Method for preparing nitric oxide donor prostaglandin analogs

Abstract

The present invention relates to a method for preparing 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-propylene-1-yl ester of formula (I). According to the present invention, by coupling bimatoprost in a boronate protected form with 6-(nitrooxy)hexanoyl chloride having high chemical purity and removing the boronate protecting group, a high-purity compound (I) can be effectively prepared. 6-(nitrooxy)hexanoyl chloride having high chemical purity is prepared from 6-(nitrooxy)hexanoic acid having high chemical purity. The present invention also relates to a method for preparing and purifying 6-(nitrooxy)hexanoic acid.

Description

Technical Field

[0001] The present invention relates to an improved process 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 obtaining said product with high chemical purity. The invention also describes the preparation of high-purity 6-(nitrooxy)hexanoic acid (VIIa), which is a key synthetic intermediate. Background of the Invention

[0003] 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) is a prostaglandin analog that has been shown to be effective as an IOP lowering agent (Impagnatiello F, Toris CB, Batugo M, Prasanna G, Borghi V, Bastia E, Ongini E, Krauss AHP; Invest Ophthalmol Vis Sci. 2015; 56:6558-64)

[0004]

[0005] WO 2009 / 136281 discloses a method for preparing the compound of formula (I).

[0006] WO 2009 / 136281 discloses in particular the synthesis of compound (I) and in general the preparation of 15-alkyl nitrate esters of bimatoprost.

[0007] WO 2009 / 136281 discloses the synthesis of a compound of formula (I) (Example B-1), which is prepared by reacting a boronate-protected form of bimatoprost (compound (II)) with 6-bromohexanoyl chloride to obtain a boronate-protected form of bimatoprost 15-(6-bromohexanoyl) ester (compound (XI)), which is converted into a nitrate derivative using silver nitrate in acetonitrile and purified by deprotection / reverse phase chromatography to obtain a compound of formula (I).

[0008]

[0009] The main disadvantages of the above synthesis are the use of an equimolar excess of 6-bromohexanoyl chloride in the esterification reaction, which presents a structural warning about potential mutagenicity, and the use of silver nitrate in the final step, which produces a large amount of silver salts in the wastewater. Another major disadvantage of this method is the formation of impurities and by-products 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 multiple purifications because they have similar polarity in the chromatography, similar lipophilicity and / or solubility as compound (I). In addition, compound (X) is predicted to be positive for bacterial in vitro mutagenicity. Removal of these impurities requires repeated purifications, which further reduces the yield and increases the cost of production on a commercial scale.

[0010]

[0011] According to the method disclosed in WO 2009 / 136281, 15-(6-bromohexanoyl) ester of bimatoprost (compound (IX)) is an impurity produced by incomplete reaction of compound (XI) with silver nitrate after removal of boronate protection. 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) is a byproduct formed by a halogen exchange reaction between the bromine atom of 15-(6-bromohexanoyl) ester of bimatoprost (compound (XI)) in boronate protected form and the free chloride anion of 4-dimethylaminopyridine hydrochloride formed during the esterification step. 15-(6-chlorohexanoyl) ester of bimatoprost (XIa) in boronate protected form (Scheme 3) does not react with silver nitrate and generates compound (X) after removal of the protecting group.

[0012] WO 2009 / 136281 also discloses an alternative method for preparing 15-acylalkyl nitrate bimatoprost derivatives (Examples N-1 and O-1). The synthesis comprises reacting bimatoprost in a boronate protected form (compound of formula (II)) with a nitrate-alkyl carboxylic acid chloride in the presence of 4-dimethylaminopyridine (DMAP) (PS-DMAP) supported on a resin, followed by removal of the boronate protecting group and purification by silica gel chromatography.

[0013] The above method avoids the use of 6-bromohexanoyl chloride and the removal of silver salts from the final product. However, this method presents another major disadvantage, namely the use of 4-dimethylaminopyridine supported on a resin, which makes the method unsuitable for commercial scale-up and expensive. In addition, the nitrate-alkyl carboxylic acid chloride is added in high excess relative to the compound of formula (II) in two consecutive steps, in fact the alkyl carboxylic acid chloride is added in an amount of about 2 to 4 equivalents.

[0014] 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 a boronate protected form with an excess of nitrate-alkyl-(p-nitrophenyl) carboxylate in the presence of 4-dimethylaminopyridine.

