentage (a / a%); the HPLC detection limit is 0.05%.

[0083] In step 1a), the ring-opening reaction of ε-caprolactone is preferably carried out in the presence of potassium hydroxide, in methanol, and at a temperature from 20°C to reflux. During the post-treatment of the reaction mixture in step 1a), the potassium 6-hydroxyhexanoate is purified as follows: the solid from step 2a)-ii is pulped in a mixture containing methyl tert-butyl ether, methanol, and 0.3-1 mol of water relative to each mole of ε-caprolactone (step 2a)-iii. This yields the potassium 6-hydroxyhexanoate solid.

[0084] The crude nitration mixture of step 5a) contains 99% or more of 6-(nitrooxy)hexanoic acid (VIIIa) and 0.2% or less of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa). The chemical yield of 6-(nitrooxy)hexanoic acid (VIIIa) is 80-85%. Chemical purity was assessed by analytical UHPLC.

[0085] The preferred chlorinating agent used in step 6a) is oxalyl chloride.

[0086] Step 2b) is carried out at a temperature ranging from 0°C to 25°C, preferably in an aprotic organic solvent selected from methyl tert-butyl ether, N,N-dimethylformamide, or dichloromethane; most preferably, the organic solvent is methyl tert-butyl ether. "Free form" of 4-dimethylaminopyridine (DMAP) means that DMAP is not bound to the resin. The molar ratio of compound (II) to 6-(nitrooxy)hexanoyl chloride (VIIIb) is preferably in the range of 1:1.4 to 1:1.6.

[0087] The molar ratio of compound (II) to 4-dimethylaminopyridine is preferably from 1:2.0 to 1:2.4.

[0088] In step 3b), the removal of the borate ester protecting group is preferably carried out with methanol at a temperature of 17°C to 25°C.

[0089] A preferred method for preparing compound (I) comprises the following steps, as described in process 1:

[0090] Step A

[0091] 1a) Reaction of ε-caprolactone with potassium hydroxide in methanol at a temperature of 20°C to the methanol reflux temperature, preferably at the methanol reflux temperature, and

[0092] 2a) Purification of the potassium 6-hydroxyhexanoate obtained in step 1a), comprising:

[0093] i. Add methyl tert-butyl ether (MTBE) to the reaction mixture of step 1a) at a ratio of 2:1 MTBE / methanol;

[0094] ii. Filtering solids;

[0095] iii. The solid is prepared into a slurry in a mixture of methyl tert-butyl ether, methanol and water in a ratio of 3:1 to 5:1, wherein the amount of water is 0.3-1 mol per mole of ε-caprolactone;

[0096] iv. Separate the solid potassium 6-hydroxyhexanoate.

[0097] 3a) Add potassium 6-hydroxyhexanoate (formula (X), M=K) to a mixture of fuming HNO3 and concentrated H2SO4 in dichloromethane at a temperature of about 0°C to 10°C, and vigorously stir the reaction mixture at a temperature of 0°C to 5°C while monitoring the reaction progress (until 99.9% conversion);

[0098] 4a) Carefully add water to the nitrification mixture and keep the temperature between 0°C and 5°C.

[0099] 5a) Separate the organic phase, dry the organic phase with sodium sulfate, and distill off the solvent to obtain crude 6-(nitrooxy)hexanoic acid (VIIIa) containing 99% or more of 6-(nitrooxy)hexanoic acid (VIIIa) and less than 0.2% of the byproduct 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (IXa).

[0100] 6a) Crude 6-(nitrooxy)hexanoic acid is reacted with oxaloyl chloride to obtain 6-(nitrooxy)hexanoyl chloride (VIIIb), which is used in step B of the method without further purification;

[0101] Step B

[0102] 1b) Bimatoprost was reacted with butylboronic acid (1.1-1.8 equivalents) in methyl tert-butyl ether (MTBE) at a temperature of about 40°C, and then water was removed by azeotropic distillation to obtain bimatoprost borate (II).

