HK40071158B - Process for the preparation of a nitric oxide donating prostaglandin analogue - Google Patents

Description

Methods for preparing nitric oxide donor prostaglandin analogs

Technical Field

This invention relates to an improved method suitable for the large-scale 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), 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 (VIIa), a key synthetic intermediate.

Background of the Invention

Hexanoic acid of formula (I), 6-(nitrooxy)-,(1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester, is a prostaglandin analog that has been proven effective as an 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).

A method for preparing compound (I) is disclosed in WO 2009/136281.

WO 2009/136281 specifically discloses the synthesis of compound (I) and generally discloses the preparation of the 15-alkyl nitrate of bimatoprost.

WO 2009/136281 discloses the synthesis of compound (I) (Example B-1), which is obtained by reacting bimatoprost in the borate protected form (compound (II)) with 6-bromohexanoyl chloride to give the 15-(6-bromohexanoyl) ester (compound (XI)) of bimatoprost in the borate protected form, which is then converted to a nitrate ester derivative in acetonitrile with silver nitrate and purified by deprotection/reversed-phase chromatography to obtain compound (I).

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 structural warnings regarding potential mutagenicity, 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 15-(6-bromohexanoyl) ester of bimatoprost (compound (IX)) and 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)), which are difficult to remove even after repeated purifications because they have similar chromatographic polarity, similar lipophilicity, and/or solubility to compound (I). Furthermore, compound (X) was predicted to be positive for in vitro bacterial mutagenicity. The need for repeated purifications to remove these impurities further reduces yield and increases the cost of commercial-scale production.

According to the method disclosed in WO 2009/136281, the 15-(6-bromohexanoyl) ester of bimatoprost (compound (IX)) is an impurity resulting from the incomplete reaction of compound (XI) with silver nitrate after the removal of the borate ester protection. The 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) is a byproduct formed by a halogen exchange reaction between the bromine atom of the borate-protected form of bimatoprost (compound (XI)) and the free chloride anion of 4-dimethylaminopyridine hydrochloride formed during the esterification step. The borate-protected form of bimatoprost (XIa) (procedure 3) does not react with silver nitrate and yields compound (X) after the protecting group is removed.

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

The above method avoids the use of 6-bromohexanoyl chloride and the removal of silver salt from the final product. However, this method presents another major drawback, namely the use of 4-dimethylaminopyridine loaded on the resin, which makes the method unsuitable for commercial scale-up and expensive. In addition, the nitrate ester-alkyl carboxylic acid chloride is added in a high excess relative to the compound of formula (II) in two consecutive steps, with the alkyl carboxylic acid chloride actually added in an amount of about 2 to 4 equivalents.

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

The main drawback of this method is the removal of unreacted nitrate esters-alkyl-(p-nitrophenyl)carboxylic acids and the byproduct p-nitrophenol formed in equimolar amounts with the compound of formula (I).

WO 2016/155906 discloses a 15-nitrooxy derivative of fluprostenol and reports the synthesis of a 15-nitrooxyhexyl ester of fluprostenol isopropyl ester. This compound is prepared by reacting the borate-protected form of fluprostenol isopropyl ester with (4-nitrophenyl)-6-nitrooxyhexyl ester in the presence of an excess of 4-dimethylaminopyridine.

As mentioned above, the main drawback of this method is the removal of unreacted nitrate esters-alkyl-(p-nitrophenyl)carboxylic acids, and especially the removal of p-nitrophenol byproducts, by chromatographic methods.

EP 3 530 649A1, filed before and published after the relevant date of this invention, discloses a method for preparing hexanoic acid, 6-(nitrooxy),(1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (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 intermediate 6-(nitrooxy)hexanoyl chloride is prepared from 6-(nitrooxy)hexanoic acid, which is prepared by the ring-opening reaction of 2-caprolactone and subsequent nitration of potassium 6-hydroxyhexanoate in dichloromethane using a mixture of _HNO3 and _{H2SO4 .} 6-(nitrooxy)hexanoic acid is used unpurified to prepare the corresponding acyl chloride.

Over the past few years, various regulatory agencies have been emphasizing purity requirements and impurity identification in active pharmaceutical ingredients (APIs). Currently, any impurity is considered an organic substance, other than the active pharmaceutical ingredient, that may affect the efficacy and safety of the drug. Therefore, the identification and quantification of each impurity, especially those with mutagenic structural warnings, has become a mandatory regulatory requirement. Furthermore, because the product is intended for pharmaceutical use, the range of industrially acceptable reagents, solvents, catalysts, etc., that can be used in the synthesis of the active ingredient is limited to those acceptable to the pharmaceutical industry.