[0015] The use of chromatography to remove unreacted nitrate-alkyl-(p-nitrophenyl)carboxylate and the by-product p-nitrophenol formed in an equimolar amount to the compound of formula (I) is a major disadvantage of this process.

[0016] WO 2016 / 155906 discloses 15-nitrooxy derivatives of fluprostenol and reports the synthesis of 15-nitrooxyhexyl ester of fluprostenol isopropyl ester. The compound is prepared by reacting fluprostenol isopropyl ester in a boronate-protected form with (4-nitrophenyl)-6-nitrooxyhexanoate in the presence of an excess of 4-dimethylaminopyridine.

[0017] As mentioned above, the removal of unreacted nitrate-alkyl-(p-nitrophenyl)carboxylate and especially the p-nitrophenol by-product by chromatographic methods is a major disadvantage of this process.

[0018] EP 3 530 649 A1, filed before the relevant date of the present invention and published thereafter, discloses a process for preparing hexanoic acid, 6-(nitrooxy), (1S, 2E)-3-[(1R, 2R, 3S, 5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (I)). Compound (I) is prepared by coupling bimatoprost in boronate protected form with 6-(nitrooxy)hexanoyl chloride and removing the boronate protecting group. The 6-(nitrooxy)hexanoyl chloride intermediate is prepared from 6-(nitrooxy)hexanoic acid, which is prepared by ring opening of 2-caprolactone and subsequent nitration of 6-hydroxyhexanoic acid potassium salt using a mixture of HNO3 and H2SO4 in dichloromethane. 6-(Nitrooxy)hexanoic acid was used without purification to prepare the corresponding acid chloride.

[0019] Over the past few years, various regulatory agencies have been emphasizing on purity requirements and impurity identification in active pharmaceutical ingredients (APIs). Currently, any impurity is considered to be an organic substance other than the API that may affect the efficacy and safety of the drug product. Therefore, identification and quantification of each impurity, especially those with mutagenic structural alerts, has become a mandatory regulatory requirement. In addition, since the product is for pharmaceutical use, the range of industrially acceptable reagents, solvents, catalysts, etc. that can be used in the synthesis of active ingredients is limited to those that are acceptable to the pharmaceutical industry.

[0020] The compound of formula (I) is in oily form. Since it cannot be crystallized, it is difficult to purify the compound in large quantities. Therefore, the presence of impurities is a key issue for large-scale production. Since the main sources of impurities are synthetic intermediates and by-products, the purity of the intermediates and the control of reaction conditions are important requirements for obtaining a final product with a pharmaceutically acceptable purity.

[0021] In summary, the prior art methods for preparing the compound of formula (I) have some disadvantages; for example, the use of 6-bromohexanoyl chloride and the reaction conditions lead to the formation of the by-product 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)), which is predicted to be positive for bacterial in vitro mutagenicity according to statistical-based methods and expert rule-based methods according to regulatory requirements; the use of silver nitrate to prepare the intermediate nitrate-alkyl carboxylic acid chloride or to nitrate the 15-(6-bromohexanoyl) ester of bimatoprost in boronate protected form (compound (XI)) leads to the need to treat large amounts of silver nitrate wastewater, and in addition, the metal content in the active pharmaceutical ingredient must meet specific acceptance criteria.

[0022] Therefore, it is necessary to develop an industrially feasible method for providing 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.

[0023] Compound (I) can be efficiently prepared by coupling bimatoprost in a boronate-protected form (compound (II)) with 6-(nitrooxy)hexanoyl chloride, which is prepared by ring-opening caprolactone with an alkali metal hydroxide solution, followed by nitration of 6-hydroxyhexanoic acid alkali metal salt with a mixture of HNO3 and H2SO4 to produce 6-(nitrooxy)hexanoic acid and converting it into 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 showed that compound (I) prepared using this method contained a "dimer impurity" 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimatoprost (compound (XII))

[0024]

[0025] Compound (I) and compound (XII) have similar polarities in chromatography, therefore, removal of compound (XII) requires repeated purifications, which reduces the yield of the process.

[0026] Compound (XII) is formed by reacting 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoyl chloride (compound (IVb)) with compound (II) during the coupling step.

[0027] It has been found that the acidic reaction conditions for the nitration of the 6-hydroxyhexanoic acid base salt (using a mixture of HNO3 and H2SO4) lead to the formation of a 6-[6-hydroxyhexanoyl]oxy}hexanoic acid impurity, which, in the presence of HNO3, is converted to the nitrate derivative 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (VIIb)).