[0103] 2b) Bimaprost borate (II) is reacted with crude 6-(nitrooxy)hexanoyl chloride (VIIIb) (1.4-1.6 equivalents) obtained in step 6a) in methyl tert-butyl ether in the presence of 4-dimethylaminopyridine (2.0-2.4 equivalents) at a temperature ranging from 0°C to 20±3°C to obtain 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohep-2-en-1-yl]-2,4-dioxa-3-borazadicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl ester (XI);

[0104] 3b) Reaction of 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borazadicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl ester (XI) with methanol at room temperature to remove the protecting group and obtain crude 6-(nitrooxy)hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I).

[0105] 4b) The crude compound (I) was purified by high performance silica gel chromatography using dichloromethane / methanol as the mobile phase to obtain 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I).

[0106] After chromatographic purification, the isolated compound (I) was quantitatively analyzed by HPLC. It had a chemical purity of over 99% and contained approximately 0.11% of bimatoprost 15-(6-chlorohexanoyl) ester (V) and 0.05% of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (VII) below the HPCL detection limit.

[0107]

[0108] Another object of the present invention is a pharmaceutical formulation comprising 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) and at least a pharmaceutically acceptable excipient, wherein 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester and at least a pharmaceutically acceptable excipient, 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 about 0.11% of bimaprost 15-(6-chlorohexanoyl) ester (compound (V)) and less than 0.05% of the dimer impurity bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (VII)).

[0109] In this application, the quantification of compounds expressed as percentage % is determined by analytical HPLC (a / a%). Example

[0110] All the following synthesis steps were carried out in a nitrogen atmosphere.

[0111] Example 1

[0112] Synthesis of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (I)) Synthesis of 6-(nitrooxy)hexanoyl chloride (compound (VIIIb))

[0113] Steps 1a) and 2a): Synthesis of potassium 6-hydroxyhexanoate (compound (X))

[0114] A solution of potassium hydroxide (141.12 g, corresponding to 122.8 g of pure substance) in methanol (1250 mL) was prepared under cooling at 15 °C to 20 °C. This solution was then added to a solution of 250.0 g of ε-caprolactone in methanol (675 mL) over 0.25 hours at 5 °C to 37 °C. The mixture was stirred at 20 °C to 63 °C for 18 hours. The solvent was then concentrated to approximately 675 mL, and methyl tert-butyl ether (1250 mL) was added at approximately 57 °C. The reaction mixture was stirred at room temperature for 48 hours, and the solid was separated by filtration to obtain 380.9 g of crude potassium 6-hydroxyhexanoate. The crude substance was re-slurryed at 22 ± 3 °C in MTBE (1475 mL), methanol (375 mL), and water (19.7 mL) for 8 hours, and then filtered. The solid was re-slurryed in methyl tert-butyl ether (500 ml), filtered again, and then dried under vacuum at 50 °C for 12 hours to obtain 354.7 g of potassium 6-hydroxyhexanoate (93.2% yield) with a purity of 98%. Melting point 208 °C.

[0115] Steps 3a) to 5a): Synthesis of 6-(nitrooxy)hexanoic acid (compound (VIIIa))

[0116] Fuming HNO3 (485.5 g, 4.6 equivalents) was added to concentrated H2SO4 (553.0 g, 3.1 equivalents) over 30 minutes at 0 to 5°C, followed by the addition of dichloromethane (5820 ml) over 23 minutes at 0 to 5°C. Potassium 6-hydroxyhexanoate (306.09 g, 1 equivalent) was added batch-by-batch over 35 minutes at a temperature below 5°C. The mixture was stirred at 0 to 5°C for 2 hours. 1 ¹H-NMR monitoring showed 99.9% conversion. Water (2.9 L) was added over 20 minutes while maintaining the temperature between 0°C and 5°C. 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) with 99.2% HPLC purity and 0.15% 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa)).

[0117] Step 6a): Synthesis of 6-(nitrooxy)hexanoyl chloride (compound (VIIIb))

[0118] 6-(nitrooxy)hexanoic acid (270.8 g, 1 equivalent) was dissolved in dichloromethane (1220 ml) and cooled to 0-5°C under nitrogen. Then, N,N-dimethylformamide (1.6 ml) and oxaloyl chloride (198.2 g) were added over 30 minutes at 0-5°C. The reaction mixture was stirred at 0-5°C for 1 hour, then at 15-20°C for 4 hours. The reaction mixture was then concentrated under vacuum at or below 40°C and co-evaporated with dichloromethane to give 6-(nitrooxy)hexanoyl chloride (323.9 g, 99.6% yield, titration AgNO3 = 92.0%).