Compound (I) is oily and cannot be crystallized, making large-scale purification difficult. Therefore, the presence of impurities is a key issue for large-scale production. Since the main sources of impurities are synthetic intermediates and byproducts, the purity of intermediates and the control of reaction conditions are important requirements for obtaining pharmaceutically acceptable final products.

In summary, the existing techniques for preparing compounds of formula (I) have several drawbacks; for example, the use of 6-bromohexanoyl chloride and the reaction conditions result in the formation of the byproduct 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)), which, according to regulatory requirements, is predicted to be positive for bacterial mutagenicity in vitro based on statistical and expert rule-based methods; the use of silver nitrate to prepare the intermediate nitrate-alkylcarboxylic acid chloride or to nitrate the borate-protected form of bimatoprost 15-(6-bromohexanoyl) ester (compound (XI)) necessitates the treatment of large quantities of silver nitrate wastewater; furthermore, the metal content in the active pharmaceutical ingredient must meet specific acceptance criteria.

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

Compound (I) can be efficiently prepared by coupling the borate-protected form of bimatoprost (compound (II)) with 6-(nitrooxy)hexanoyl chloride, which is prepared by ring-opening caprolactone with an alkali metal hydroxide solution, followed by nitration of the alkali metal salt of 6-hydroxyhexanoic acid with a mixture of _HNO3 and _H2SO4 to generate 6-(nitrooxy)hexanoic acid and then converting it to _6- (nitrooxy)hexanoyl chloride; 6-(nitrooxy)hexanoic acid and 6-(nitrooxy)hexanoyl chloride are used directly without further purification. However, experiments conducted by the applicant show that compound (I) prepared using this method contains the "dimeric impurity" 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimatoprost (compound (XII)).

Compounds (I) and (XII) have similar polarity in chromatography; therefore, removing compound (XII) requires repeated purification, which reduces the yield of the method.

Compound (XII) is formed by the reaction of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoyl chloride (compound (IVb)) with compound (II) during the coupling step.

It has been found that the acidic reaction conditions _for the nitration of 6-hydroxyhexanoic acid base salts (using a mixture of _HNO3 and _H2SO4 ) lead to the formation of the 6-[6-hydroxyhexanoyl]oxy}hexanoic acid impurity, which, in the presence of _HNO3 , is converted into the nitrate ester derivative 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (VIIb)).

Therefore, reacting the crude product of the nitration reaction without any purification with a chlorinating agent results in the formation of 6-(nitoxy)hexanoyl chloride (IVa) and 6-[6-hydroxyhexanoyl]oxy}hexanoyl chloride (IVb):

It has been found that the key factor in obtaining compound (I) in high yield and high purity is the use of high-purity 6-(nitrooxy)hexanoic acid intermediate (VIIa).

It can be obtained by purifying the crude product of the nitration reaction using reversed-phase chromatography.

This invention provides a method suitable for large-scale production that allows for the good yield 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)), which contains below safe levels of the predicted genotoxic impurity bimaprost 15-(6-chlorohexanoyl) ester (X) and below 0.1% w/w of the “dimeric impurity” bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)).

The “safe level” is calculated based on the acceptable intake of 1.5 μg/day at the toxicological concern threshold (TTC) for long-term treatment.

Furthermore, the present invention relates to a method for preparing a high-purity 6-(nitrooxy)hexanoic acid intermediate of formula (VIIa).

Invention Details

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

The method includes the following steps:

1) To make compound (II):

With 6-(nitrooxy)hexanoyl chloride (IVa)

The reaction, in the presence of free 4-dimethylaminopyridine, yields compound (III).

2) Remove the borate ester protecting group from compound (III) to obtain compound (I);

3) Purify compound (I);

The method is characterized in that 6-(nitrooxy)hexanoyl chloride (compound (IVa)) is prepared by a method comprising the following steps:

4) Make 2-caprolactone (V):

The 6-hydroxyhexanoate of formula (VI) is obtained by reacting with an inorganic base selected from KOH, NaOH, and LiOH:

Where M is K, Na, or Li.

5) Nitration of compound ( _VI ) with a mixture of _HNO3 and _H2SO4 yields 6-(nitrooxy)hexanoic acid (VIIa).

The resulting product (VIIa) may contain 1% w/w of the byproduct 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb).