[0028]

[0029] Thus, reacting the crude product of the nitration reaction without any purification with a chlorinating agent results in the formation of 6-(nitrooxy)hexanoyl chloride (IVa) and 6-[6-hydroxyhexanoyl]oxy}hexanoyl chloride (IVb):

[0030]

[0031] It has been found that the key factor in obtaining compound (I) in high yield and high purity is to use high purity 6-(nitrooxy)hexanoic acid intermediate (VIIa)

[0032]

[0033] It can be obtained by purifying the crude product of the nitration reaction using reverse phase chromatography.

[0034] The present invention provides a method suitable for large-scale production, which allows obtaining 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, which contains the predicted genotoxic impurity bimatoprost 15-(6-chlorohexanoyl) ester (X) in an amount below the safe level and the "dimer impurity" bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)) in an amount below 0.1% w / w.

[0035] The “safe level” was calculated based on the acceptable intake of 1.5 μg / day, the threshold of toxicological concern (TTC), for long-term treatment.

[0036] Furthermore, the present invention relates to a process for preparing a high-purity 6-(nitrooxy)hexanoic acid intermediate of the formula (VIIa).

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0039] The object of the present 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):

[0040]

[0041] The method comprises the following steps:

[0042] 1) Compound (II):

[0043]

[0044] With 6-(nitrooxy)hexanoyl chloride (IVa)

[0045]

[0046] The reaction is carried out in the presence of free 4-dimethylaminopyridine to obtain compound (III)

[0047]

[0048] 2) removing the borate protecting group of compound (III) to obtain a compound of formula (I);

[0049] 3) purifying compound (I);

[0050] The method is characterized in that 6-(nitrooxy)hexanoyl chloride (compound (IVa)) is prepared by a method comprising the following steps:

[0051] 4) Make 2-caprolactone (V):

[0052]

[0053] Reaction with an inorganic base selected from KOH, NaOH and LiOH to obtain a 6-hydroxyhexanoate of formula (VI):

[0054]

[0055] wherein M is K, Na or Li.

[0056] 5) Nitrate the compound of formula (VI) with a mixture of HNO3 and H2SO4 to obtain 6-(nitrooxy)hexanoic acid (VIIa)

[0057]

[0058] The obtained product (VIIa) may contain the by-product 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb) in an amount of 1% w / w.

[0059]

[0060] 6) Purify the nitration mixture by reverse phase chromatography using aqueous formic acid (H2O+HCOOH) and acetonitrile as eluents; to obtain pure 6-(nitrooxy)hexanoic acid of formula (VIIa) having a content of compound (VIIb) below the HPLC detection limit (<0.05%)

[0061] 7) Pure 6-(nitrooxy)hexanoic acid (VIIa) is reacted with a chlorinating agent to obtain 6-(nitrooxy)hexanoyl chloride (IVa).

[0062] Preferably, the 6-(nitrooxy)hexanoyl chloride obtained in step 7) is directly reacted with the compound of formula (II) in step 1) without further purification.

[0063] Step 1) is carried out at a temperature ranging from 0°C to room temperature, and preferably, it is carried out 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. The free form of 4-dimethylaminopyridine (DMAP) means that DMAP is not bound to the resin.

[0064] 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.

[0065] The molar ratio of compound (II) to 4-dimethylaminopyridine is preferably in the range of 1:2.0 to 1:2.4.

[0066] In step 2), the removal of the boronate protecting group is preferably carried out using methanol at a temperature of 17°C to 24°C.

[0067] In step 4), the inorganic base used is preferably potassium hydroxide.

[0068] Step 4) is preferably carried out in a solvent selected from methanol, ethanol or isopropanol, most preferably methanol.

[0069] Steps 5) and 7) are carried out in dichloromethane.

[0070] In step 6), the concentration of the aqueous formic acid 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.

[0071] The preferred chlorinating agent used in step 7) is oxalyl chloride.

[0072] Compound (II) is obtained by reacting bimatoprost with butylboronic acid. Preferably, the reaction is carried out in methyl tert-butyl ether as a solvent.