[0119] Step 1b): (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-ene] The synthesis of [3.2.1)octane-7-yl]-N-ethyl-hept-5-enamide (compound (II)) become

[0120] Bimatoprost (249.0 g, 1 equivalent) was suspended in methyl tert-butyl ether (4 L), and butylboronic acid (69.0 g, 1.13 equivalent) was added. The mixture was heated to 40 °C for 1 hour. 1 H NMR monitoring of the reaction continued until conversion >97%.

[0121] Cool the reaction mixture to approximately 20°C, filter, and rinse with methyl tert-butyl ether (250 ml). Concentrate the mixture under vacuum at approximately 40°C by azeotropic distillation. Rinse with methyl tert-butyl ether and continue azeotropic distillation until 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-boronabicyclo[3.2.1)octane-7-yl]-N-ethyl-hept-5-enamide (compound (II)) is equal to or less than 0.25%.

[0122] The crude product (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-boronabicyclo[3.2.1)octan-7-yl]-N-ethyl-hept-5-enamide was isolated in quantitative yield (313.1 g, corresponding to 298.6 g of pure substance).

[0123] Step 2b): 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino) [3.2.1]oct-6-yl]-1-(2-phenylethyl)- Synthesis of prop-2-en-1-yl ester (compound (XI))

[0124] (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borazadicyclo[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 atmosphere and cooled to 0°C to 5°C. 4-Dimethylaminopyridine (162.7 g, 2.27 equivalent) was added in a single addition. A solution of 6-(nitrooxy)hexanoyl chloride (compound (VIIIb)) (172.1 g, 1.5 equivalent) in methyl tert-butyl ether (600 mL) was added dropwise over 40 minutes at 0°C to 5°C. After stirring at 0°C to 5°C for 2 hours and 30 minutes and at 15°C to 20°C for 16.5 hours, the reaction mixture was cooled at 0°C to 5°C and deionized water was added over 20 minutes at a maximum temperature of 10°C.

[0125] Stir the mixture for 5 minutes. Separate and discard the aqueous layer. Wash the organic layer with 1N hydrochloric acid solution, then with deionized water, and finally with brine.

[0126] The organic layer was dried with sodium sulfate and concentrated under vacuum to give 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohep-2-en-1-yl]-2,4-dioxa-3-boronabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl ester (compound (XI)) (402.9 g, 94.9% yield).

[0127] Steps 3b) and 4b): 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethyl) [[(2-phenylethyl)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (chemical) Synthesis of compound (I)

[0128] Dissolve 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohep-2-en-1-yl]-2,4-dioxa-3-borazadicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl ester (compound (XI)) (396 g crude product, 1 equivalent) in methanol (4.20 L). Stir the resulting solution at room temperature for 24 hours. 1The reaction was monitored by ¹H-NMR. Methanol was then removed under vacuum at 35-40°C. The residue was dissolved in methanol, stirred for 14 hours, and evaporated under vacuum at 35-40°C. The residue was dissolved in methyl tert-butyl ether, washed with deionized water, and then washed with brine. The organic layer was dried over sodium sulfate and concentrated under vacuum at a temperature below 40°C to give crude 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (352.3 g, 96.5% yield).

[0129] The residue was fractionated into three equal portions (approximately 107 g each) and purified by chromatography on a Biotage Isolera LS system using a high-performance silica column (SNAP Ultra column, 1500 g silica) with dichloromethane / methanol as eluent at a gradient of 100:0 to 95:5 v / v. The fractions were monitored by TLC and UHPLC. The fractions were concentrated under vacuum at or below 50 °C to give an oily compound (I) (242.5 g, 90.3% yield).