6) The nitrated mixture was purified by reversed-phase chromatography using an aqueous formic acid solution ( _H₂O + HCOOH) and acetonitrile as eluents to obtain pure 6-(nitrooxy)hexanoic acid of formula (VIIa) with a content of compound (VIIb) below the HPLC detection limit (<0.05%).

7) Reaction of pure 6-(nitrooxy)hexanoic acid (VIIa) with a chlorinating agent to obtain 6-(nitrooxy)hexanoyl chloride (IVa).

Preferably, the 6-(nitrooxy)hexanoyl chloride obtained in step 7) is reacted directly with the compound of formula (II) in step 1) without further purification.

Step 1) is carried out at a temperature in the range of 0°C to room temperature, and preferably in an aprotic organic solvent preferably 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) indicates that DMAP is not bound to the resin.

The molar ratio of compound (II) to 6-(nitrooxy)hexanoyl chloride (IVa) is preferably in the range of 1:1.4 to 1:1.6.

The molar ratio of compound (II) to 4-dimethylaminopyridine is preferably in the range of 1:2.0 to 1:2.4.

In step 2), the removal of the borate ester protecting group is preferably carried out using methanol at a temperature of 17°C to 24°C.

In step 4), the inorganic base used is preferably potassium hydroxide.

Step 4) is preferably carried out in a solvent selected from methanol, ethanol or isopropanol, with methanol being the most preferred.

Steps 5) and 7) are carried out in dichloromethane.

In step 6), the concentration of the formic acid aqueous solution ( _H₂O + HCOOH) is 0.1%. Preferably, pure 6-(nitrooxy)hexanoic acid of formula (VIIa) is obtained by extracting the fraction containing compound (VIIa) with _{CH₂Cl₂ ,} drying with _MgSO₄ , and evaporating the solvent.

The preferred chlorinating agent used in step 7) is oxalyl chloride.

Compound (II) is obtained by reacting bimatoprost with butylboronic acid. Preferably, the reaction is carried out in methyl tert-butyl ether as a solvent.

Another object of the present invention is a method for preparing compound (VIIa) as described below and depicted in process 1 (the reference numbers of the steps reported in the process correspond to those reported above), the method comprising:

Step 4) React 2-caprolactone (V) with potassium hydroxide in methanol to obtain potassium 6-hydroxyhexanoate (where M is a potassium compound of formula (VI)).

Step 5) Preferably, at a temperature of about 5°C to 10°C, preferably 10°C, potassium 6-hydroxyhexanoate is reacted with a mixture of _HNO3 and _H2SO4 in dichloromethane to obtain crude 6-(nitrooxy)hexanoic acid (VIIa), which contains the byproduct ₆ -{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb));

Step 6) Purify the crude mixture containing 6-(nitrooxy)hexanoic acid (VIIa) and 6-{[ _6- (nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb) by reversed-phase chromatography (e.g., C18, 25 μ-Standard Type Flash Cartridge), preferably using H₂O + HCOOH 0.1%/acetonitrile at a ratio of 90:10 to _75:25 as the eluent, and extract the fraction containing compound (VIIa) using _CH₂Cl₂ to obtain pure 6-(nitrooxy)hexanoic acid (VIIa) containing a byproduct 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb) in an amount below the HPLC detection limit (<0.05%).

The pure compound (VIIa) obtained in step 6) above can be used in step 7) to obtain 6-(nitoxy)hexanoic acid (IVa) by reacting pure 6-(nitoxy)hexanoic acid with oxaloyl chloride. It is used in the method described above to obtain compound (I) without further purification.

A preferred method for preparing a compound of formula (I) is described in process 2, the preferred method comprising the following steps:

Step 1a) Bimatoprost is 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 is removed by azeotropic distillation to obtain bimatoprost borate (II).

Step 1) Bimaprost borate (II) is reacted with 6-(nitrooxy)hexanoyl chloride (IVa) (1.4-1.6 equivalents) in methyl tert-butyl ether in the presence of 4-dimethylaminopyridine (2.0-2.4 equivalents) at a temperature ranging from 0°C to approximately room temperature to obtain (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 6-(nitrooxy)hexanoate (III);

Step 2) React (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 6-(nitrooxy)hexanoate (III) with methanol at room temperature to remove the protecting group and obtain crude hexanoic acid, 6-(nitrooxy)-,(1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-2-[(2Z)-7-(ethylamino)-2-en-1-yl]-2,4-dioxa-3-borazadicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (III). [I]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I); HPLC quantitative analysis of the crude reaction mixture detected approximately 70% of compound (I), approximately 0.12% of 15-(6-chlorohexanoyl) ester of bimaprost (compound (X)), and less than 0.05% of the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimaprost (compound (XII)) (see Table 1).