[0073] Another object of the present invention is a process for preparing compound (VIIa) described below and depicted in Scheme 1 (the reference numbers of the steps reported in the scheme correspond to those reported above), which comprises:

[0074] Step 4) reacting 2-caprolactone (V) with potassium hydroxide in methanol to obtain 6-hydroxyhexanoic acid potassium salt (compound of formula (VI) wherein M is potassium);

[0075] Step 5) reacting 6-hydroxyhexanoic acid potassium salt with a mixture of HNO3 and H2SO4 in dichloromethane, preferably at a temperature of about 5°C to 10°C, preferably 10°C, to obtain crude 6-(nitrooxy)hexanoic acid (VIIa) containing a by-product 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb));

[0076] Step 6) Purify the crude mixture containing 6-(nitrooxy)hexanoic acid (VIIa) and 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb) by reverse phase chromatography (e.g. C18, 25μ-Standard Type Flash Cartridge), preferably using 90:10 to 75:25 H2O+HCOOH 0.1% / acetonitrile as eluent, and extract the fraction containing compound (VIIa) with CH2Cl2 to obtain pure 6-(nitrooxy)hexanoic acid (VIIa) containing the by-product 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb) in an amount below the HPLC detection limit (<0.05%).

[0077] The pure compound (VIIa) obtained in step 6) above can be used in step 7) by reacting pure 6-(nitrooxy)hexanoic acid with oxalyl chloride to obtain 6-(nitrooxy)hexanoyl chloride (IVa), which is used in the method for obtaining the compound of formula (I) as described above without further purification.

[0078] A preferred method for preparing the compound of formula (I) is depicted in Scheme 2, which comprises the following steps:

[0079] Step 1a) reacting bimatoprost with butylboronic acid (1.1-1.8 equivalents) in methyl tert-butyl ether (MTBE) at a temperature of about 40° C., and then removing water by azeotropic distillation to obtain bimatoprost boronate (II);

[0080] Step 1) reacting bimatoprost boronate (II) 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 about room temperature to obtain (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (III);

[0081] Step 2) (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (III) was reacted 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)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (III). HPLC quantitative analysis of the crude reaction mixture detected a content of about 70% of compound (I), a content of about 0.12% of bimatoprost 15-(6-chlorohexanoyl) ester (compound (X)), and a dimer impurity of less than 0.05% of bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)) (see Table 1).

[0082] Step 3) The crude reaction mixture of step 2 is 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) having a chemical purity higher than 98%, a content of about 0.12% of bimatoprost 15-(6-chlorohexanoyl) ester (compound (X)) and an amount of the dimer impurity bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)) lower than 0.05% of the HPLC detection limit.

[0083] Process 1

[0084]

[0085] Process 2

[0086]

[0087] The experimental procedures of the method steps of the present invention are described in detail as follows.

[0088] Another object of the present invention is hexanoic acid, 6-(nitrooxy)-, (1S, 2E)-3-[(1R, 2R, 3S, 5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester of formula (I), which has a chemical purity higher than 98% and contains a content of about 0.12% of bimatoprost 15-(6-chlorohexanoyl) ester (compound (X))

[0089]

[0090] and 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimatoprost (Compound (XII)) in an amount less than 0.05%

[0091]

[0092] Another object of the present invention is a pharmaceutical preparation 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 hexanoic acid, 6-(nitrooxy)-, (1S, 2E)-3-[(1R, 2R, 3S ,5R)-2-[(2Z)-7-(ethylamino)-7-oxo-2-hepten-1-yl]-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester has a chemical purity higher than 98%, and contains about 0.12% of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) and less than 0.05% of the dimer impurity 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimatoprost (compound (XII)).

[0093] General Synthesis

[0094] Preparation of 6-(Nitrooxy)hexanoyl chloride (IVa)

[0095] All steps were performed under nitrogen atmosphere.

[0096] Preparation of 6-hydroxyhexanoic acid potassium salt (compound (VI))

[0097] 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 complete; the solvent was removed (temperature equal to or below 40°C), the crude product was slurried in methyl tert-butyl ether, the potassium salt of 6-hydroxyhexanoic acid was filtered, washed with methyl tert-butyl ether and dried. The product was prepared in 95% yield and 98.5% purity ( 1 H-NMR and HCl analysis) to obtain 6-hydroxyhexanoic acid potassium salt (VI).