[0130] 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (I)) was dissolved in ethanol, treated with charcoal for 30 minutes, filtered, and the solvent was evaporated under vacuum at a temperature below 50°C. The oily compound (I) (212.7 g, 92.4% yield) was isolated. The purity of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (I)) was 99.89% (evaluated by UHPLC), the content of bimaprost 15-(6-chlorohexanoyl) ester (compound (V)) was 0.11%, and the content of the dimer impurity bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (VII)) was less than 0.05%.

[0131] Example 2 (Comparative Example)

[0132] The synthesis described below was performed according to the method disclosed in WO 2019 / 162149.

[0133] Synthesis of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I)

[0134] Synthesis of 6-(nitrooxy)hexanoyl chloride (VIIIb)

[0135] Synthesis of potassium 6-hydroxyhexanoate (compound (X))

[0136] A solution of 90% (89.7 g) potassium hydroxide in methanol (830 ml) was prepared under cooling at 10-20 °C. 165.2 g of ε-caprolactone (1 equivalent) and methanol (415 ml) were introduced into a 4 L three-necked round-bottom flask. The mixture was stirred until dissolved. The potassium hydroxide solution in methanol was then added over 25 minutes at 0-30 °C. The mixture was stirred at 15-20 °C for 20 hours. The reaction mixture was concentrated under vacuum (at a temperature equal to or below 40 °C) to give crude potassium 6-hydroxyhexanoate. The crude solid was suspended in methyl tert-butyl ether (830 ml) at 15-20 °C for 2 hours, filtered through a pore size 3 filter, washed with methyl tert-butyl ether (2 × 165 ml), and dried under vacuum at 35 °C to give a white powder of potassium 6-hydroxyhexanoate (228.1 g, 98.3% HCl analysis), yield 92.7%.

[0137] Synthesis of 6-(nitrooxy)hexanoic acid (VIIIa)

[0138] The nitration reaction was carried out on a scale of approximately 100 g to control the reaction temperature and minimize the time of addition of the nitration mixture. The reaction was performed twice. The nitration mixture was quenched by adding a saturated aqueous sodium chloride solution (brine). The results of the two separate nitration reactions are reported in Table 1.

[0139] Nitrification 1 Concentrated sulfuric acid (200.9 g) was added to a 6 L glass reactor under nitrogen atmosphere and cooled to 0-5 °C. Fuming HNO3 (187.9 g) was carefully added dropwise over 20 minutes at 0-10 °C. Then dichloromethane (2.23 L) was added, and the reaction mixture was stirred for 45 minutes. Potassium 6-hydroxyhexanoate (110.1 g, 1 equivalent) was added fractionally over 30 minutes at -5-5 °C. The mixture was stirred at -5-5 °C for 2.5 hours, then stirred overnight at 20 °C. 1The reaction was monitored by ¹H-NMR, showing 99.6% conversion. A saturated aqueous sodium chloride solution (315.3 g in 1.0 L) was carefully added over 25 minutes at or below 10 °C. A large amount of inorganic salt precipitate was observed. The reaction mixture was transferred to a separatory funnel, taking care not to transfer the inorganic salt. The organic layer was decanted, dried over sodium sulfate, and concentrated under vacuum (at or below 40 °C) to give crude 6-(nitrooxy)hexanoic acid (105.4 g, 85.2%). HPLC purity = 83.7%, amount of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa) 8.8%).

[0140] Nitration 2 : 109.97 g of potassium 6-hydroxyhexanoate was used to react and 101.6 g of crude 6-(nitrooxy)hexanoic acid (VIIIa) (82%) was obtained, which had an HPLC purity of 89.7% and a content of 5.8% of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (IXa)).

[0141] The two batches were combined and proceeded to the following steps without further purification.

[0142] Synthesis of 6-(nitrooxy)hexanoyl chloride (VIIIb)

[0143] 193 g of 6-(nitrooxy)hexanoic acid was dissolved in 870 mL of dichloromethane. The resulting turbid solution was filtered through a glass fiber filter, washed with 130 mL of dichloromethane, and the clear solution (water content = 0.018%) was analyzed by Karl Fischer. The solution was cooled to 0-5 °C under nitrogen. N,N-dimethylformamide (1.2 mL) and oxaloyl chloride (141.4 g) were then added over 30 minutes at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 1 hour, and then at 15-20 °C for 14 hours. TLC monitoring showed that the reaction was complete. The mixture was concentrated under vacuum (temperature equal to or below 40 °C) and co-evaporated with 4 × 870 mL of dichloromethane to give 212.4 g of 6-(nitrooxy)hexanoyl chloride in 91.9% yield.