Step 3) The crude reaction mixture from Step 2 was purified by 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), which has a chemical purity greater than 98%, a content of about 0.12% of bimaprost 15-(6-chlorohexanoyl) ester (compound (X)) and a dimeric impurity of bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)) below the HPLC detection limit.

Process 1

Process 2

The experimental procedure of the method steps of this invention is described in detail below.

Another object of the present invention is a 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), having a chemical purity higher than 98% and containing about 0.12% of bimaprost in a 15-(6-chlorohexanoyl) ester (compound (X)).

And less than 0.05% of bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)).

Another object of the present invention is a pharmaceutical formulation comprising hexanoic acid, 6-(nitrooxy)-,(1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I) and at least one pharmaceutically acceptable excipient, wherein the hexanoic acid, 6-(nitrooxy)-,(1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester is a pharmaceutically acceptable excipient. The 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 over 98% and contains approximately 0.12% of bimaprost 15-(6-chlorohexanoyl) ester (compound (X)) and less than 0.05% of the dimer impurity bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)).

General Synthesis

Preparation of 6-(nitrooxy)hexanoyl chloride (IVa)

All steps were performed under a nitrogen atmosphere.

Preparation of potassium 6-hydroxyhexanoate (compound (VI))

A solution of potassium hydroxide (1 equivalent) in methanol was added dropwise to a solution of 2-caprolactone (1 equivalent) in methanol; the mixture was cooled at about 5°C to 20°C and stirred at 15°C to 20°C for about 5 hours after the addition was completed; the solvent was removed (temperature equal to or below 40°C), the crude product was slurried in methyl tert-butyl ether, the potassium 6-hydroxyhexanoate was filtered, washed with methyl tert-butyl ether and dried. Potassium 6-hydroxyhexanoate (VI) was obtained in 95% yield and with a purity of 98.5% ( ^1H -NMR and HCl analysis).

Preparation of 6-(nitrooxy)hexanoic acid (compound (VIIa))

Potassium 6-hydroxyhexanoate (VI) (1 equivalent) was added fractionally over approximately 30 minutes under nitrogen atmosphere to a mixture of _HNO3 (4.6 equivalents) and _H2SO4 ( _3.1 equivalents) cooled to dichloromethane at 0-5°C, while maintaining the temperature below 10°C. The resulting mixture was stirred at 0-10°C for 2-3 hours, and the reaction endpoint was monitored by ^1H -NMR analysis. The mixture was then cooled to 0-5°C, and a saturated aqueous sodium chloride solution was added dropwise over approximately 20 minutes. The reaction mixture was maintained at a temperature below 10°C. The organic layer was dried over anhydrous sodium sulfate to remove the solvent, yielding 6-(nitrooxy)hexanoic acid (VIIa) in 86-88% yield and with HPLC purity of 96.2%. The major impurity was the dimer compound 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (VIIb)) (approximately 1%).

Crude 6-(nitrooxy)hexanoic acid (compound (VIIa)) (1 equivalent) was loaded onto a SpheraPlus Flash Cartridges-C18, 25μ-Standard Type Flash Cartridge, 48g, and eluted with _H₂O + HCOOH 0.1%/acetonitrile at a _ratio of 90:10 to 75:25. The fraction containing compound (VIIa) was collected, extracted with _CH₂Cl₂ , dried, and the solvent was evaporated to give pure 6-(nitrooxy)hexanoic acid in 80-90% yield. The HPLC purity of compound (VIIa) was 99%, and the content of compound (VIIb) was less than 0.05%.

Preparation of 6-(nitrooxy)hexanoyl chloride (compound (IVa))

N,N-dimethylformamide and oxalyl chloride were added dropwise to a dichloromethane solution of 6-(nitrooxy)hexanoic acid, and the solution temperature was maintained at 0°C to 5°C for 1 hour. The mixture was then stirred at 15°C to 30°C for 24 hours. The solvent was removed by evaporation, and 6-(nitrooxy)hexanoic acid chloride was obtained in a yield of 88-97% w/w and was used without further purification.

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

All steps were performed under a nitrogen atmosphere.