[0098] Preparation of 6-(Nitrooxy)hexanoic acid (Compound (VIIa))

[0099] To a mixture of HNO3 (4.6 eq.) and H2SO4 (3.1 eq.) in dichloromethane cooled at 0-5°C was added 6-hydroxyhexanoic acid potassium salt (VI) (1 eq.) portionwise under nitrogen over about 30 min while keeping the temperature below 10°C; the resulting mixture was stirred at 0-10°C for 2-3 h and then heated to room temperature by 1 The reaction endpoint was monitored by H-NMR analysis; the mixture was cooled at a temperature of 0°C to 5°C and a saturated aqueous sodium chloride solution was added dropwise over about 20 minutes. The reaction mixture was kept at a temperature below 10°C; the organic layer was dried over anhydrous sodium sulfate, the solvent was removed, and 6-(nitrooxy)hexanoic acid (VIIa) was obtained in a yield of 86-88% and a HPLC purity of 96.2%. The main impurity was a dimer compound 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (VIIb)) (about 1%).

[0100]

[0101] Crude 6-(nitrooxy)hexanoic acid (Compound (VIIa)) (1 equivalent) was loaded onto -SpheraPlusFlash Cartridges-C18,25μ-Standard Type Flash Cartridge,48g, eluted with 90:10 to 75:25 H2O+HCOOH 0.1% / acetonitrile. The fractions containing compound (VIIa) were collected, extracted with CH2Cl2 and dried, and the solvent was evaporated to obtain 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%.

[0102] Preparation of 6-(Nitrooxy)hexanoyl chloride (Compound (IVa))

[0103] N,N-dimethylformamide and oxalyl chloride were added dropwise to a solution of 6-(nitrooxy)hexanoic acid in dichloromethane, maintaining the solution temperature at 0 to 5°C for 1 hour, and then the mixture was stirred at 15 to 30°C for 24 hours; the solvent was removed by evaporation to obtain 6-(nitrooxy)hexanoyl chloride in a yield of 88-97% w / w, which was used without further purification.

[0104] Hexanoic acid, 6-(nitrooxy)-, (1S,2E)-3-[(1R,2R,3S,5R)-2-[(2Z)-7-(ethylamino)-7-oxy Preparation of [2-(2-hepten-1-yl)-3,5-dihydroxycyclopentyl]-1-(2-phenylethyl)-2-propen-1-yl ester (compound (I)) Preparation

[0105] All steps were performed under nitrogen atmosphere.

[0106] Bimatoprost was added to methyl tert-butyl ether and the resulting solution was cooled to about 15°C to 18°C; then n-butylboric acid (1.11-1.18 equivalents) was added in one portion and the mixture was stirred at 40°C for about 1-1.5 hours. 1 The reaction endpoint was monitored by H NMR analysis; the reaction mixture was cooled to about 20°C to 25°C, filtered and the generated water was removed by azeotropic distillation of methyl tert-butyl ether at a temperature equal to or lower than 40°C until the water content was lower than or equal to 0.25% to obtain crude bimatoprost boronate (compound (II)), which was used in the next step without further purification.

[0107] The crude bimatoprost boronate (compound (II)) is added to methyl tert-butyl ether and the resulting solution is cooled to about 0°C to 5°C, 4-dimethylaminopyridine (about 2.1-2.3 equivalents) is added, and 6-(nitrooxy)hexanoyl chloride (IVa) (1.5 equivalents) dissolved in methyl tert-butyl ether is added dropwise, maintaining the temperature of the mixture at about 0°C to 5°C. After the addition, the mixture is stirred at about 0°C to 5°C for up to 4 hours and then overnight to 15°C to 20°C; the reaction endpoint is monitored by HPLC analysis; (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl-6-(nitrooxy)hexanoate (Compound (III)) is isolated by standard work-up methods (examples of work-up are described in Example 1);

[0108] (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl-6-(nitrooxy)hexanoate (Compound (III)) was dissolved in methanol and the resulting solution was stirred at 17° C. to 25° C. for about 18 hours; 1 The conversion of compound (III) to compound (I) was monitored by HNMR. In the case of cessation of the reaction, the mixture was evaporated and redissolved in fresh methanol until complete conversion. The reaction mixture was then concentrated in vacuo at a temperature below 40°C and the crude 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-propene-1-yl ester (compound (I)) was isolated by standard work-up methods.

[0109] HPLC quantitative analysis of the crude reaction mixture detected a content of about 70% of compound (I), a content of about 0.12% of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)), and a dimer impurity (compound (XII)) in an amount below the HPLC detection limit of 0.05% (see Table 1).