[0144] (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4- Preparation of dioxa-3-boronabicyclo[3.2.1)octane-7-yl]-N-ethyl-hept-5-enamide (compound (II))

[0145] Add methyl tert-butyl ether (3900 mL, 14 volumes) to the flask. Add bimatoprost (249.1 g, 1 equivalent), and rinse the apparatus with methyl tert-butyl ether (250 mL, 1 volume). Add butylboronic acid (70.66 g, 69.2 g pure substance, 1.13 equivalents) to the resulting suspension in one addition, and rinse the apparatus with methyl tert-butyl ether (250 mL, 1 volume). Heat the mixture to 40°C for 2 hours. 1H NMR monitoring of the reaction continued until conversion >97%.

[0146] 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 volume). The filtrate was concentrated under vacuum by azeotropic distillation at approximately 40°C. Washing with methyl tert-butyl ether and azeotropic distillation continued until 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-boronabicyclo[3.2.1)octane-7-yl]-N-ethyl-hept-5-enamide (compound (II)) was 0.2%. 1 H NMR analysis of compound (II) was used to determine residual MTBE.

[0147] (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-boronabicyclo[3.2.1)octane-7-yl]-N-ethyl-heptane-5-enamide (compound (II)) was obtained in quantitative yield (386.7 g crude, uncorrected).

[0148] 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxo [3.2.1]oct-6-yl]-1-(2-phenylethyl)-prop-2-ene-1- Preparation of basic ester (XI)

[0149] Methyl tert-butyl ether (4100 mL, 11.6 V) was added to a 4 L three-necked round-bottom flask under nitrogen atmosphere. (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-boronabicyclo[3.2.1)octane-7-yl]-N-ethyl-heptane-5-enamide (compound (II)) (378 g crude, 1 equivalent) was added, and the apparatus was rinsed with methyl tert-butyl ether (510 mL, 1.44 V). The resulting solution was cooled to 0°C to 5°C.

[0150] A single addition of 177.5 g (2.27 equivalents) of 4-dimethylaminopyridine was made. A solution of 204.4 g (corresponding to 188 g of pure substance, 1.5 equivalents) of 6-(nitrooxy)hexanoyl chloride in 650 mL (1.84 volume) was added dropwise over 45 minutes at 0°C to 5°C. The dropping funnel was rinsed with 40 mL (0.12 volume) of methyl tert-butyl ether. The mixture was stirred at 0°C to 5°C for 2 hours. The mixture was then stirred at 15°C to 20°C for 17.5 hours. HPLC monitoring showed 99.0% conversion. Deionized water (1730 mL, 4.89 volume) was added over 9 minutes at a maximum temperature of 25°C.

[0151] Decant the mixture. Analyze the aqueous layer and discard it. Wash the organic layer with 1M hydrochloric acid solution. Analyze the aqueous layer and discard it. Wash the organic layer first with deionized water (1351 mL, 5 volumes), then with saturated sodium chloride solution (3 × 1177 mL, 3 × 4.25 volumes).

[0152] The aqueous layer (pH = 4 after the last wash) was analyzed and discarded. The organic layer was dried over sodium sulfate (240 g, 86.8% w / w), washed with methyl tert-butyl ether (616 mL, 2 volumes), and concentrated under vacuum to give a quantitative yield of 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohep-2-en-1-yl]-2,4-dioxa-3-boronabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl ester (compound (XI)) (417.4 g, corresponding to 322.1 g of pure product, yield 85.8%).