Bimatoprost was added to methyl tert-butyl ether, and the resulting solution was cooled to approximately 15°C to 18°C. Then, butylboronic acid (1.11–1.18 equivalents) was added in a single batch, and the mixture was stirred at 40°C for approximately 1–1.5 hours. The reaction endpoint was monitored by ^1H NMR analysis. The reaction mixture was cooled to approximately 20°C to 25°C, filtered, and the water generated was removed by azeotropic distillation of methyl tert-butyl ether at a temperature equal to or below 40°C until the water content was less than or equal to 0.25%, yielding crude bimatoprost borate ester (compound (II)), which was used in the next step without further purification.

Crude bimaprost borate ester (compound (II)) was added to methyl tert-butyl ether and the resulting solution was cooled to about 0°C to 5°C. 4-Dimethylaminopyridine (about 2.1-2.3 equivalents) was added, followed by dropwise addition of 6-(nitrooxy)hexanoyl chloride (IVa) (1.5 equivalents) dissolved in methyl tert-butyl ether, while maintaining the temperature of the mixture at about 0°C to 5°C. After addition, the mixture was stirred at about 0°C to 5°C for up to 4 hours and then overnight at 15°C to 20°C; the reaction endpoint was monitored by HPLC analysis; (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-6-(nitrooxy)hexanoate (compound (III)) was separated by standard post-treatment methods (examples of post-treatment are described in Example 1);

(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-6-(nitrooxy)hexanoate (compound (III)) was dissolved in methanol and the resulting solution was stirred at 17°C to 25°C for about 18 hours; the conversion of compound (III) to compound (I) was monitored by ^1H NMR. When the reaction was stopped, the mixture was evaporated and redissolved in fresh methanol until complete conversion. The reaction mixture was then concentrated in a vacuum at a temperature below 40°C and the crude product, hexanoic acid, 6-(nitrooxy)-,(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 (compound (I)), was separated by a standard post-treatment method.

HPLC quantitative analysis of the crude reaction mixture detected approximately 70% of compound (I), approximately 0.12% of bimatoprost 15-(6-chlorohexanoyl) ester (compound (X)), and a dimer impurity (compound (XII)) below the HPLC detection limit (see Table 1).

The crude reaction mixture was purified by column chromatography using a silica gel column and a mixed solvent of dichloromethane and methanol, and compound (I) was given in an overall yield of more than 60% w/w, based on bimatoprost.

The HPLC quantitative analysis showed that the purity of compound (I) was higher than 98%, the amount of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) was less than 0.15%, and the content of dimer impurity (compound (XII)) was less than 0.05%.

The method of the present invention provides hexanoic acid, 6-(nitrooxy)-,(1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (I) in high yield and high purity, and has reduced amounts of byproducts, especially (S,E) )-1-((1R,2R,3S,5R)-2-((Z)-7-(ethylamino)-7-oxohept-2-enyl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-chlorohexanoate (compound (X)) (predicted to be positive for bacterial in vitro mutagenicity), and 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimaprost (compound (XII)).

The above advantages make the method of the present invention a cost-effective method that can be easily scaled up to industrial scale.

Experimental Examples

All the synthesis steps described below were performed under a 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 (IV))

Step 1: Synthesis of potassium 6-hydroxyhexanoate (compound (VI))

A solution of potassium hydroxide (1.98 g, 1 equivalent) in methanol (10 ml) was prepared under cooling at 15°C to 20°C and added to a solution of 3.65 g of 2-caprolactone (1 equivalent) in methanol (6 ml) over 0.5 hours at 5°C to 20°C. The mixture was stirred at 15°C to 20°C for 4.5 hours. The reaction mixture was concentrated under vacuum (at a temperature equal to or below 40°C) to give crude potassium 6-hydroxyhexanoate (6.10 g). The crude product was re-slurried in methyl tert-butyl ether (10 ml) at 20°C to 25°C for 4 hours, filtered, washed with methyl tert-butyl ether (2 x 25 ml), and dried under vacuum (at a temperature equal to or below 40°C) to give potassium 6-hydroxyhexanoate (5.16 g) in 93.7% yield. Melting point 208°C.