[0110] The crude reaction mixture was purified by column chromatography using a silica gel column and a mixed solvent of dichloromethane and methanol to obtain Compound (I) in an overall yield of more than 60% w / w based on bimatoprost.

[0111] The HPLC quantitative analysis purity of compound (I) was higher than 98%, the amount of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) was lower than 0.15%, and the content of dimer impurity (compound (XII)) was lower than 0.05%.

[0112] The process 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 a reduced amount of by-products, 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 mutagenicity in vitro), and 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimatoprost (Compound (XII)).

[0113] The above advantages make the process of the present invention a cost-effective process that can be easily converted to industrial scale.

[0114] Experimental Examples

[0115] All synthetic steps described below were performed under nitrogen atmosphere.

[0116] Example 1

[0117] 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))

[0118] Synthesis of 6-(Nitrooxy)hexanoyl chloride (Compound (IV))

[0119] Step 1: Synthesis of 6-hydroxyhexanoic acid potassium salt (Compound (VI))

[0120] A solution of potassium hydroxide (1.98 g, 1 eq) 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 eq) in methanol (6 ml) at 5°C to 20°C over 0.5 hours. The mixture was stirred at 15°C to 20°C for 4.5 hours. The reaction mixture was concentrated in vacuo (at a temperature equal to or lower than 40°C) to give the crude potassium 6-hydroxyhexanoate salt (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 in vacuo (at a temperature equal to or lower than 40°C) to give potassium 6-hydroxyhexanoate salt (5.16 g) in 93.7% yield. Melting point 208°C.

[0121] Step 2: Synthesis of 6-(nitrooxy)hexanoic acid (Compound (VIIa))

[0122] Fuming HNO3 (4.6 eq) was added to concentrated H2SO4 (3.1 eq) at 0°C to 5°C over 14 min, followed by CH2Cl2 (20 ml) at 0°C to 5°C over 12 min. Potassium 6-hydroxyhexanoate (10.13 g, 1 eq) was added portionwise at below 10°C over 28 min. The mixture was stirred at 0°C to 10°C for 2.2 h and stirred by 1 The reaction was monitored by H-NMR, showing 99.9% conversion. The mixture was cooled to 0°C to 5°C and a saturated aqueous sodium chloride solution was carefully added at a temperature equal to or lower than 10°C over 17 minutes. After filtration, the organic layer was decanted, dried over sodium sulfate and concentrated in vacuo (at a temperature equal to or lower than 40°C) to give 9.15 g of 6-(nitrooxy)hexanoic acid (87.7% yield) with an HPLC purity of 96.2% and a content of 0.75% of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (compound (VIIb)).

[0123] Step 3: Purification of crude 6-(nitrooxy)hexanoic acid (Compound (VIIa)) containing 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (Compound (VIIb))

[0124] 1.5 g of the crude 6-(nitrooxy)hexanoic acid (VIIa) obtained in step 2 was loaded onto -Sphera Plus Flash Cartridges-C18, 25μ-Standard Type Flash Cartridge, 48g, eluted with H2O+HCOOH 0.1% / acetonitrile from 90:10 to 75:25. The fractions containing compound (VIIa) eluted at 78:22 were collected, then extracted with CH2Cl2 (3x150mL), dried over sodium sulfate and concentrated in vacuo at a temperature below 40°C to give 6-(nitrooxy)hexanoic acid 1.04g (69% yield) with an HPLC purity of >99.5% and an amount of compound (VIIb) <0.05%.

[0125] Step 4: Synthesis of 6-(nitrooxy)hexanoyl chloride (Compound (IVa))

[0126] 6-(Nitrooxy)hexanoic acid (2.61 g, 1 equivalent) was dissolved in dichloromethane (15 ml) and cooled to 0°C to 5°C under nitrogen. N,N-dimethylformamide (0.16 ml) and oxalyl chloride (1.93 g) were then added at 0°C to 5°C over 34 minutes. The reaction mixture was stirred at 0°C to 5°C for 3.5 hours and then at 15°C to 20°C for 14 hours. The reaction mixture was then concentrated in vacuo (temperature equal to or lower than 40°C) and co-evaporated with dichloromethane to give 6-(nitrooxy)hexanoyl chloride (compound (IVa)) (2.8 g) in 97% yield.