[0153] 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo [2-Heptene-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I) (crude compound) synthesis

[0154] Dissolve 6-(nitrooxy)hexanoic acid (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohep-2-en-1-yl]-2,4-dioxa-3-boronabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl ester (compound (XI)) (407.3 g crude, 1 equivalent) in methanol (3550 mL, 8.9 v / v). Add the resulting solution to a flask and rinse the apparatus with methanol (1140 mL, 2.8 v / v). Stir the mixture at 15°C to 25°C for 15 hours. 1The reaction was monitored by ¹H-NMR. Methanol was removed under vacuum at 35-40°C. Methanol (3550 mL, 8.8 V) was added, and the solution was transferred to the reactor. The reaction mixture was stirred at 20-22°C for 16 hours. IPC was performed by NMR, showing that the reaction was complete. 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 V). The resulting solution was washed with deionized water (2190 mL). The aqueous layer (pH = 7) was discarded. The organic layer was washed with sodium chloride solution (2 × 1930 mL, 2 × 4.8 V). The aqueous layer was discarded. The organic layer was dried over sodium sulfate (395 g, 1 equivalent w / w), washed with methyl tert-butyl ether (790 mL, 2 volumes), and concentrated under vacuum at a temperature below 40 °C to give crude 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (I)) (367.1 g, 98.3% yield) with an HPLC purity of 80.4%.

[0155] The mixture was purified on a Combiflash column using silica gel columns (750 g × 6, 10.34 V) and dichloromethane / methanol as eluent, employing a gradient from 100:0 to 95:5. Fractions were monitored by TLC and analyzed by HPLC (area %). The fraction was concentrated under vacuum at a temperature equal to or below 50°C to obtain 206 g of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (I)). This was added to a 4 L three-necked round-bottom flask and dissolved in absolute ethanol (2100 mL, 10 volumes). Activated carbon (21 g, 10% / w) was added, and the mixture was stirred at 20°C to 25°C for 0.5 hours. The charcoal was filtered off, and the mixture was washed with absolute ethanol (210 mL, 1 volume). The filtrate was concentrated under industrial vacuum at 45°C to 50°C for 4 hours, followed by concentration under high vacuum at 45°C to 50°C for 8 hours. 1 ¹H NMR monitoring in DMSO-d6 showed no residual solvent.

[0156] 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (192.9 g) was obtained from compound (II) in an overall yield of 62%. The HPLC purity was 99.13%.

[0157]

[0158]

[0159] Table 1 reports the quantitative analysis and chemical yield results of 6-(nitrooxy)hexanoic acid (VIIIa) prepared by nitration according to the method disclosed in WO 2019 / 162149. The results show that, during the post-treatment of the nitration of the alkali metal salt of 6-hydroxyhexanoic acid, the addition of water instead of the NaCl saturated aqueous solution (brine) as described in WO 2019 / 162149 produced a crude nitration mixture containing almost pure compound (VIIIa) (purity 99.2% (a / a%)) and 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 yields, such as 99%. The use of the crude nitration mixture in step 5a) allows for the acquisition of the final product 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I) containing less than 0.05% of the impurity bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (VII). Indeed, the results in Table 2 report the quantitative analysis of compound (I) and major impurities prepared according to the methods of the present invention (Example 1) and the methods disclosed in WO 2019 / 162149 (Example 2 - Comparative Example), showing that the method of the present invention provides compound (I) with a content of the "dimeric impurity" bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (VII)) below the detection limit of 0.05% and a content of bimatoprost 15-(6-chlorohexanoyl) ester (compound (V)) of 0.11%. The methods disclosed in the prior art produce compound (I) with lower chemical purity and a content of compound (VII) of 0.1% to 0.4%.

[0160] The results demonstrate that the method of the present invention represents an improved method that can be easily transferred to industrial scale.