Step 2: Synthesis of 6-(nitrooxy)hexanoic acid (compound (VIIa))

Fuming _HNO3 (4.6 equivalents) was added to concentrated _H2SO4 ( _3.1 equivalents) over 14 minutes at 0 to 5°C, followed by _CH2Cl2 (20 ml) over 12 minutes at 0 to 5°C. Potassium 6-hydroxyhexanoate (10.13 g, ₁ equivalent) was added fractionally over 28 minutes at a temperature below 10°C. The mixture was stirred at 0 to 10°C for 2.2 hours, and the reaction was monitored by ^1H -NMR, showing 99.9% conversion. The mixture was cooled to 0 to 5°C and a saturated aqueous sodium chloride solution was carefully added over 17 minutes at a temperature equal to or below 10°C. After filtration, the organic layer was decanted, dried over sodium sulfate, and concentrated under vacuum (at a temperature equal to or below 40°C) to give 9.15 g of 6-(nitrooxy)hexanoic acid (87.7% yield) with an HPLC purity of 96.2%, and the content of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (VIIb)) was 0.75%.

Step 3: Purification of crude 6-(nitrooxy)hexanoic acid (compound (VIIa)) containing 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (VIIb))

1.5 g of crude 6-(nitrooxy)hexanoic acid (VIIa) obtained in step 2 was loaded onto a Sphera ^Plus Flash Cartridges-C18, 25 μ-Standard Type Flash Cartridge, 48 g, and eluted with _H₂O + HCOOH 0.1%/acetonitrile at a ratio of 90:10 to 75:25. The fraction containing compound (VIIa) eluted at 78:22 was collected, extracted with _CH₂Cl₂ ( ₃ x 150 mL), dried over sodium sulfate, and concentrated under vacuum at a temperature below 40 °C to give 1.04 g (69% yield) of 6-(nitrooxy)hexanoic acid with an HPLC purity >99.5% and an amount of compound (VIIb) <0.05%.

Step 4: Synthesis of 6-(nitrooxy)hexanoyl chloride (compound (IVa))

6-(nitrooxy)hexanoic acid (2.61 g, 1 equivalent) was dissolved in dichloromethane (15 ml) and cooled to 0-5 °C under nitrogen. N,N-dimethylformamide (0.16 ml) and oxaloyl chloride (1.93 g) were then added over 34 minutes at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 3.5 h and then at 15-20 °C for 14 h. The reaction mixture was then concentrated under vacuum (temperature equal to or below 40 °C) and co-evaporated with dichloromethane to give 6-(nitrooxy)hexanoyl chloride (compound (IVa)) (2.8 g) in 97% yield.

Step 5: Synthesis 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]oct-7-yl]-N-ethyl-hept-5-enamide (compound (II))

Bimatoprost (2.3 g, 1 equivalent) was dissolved in methyl tert-butyl ether (30 ml), and butylboronic acid (0.62 g, 1.13 equivalent) was added. The mixture was heated to 40 °C for 1 hour. The reaction was monitored by ¹ H NMR until the conversion was >97%.

The reaction mixture was washed with methyl tert-butyl ether (10 ml) and the mixture was heated in a vacuum at about 40 °C for azeotropic distillation. Washing with methyl tert-butyl ether and azeotropic distillation were 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]oct-7-yl]-N-ethyl-hept-5-enamide (compound (II)) was equal to or less than 0.25%. Compound (II) was obtained in quantitative yield (2.84 g).

Step 6: Synthesis of (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 6-(nitrooxy)hexanoate (compound (III))

(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]oct-7-yl]-N-ethyl-hept-5-enamide (compound (II)) (3.21 g, 1 equivalent) was dissolved in methyl tert-butyl ether (30 ml) and cooled to 0°C to 5°C. 4-Dimethylaminopyridine was added in a single step. Pyridine (1.85 g, 2.27 equivalents). A solution of 6-(nitrooxy)hexanoyl chloride (compound (IVa)) (1.96 g, 1.5 equivalents) was added dropwise over 1 hour at 0 to 5 °C. After stirring at 0 to 5 °C for 24 minutes and then at 15 to 20 °C for 14.5 hours, the reaction mixture was cooled at 0 to 5 °C and deionized water was added over 20 minutes at the maximum temperature of 15 °C.

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

The organic layer was dried with sodium sulfate and concentrated under vacuum to give (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 6-(nitrooxy)hexanoate (compound (III)) (4.1 g, 94% yield).

Step 7: 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) (compound (I)).