[0127] 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-borabicyclo[3.2.1]oct-7-yl]-N-ethyl-hept-5-enamide (Compound (II))

[0128] Bimatoprost (2.3 g, 1 eq) was dissolved in methyl tert-butyl ether (30 ml) and butylboronic acid (0.62 g, 1.13 eq) was added. The mixture was heated to 40°C for 1 hour. 1 The reaction was monitored by H NMR until the conversion was >97%.

[0129] The reaction mixture was rinsed with methyl tert-butyl ether (10 ml) and the mixture was heated at a temperature of about 40° C. in a vacuum for azeotropic distillation. The rinsing with methyl tert-butyl ether and the 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-borabicyclo[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).

[0130] Step 6: Synthesis of (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (Compound (III))

[0131] (Z)-7-[(1S,5R,6R,7R)-3-butyl-6-[((E,3S)-3-hydroxy-5-phenyl-pent-1-enyl]-2,4-dioxa-3-borabicyclo[3.2.1]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-Dimethylaminopyrrolidone was added in one portion. Pyridine (1.85 g, 2.27 eq.). A solution of 6-(nitrooxy)hexanoyl chloride (compound (IVa)) (1.96 g, 1.5 eq.) in methyl tert-butyl ether (5 ml) was added dropwise at 0 to 5°C over 1 hour. After stirring at 0 to 5°C for 24 minutes and at 15 to 20°C for 14.5 hours, the reaction mixture was cooled at 0 to 5°C and deionized water was added at a maximum temperature of 15°C over 20 minutes.

[0132] The mixture was stirred for 5 minutes. The aqueous layer was discarded. The organic layer was washed with 1N hydrochloric acid solution, then with deionized water, and finally with brine.

[0133] The organic layer was dried over sodium sulfate and concentrated in vacuo to give (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (Compound (III)) (4.1 g, 94% yield).

[0134] 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)).

[0135] (1S,2E)-3-{(6R,7R)-3-butyl-7[(2Z)-7-(ethylamino)-7-oxohept-2-en-1-yl]-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl}-1-(2-phenylethyl)-prop-2-en-1-yl 6-(nitrooxy)hexanoate (Compound (III)) (4 g crude product, 1 equivalent) was dissolved in methanol (30 ml) and the resulting solution was stirred at room temperature for 24 hours. 1The reaction was monitored by H-NMR. Methanol was then removed in vacuo at 35°C to 40°C. The residue was dissolved in methyl tert-butyl ether and washed with deionized water and then with brine. The organic layer was dried over sodium sulfate and concentrated in vacuo at a temperature below 40°C to give a crude reaction mixture (3.8 g), which showed after reverse phase quantitative analysis by HPLC (% area) that the amount of compound (I) in the mixture was 73%, the content of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) was 0.11%, and the content of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate of bimatoprost (compound (XII)), a dimer impurity, was less than 0.05% (see Table 1).

[0136] The residue was chromatographed on a silica gel column with dichloromethane / methanol 95:5 as eluent. The fractions were monitored by TLC, only the pure fractions were combined and concentrated in vacuo at a temperature equal to or lower than 40° C. to give Compound (I) with an HPLC purity of >98%, a content of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) of 0.11% and a content of the dimeric impurity bimatoprost 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)) of less than 0.05%.

[0137] Example 2 (Comparative Example)

[0138] 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 (Scheme 3 below).

[0139] 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-borabicyclo[3.2.1]oct-7-yl]-N-ethyl-hept-5-enamide (Compound (II))

[0140] Butylboronic acid (1.129 eq) was added to a solution of bimatoprost (1 g, 1 eq) in dichloromethane (16 ml). The mixture was heated to 40 °C for 1 hour and stirred for 2 hours. 1The reaction progress was monitored by 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 at 40 ° C for 3 hours in a high vacuum to obtain compound (II) in quantitative yield, which was used in the next step without any further purification.

[0141] MS: m / z = 438 [M+H] +

[0142] Step B: Synthesis of (S,E)-1-((1S,5R,6R,7S)-3-butyl-7-((Z)-7-(ethylamino)-7-oxohept-2-en-1-yl)-2,4-dioxa-3-borabicyclo[3.2.1]oct-6-yl)-5-phenylpent-1-en-3-yl 6-bromohexanoate (Compound (XI)).

[0143] 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° C. to 5° C. for 0.5 hours and then at 20° C. to 25° C. for 16 hours.

[0144] 4-Dimethylaminopyridine (0.25 eq.) and 6-bromohexanoyl chloride (0.25 eq.) were added and the mixture was stirred for a further 19 h. 1 The reaction was monitored by H-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 in vacuo to give compound (XI) as a light yellow oil (calculated as quantitative yield), which was used in the next step without further purification.