Claims

1. A method for preparing 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I), The method includes the following steps: Step A) Prepare 6-(nitrooxy)hexanoyl chloride (VIIIb) as follows: 1a) ε-caprolactone The 6-hydroxyhexanoate of formula (X) is obtained by reacting an inorganic base selected from KOH, NaOH, and LiOH in a solvent selected from methanol, ethanol, or isopropanol at a temperature from 20°C to the solvent reflux temperature. Where M is K, Na, or Li; 2a) Purification of the 6-hydroxyhexanoate of formula (X) obtained in step 1a), comprising: i. Add methyl tert-butyl ether to the reaction mixture of step 1a) at a volume ratio of 2:1 (methyl tert-butyl ether / reaction mixture); ii. Filtering solids; iii. Prepare a slurry from the solid using a mixture of methyl tert-butyl ether, methanol, and water in a ratio of 3:1 to 5:1, wherein the amount of water is 0.3-1 mol per mole of ε-caprolactone; iv. Separate 6-hydroxyhexanoate solid of formula (X); 3a) React 6-hydroxyhexanoate with a mixture of fuming HNO3 and concentrated H2SO4 in dichloromethane at a temperature ranging from 0°C to 10°C; 4a) Add water to the nitrification mixture from step 3a) while maintaining the temperature between 0°C and 5°C; 5a) Separate the organic phase, dry the organic phase with sodium sulfate, and distill off the solvent to obtain crude 6-(nitrooxy)hexanoic acid (VIIIa). 6a) Crude 6-(nitrooxy)hexanoic acid (VIIIa) is reacted with a chlorinating agent in dichloromethane to obtain crude 6-(nitrooxy)hexanoyl chloride (VIIIb); Step B) Preparation of 6-(nitrooxy)-hexanoic acid (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I), comprising the following steps: 1b) Bimatoprost was reacted with butylboronic acid in methyl tert-butyl ether at 40°C to obtain compound (II): 2b) Compound (II) is reacted with crude 6-(nitrooxy)hexanoyl chloride (VIIIb) from step 6a) in an aprotic organic solvent in the presence of free 4-dimethylaminopyridine to obtain compound (XI). 3b) Remove the borate ester protecting group to obtain the crude compound of formula (I); 4b) Purify crude compound (I) by column chromatography.

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

3. The method according to claim 2, wherein in step 3a), potassium 6-hydroxyhexanoate is added to the mixture of fuming HNO3 and concentrated H2SO4 in dichloromethane at a temperature of 0°C to 10°C, and the reaction mixture is vigorously stirred at a temperature of 0°C to 5°C for 60 ± 15 minutes.

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

5. The method according to claim 2, wherein in step 6a), the chlorinating agent is oxalyl chloride.

6. The method according to claim 3, wherein in step 6a), the chlorinating agent is oxaloyl chloride.

7. The method according to any one of claims 1 to 6, wherein in step 2b), the aprotic solvent is methyl tert-butyl ether, and the temperature is in the range of 0°C to 20±3°C.

8. The method according to any one of claims 1 to 6, wherein in step 2b), the molar ratio of compound (II) to 4-dimethylaminopyridine is 1:2.0 to 1:2.

4.

9. The method according to any one of claims 1 to 6, wherein in step 3b), the solvent used in the reaction is methanol, and the reaction is carried out at room temperature.

10. The method according to any one of claims 1 to 6, wherein in step 4b), the crude compound (I) is purified using high performance silica gel chromatography and dichloromethane / methanol as the mobile phase.

11. A method for synthesizing 6-(nitrooxy)hexanoic acid, comprising: 1a) ε-caprolactone It reacts with KOH in methanol at the reflux temperature of the solvent; 2a)-i. Add methyl tert-butyl ether to the reaction mixture of step 1a) at a volume ratio of 2:1 (methyl tert-butyl ether / reaction mixture); 2a)-ii. Filter the solid potassium 6-hydroxyhexanoate; 2a)-iii. The solid is prepared into a slurry with a mixture of methyl tert-butyl ether, methanol and water in a ratio of 3:1 to 5:1, wherein the amount of water is 0.3-1 mol per mole of ε-caprolactone; 2a)-iv. Separate the solid potassium 6-hydroxyhexanoate; 3a) Add the potassium 6-hydroxyhexanoate from step 2a) to a mixture of fuming HNO3 and concentrated H2SO4 in dichloromethane at a temperature ranging from 0°C to 10°C, and stir the reaction mixture vigorously at a temperature ranging from 0°C to 5°C. 4a) Add water to the nitrification mixture from step 3a) while maintaining the temperature between 0°C and 5°C; 5a) Separate the organic phase, dry the organic phase with sodium sulfate, distill off the solvent, and obtain 6-(nitrooxy)hexanoic acid characterized by a chemical purity of 99% or greater and a content of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid of 0.2% or less.

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