(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 6-(nitrooxy)hexanoate (compound (III)) (4 g crude, 1 equivalent)) was dissolved in methanol (30 ml) and the resulting solution was stirred at room temperature for 24 hours, with the reaction monitored by ¹ H-NMR. The methanol was then removed under vacuum at 35-40 °C. The residue was dissolved in methyl tert-butyl ether and washed with deionized water, followed by washing with brine. The organic layer was dried with sodium sulfate and concentrated under vacuum at a temperature below 40°C to obtain a crude reaction mixture (3.8 g). HPLC (% area) reversed-phase quantitative analysis showed that the mixture contained 73% of compound (I), 0.11% of 15-(6-chlorohexanoyl) ester of bimaprost (compound (X)), and less than 0.05% of the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimaprost (compound (XII)) (see Table 1).

The residue was chromatographically analyzed on a silica gel column using dichloromethane/methanol 95:5 as the eluent. The fractions were monitored by TLC, and only the pure fractions were mixed and concentrated under vacuum at a temperature equal to or below 40°C to obtain compound (I) with an HPLC purity >98%, containing 0.11% of bimaprost 15-(6-chlorohexanoyl) ester (compound (X)) and less than 0.05% of the dimer impurity bimaprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)).

Example 2 (Comparative Example)

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 synthesized according to the method disclosed in WO 2009/136281 (procedure 3 below).

Step A: Preparation 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]oct-7-yl]-N-ethyl-hept-5-enamide (compound (II))

Butylboronic acid (1.129 equivalents) was added to a solution of bimatoprost (1 g, 1 equivalent) in dichloromethane (16 mL). The mixture was heated to 40 °C for 1 hour, and the reaction progress was monitored by ¹ H-NMR. The solvent was removed under reduced pressure for 2 hours. Dichloromethane (16 mL) was added, and the mixture was heated to 40 °C for another hour. The solvent was removed under pressure for 40 minutes. Dichloromethane (16 mL) was added, and the mixture was heated to 40 °C for 16 hours. The solvent was evaporated, and the crude product was dried under high vacuum at 40 °C for 3 hours to give compound (II) in quantitative yield, which was used in the next step without any further purification.

MS: m/z = 438[M+H]+

Step B: Synthesis of (S,E)-1-((1S,5R,6R,7S)-3-butyl-7-((Z)-7-(ethylamino)-7-oxohep-2-en-1-yl)-2,4-dioxa-3-boronabicyclo[3.2.1]oct-6-yl)-5-phenylpent-1-en-3-yl 6-bromohexanoate (compound (XI)).

4-Dimethylaminopyridine (1.1 equivalents) and 6-bromohexanoyl chloride (1.15 equivalents) were added to a solution of compound (II) (0.8 g, 1 equivalent) in dichloromethane (15.3 ml) cooled to 0–5 °C. The mixture was stirred at 0–5 °C for 0.5 h, and then at 20–25 °C for 16 h.

4-Dimethylaminopyridine (0.25 equivalents) and 6-bromohexanoyl chloride (0.25 equivalents) were added, and the mixture was stirred for another ₁₉ hours. The reaction was monitored by ^1H -NMR until complete conversion. The mixture was diluted with dichloromethane (15.3 mL), and the organic solution was washed with deionized water (6.25 mL) and brine (6.25 mL). The organic phase was dried over _Na₂SO₄ and concentrated under vacuum to give a pale yellow oily compound (XI) (calculated as quantitative yield), which was used in the next step without further purification.

Step C: 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))

Silver nitrate (3.72 equivalents) was added to a solution of compound (VIII) (0.8 g, 1 equivalent) in acetonitrile (9.4 ml). The mixture was stirred at 20°C to 25°C for 18 hours. The conversion was monitored by ^1H -NMR in DMSO.

Add silver nitrate (0.5 equivalents) and stir the mixture for another 20 hours until HPLC analysis shows a conversion rate of 99.7%.

The mixture was filtered through a Whatman filter. The filtrate was concentrated under vacuum. The residue was dissolved in ethyl acetate (30 ml). The organic phase was washed with deionized water (5 ml) and brine (5 ml). After drying _{with Na₂SO₄} , the organic layer was concentrated under vacuum. The residue was chromatographically analyzed on a silica gel column using dichloromethane/methanol 95:5 as the eluent. The fractions were monitored by TLC, and the pure fractions were mixed and concentrated under vacuum at a temperature equal to or below 40 °C to give compound (I) in an overall yield of 86%, together with 4.27% bimatoprost.

HPLC (% area) reversed-phase quantitative analysis showed that the purity of compound (I) after chromatography was 77%, and the content of 15-(6-chlorohexanoyl) ester of bimaprost (compound (X)) was 8.34%.