[0145] 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))

[0146] Silver nitrate (3.72 eq.) was added to a solution of compound (VIII) (0.8 g, 1 eq.) in acetonitrile (9.4 ml). The mixture was stirred at 20°C to 25°C for 18 hours. 1 The conversion was monitored by H-NMR.

[0147] Silver nitrate (0.5 eq.) was added and the mixture was stirred for an additional 20 h until HPLC analysis showed 99.7% conversion.

[0148] The mixture was filtered on a Whatman filter. The filtrate was concentrated in vacuo. 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 over Na2SO4, the organic layer was concentrated in vacuo. The residue was chromatographed on a silica gel column using dichloromethane / methanol 95:5 as eluent. The fractions were monitored by TLC, the pure fractions were mixed and concentrated in vacuo at a temperature equal to or lower than 40°C, and compound (I) was obtained with 4.27% bimatoprost in an overall yield of 86%.

[0149] HPLC (% area) reverse phase quantitative analysis showed that the purity of compound (I) after chromatography was 77%, and the content of 15-(6-chlorohexanoyl) ester of bimatoprost (compound (X)) was 8.34%.

[0150]

[0151] #: Amount of compound (I) in the crude reaction mixture of step 7 of Example 1

[0152] *: Compound was chromatographed on silica gel at the end of step C

[0153] Table 1 reports the results of HPLC quantitative analysis of compound (I) and the main impurities formed during the preparation of compound (I) according to the process of the present invention (Example 1) and according to the process disclosed in WO 2009 / 136281 (Example 2).

[0154] Compound (X) is the 15-(6-chlorohexanoyl) ester of bimatoprost which is predicted to be positive for bacterial in vitro mutagenicity. The results show that the method of the present invention provides compound (I) with a content of 0.11% of compound (X) and a content of 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoate (compound (XII)) of bimatoprost as a "dimer impurity" with a detection limit of less than 0.05%. The method disclosed in the prior art produces compound (I) with a lower chemical purity and a content of 8.34% of compound (X), i.e., more than 30 times higher than the amount of compound (X) produced in the method of the present invention.

[0155] The results demonstrate that the method of the present invention represents an improvement over methods described in the prior art.

[0156] Process 3

[0157]

Claims

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 comprises the following steps: 1) Compound (II): With 6-(nitrooxy)hexanoyl chloride (IVa) The reaction is carried out in the presence of free 4-dimethylaminopyridine to obtain compound (III) 2) removing the borate protecting group of compound (III) to obtain a compound of formula (I); 3) purifying compound (I); The method is characterized in that 6-(nitrooxy)hexanoyl chloride (IVa) is prepared by a method comprising the following steps: 4) 2-caprolactone (V) Reaction with an inorganic base selected from KOH, NaOH and LiOH to obtain 6-hydroxyhexanoate of formula (VI) wherein M is K, Na or Li; 5) Nitration of the compound of formula (VI) with a mixture of HNO3 and H2SO4 to obtain 6-(nitrooxy)hexanoic acid (VIIa); 6) purifying the nitration mixture by reverse phase chromatography using aqueous formic acid H2O+HCOOH and acetonitrile as eluents to obtain pure 6-(nitrooxy)hexanoic acid of formula (VIIa) having a 6-{[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb) content below the HPLC detection limit of <0.05%; and 7) Pure 6-(nitrooxy)hexanoic acid (VIIa) is reacted 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 ranging from 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 of 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 aqueous formic acid 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 with 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) was used without further purification.

13. A method for preparing 6-(nitrooxy)hexanoic acid (VIIa) having a purity of 99% and a content of {[6-(nitrooxy)hexanoyl]oxy}hexanoic acid (VIIb) of less than 0.05%, the method comprising the following steps: 4) 2-caprolactone (V) Reaction with KOH in methanol to obtain 6-hydroxyhexanoate of formula (VI) Where M is K; 5) nitrating the compound of formula (VI) with a mixture of HNO3 and H2SO4 in dichloromethane at a temperature of 5°C to 10°C; 6) The nitration mixture was purified by reverse phase chromatography using 0.1% aqueous formic acid and acetonitrile as eluents, followed by extraction of the fractions with dichloromethane to obtain 6-(nitrooxy)hexanoic acid (VIIa)

Images