#: Amount of compound (I) in the crude reaction mixture of step 7 in Example 1

*: The compound was subjected to silica gel chromatography at the end of step C.

Table 1 reports the HPLC quantitative analysis results of compound (I) and major impurities formed during the preparation of compound (I) according to the method of the present invention (Example 1) and the method disclosed in WO 2009/136281 (Example 2).

Compound (X) is the 15-(6-chlorohexanoyl) ester of bimatoprost, which is predicted to be positive for bacterial mutagenicity in vitro. Results show that the method of the present invention provides compound (I) with a content of 0.11% of compound (X) and a detection limit below 0.05% for the "dimeric impurity" bimatoprost, namely 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)). Prior art methods produce compound (I) with lower chemical purity and a content of 8.34% of compound (X), which is more than 30 times higher than the amount of compound (X) produced by the method of the present invention.

The results confirm that the method of the present invention represents an improvement over the methods described in the prior art.

Process 3

Claims (13)

1. A method for preparing hexanoic acid, 6-(nitrooxy)-,(1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I), The method includes the following steps: 1) To make compound (II): With 6-(nitrooxy)hexanoyl chloride (IVa) The reaction, in the presence of free 4-dimethylaminopyridine, yields compound (III). 2) Remove the borate ester protecting group from compound (III) to obtain compound (I); 3) Purify compound (I); The method is characterized in that 6-(nitrooxy)hexanoyl chloride (IVa) is prepared by a method comprising the following steps: 4) Make 2-caprolactone (V) The 6-hydroxyhexanoate of formula (VI) is obtained by reacting with an inorganic base selected from KOH, NaOH, and LiOH. Where M is K, Na, or Li; 5) Nitrate compound ( _VI ) with a mixture of _HNO3 and _H2SO4 to obtain 6-(nitrooxy)hexanoic acid (VIIa); 6) The nitrated mixture was purified by reversed-phase chromatography using an aqueous formic acid solution ( _H₂O + HCOOH) and acetonitrile as eluents to obtain pure 6-(nitoxy)hexanoic acid of formula (VIIa) with a content of <0.05% below the HPLC detection limit of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb). 7) Reaction of pure 6-(nitrooxy)hexanoic acid (VIIa) with a chlorinating agent to obtain 6-(nitrooxy)hexanoyl chloride (IVa). 2. The method of claim 1, wherein step 1) is carried out in an aprotic organic solvent at a temperature in the range of 0°C to room temperature. 3. The method of claim 2, wherein the aprotic organic solvent is methyl tert-butyl ether. 4. The method of claim 1, wherein, in step 1), the molar ratio of compound (II) to 6-(nitrooxy)hexanoyl chloride (IVa) is 1:1.4 to 1:1.6, and the molar ratio of compound (II) to 4-dimethylaminopyridine is 1:2.0 to 1:2.4. 5. The method according to claim 1, wherein the inorganic base in step 4) is potassium hydroxide. 6. The method of claim 1, wherein step 4) is carried out in a solvent selected from methanol, ethanol or isopropanol. 7. The method of claim 6, wherein the solvent is methanol. 8. The method of claim 1, wherein step 5) is carried out in dichloromethane. 9. The method of claim 1, wherein, in step 6), the concentration of the formic acid aqueous solution _H₂O + HCOOH is 0.1% w/w. 10. The method of claim 1, wherein, in step 6), the chromatographic fraction containing _compound (VIIa) is extracted using _CH₂Cl₂ , and the solvent is dried and evaporated. 11. The method of claim 1, wherein step 7) is carried out in dichloromethane and the chlorinating agent is oxalyl chloride. 12. The method according to any one of claims 1 to 11, wherein the 6-(nitrooxy)hexanoyl chloride obtained in step 7) is used without further purification. 13. A method for preparing 6-(nitrooxy)hexanoic acid (VIIa) having a purity of 99% and a content of less than 0.05% {[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb), the method comprising the following steps: 4) Make 2-caprolactone (V) The reaction with KOH in methanol yields the 6-hydroxyhexanoate of formula (VI). Where M is K; 5) Nitrate ( _VI ) compounds in dichloromethane at a temperature of 5°C to 10°C using a mixture of _HNO3 and _H2SO4 ; 6) The nitrated mixture was purified by reversed-phase chromatography using 0.1% formic acid aqueous solution and acetonitrile as eluent, followed by extraction fraction with dichloromethane to obtain 6-(nitrooxy)hexanoic acid (VIIa).