HK1208869A1 - A tenofovir prodrug purification method - Google Patents

Description

Purification method of tenofovir prodrug

Technical Field

The invention relates to a purification method of a novel tenofovir prodrug.

Background

Tenofovir (tenofovir) is a novel nucleotide reverse transcriptase inhibitor, effective against various viruses, and useful for treating viral infectious diseases such as hepatitis B virus. Because tenofovir is a phosphonic acid group of a double anion under the physiological pH condition, the tenofovir is not easy to permeate a cell membrane for absorption, the bioavailability is very low, and dose-dependent nephrotoxicity exists, so that the treatment effect of the tenofovir is limited, and therefore, the phosphonate prodrug is prepared by means of esterification, salification and the like and can be clinically used. For example, Tenofovir disoproxil fumarate (Tenofovir fumarate) was the first generation of orally active Tenofovir prodrugs developed by giread Science, for the treatment of aids infections and hepatitis b.

Because tenofovir disoproxil fumarate is highly sensitive to a seroenzyme-mediated hydrolysis reaction, the concentration of a medicament at an action part cannot be effectively increased, two equivalents of potentially toxic formaldehyde are released in a metabolic process, and side effects such as lactic acidosis, severe hepatomegaly, lipodystrophy and the like are found in a clinical treatment process. In order to improve the stability of the tenofovir prodrug in plasma and reduce the concentration of a metabolite, namely tenofovir in plasma so as to reduce the drug toxicity, Girride company develops a tenofovir phosphoramidate prodrug (GS-7340) shown as a formula V and discloses a preparation method of GS-7340 (WO 2002008241).

On the basis of the research on GS-7340, Chinese patent application 2012100041647.4 reports a novel tenofovir prodrug represented by formula I, which is more stable in blood and has a higher concentration of tenofovir, an active metabolite in Peripheral Blood Mononuclear Cells (PBMCs), compared with GS-7340, thereby possibly being a novel tenofovir prodrug with better curative effect and less toxic and side effects.

When the compound shown as the formula I is prepared, a phosphorus chiral center is generated in the reaction process, so that a mixture of two compounds shown as the formula I and the formula II is obtained. The compound shown as the formula I and the compound shown as the formula II are diastereoisomers, the separation and purification of the compound shown as the formula I are difficult to realize by using a common recrystallization method, the separation of the two compounds can be realized by using a preparation liquid phase to obtain the compound shown as the formula I, but the method has the disadvantages of complex operation, high cost and unsuitability for large-scale preparation, and the Chinese patent application 2012100041647.4 reports the separation and purification method.

Disclosure of Invention

Through repeated research, the inventor successfully utilizes a salt-forming crystallization method to realize separation and purification of a mixture of a compound shown in a formula I and a compound shown in a formula II, thereby preparing the compound shown in the formula I with the purity of more than 95%.

The invention provides a method for purifying a compound shown as a formula I,

the method comprises the steps of adding D- (+) -dibenzoyl tartaric acid into a mixture of a compound shown as a formula I and a compound shown as a formula II to form a salt, separating after one or more times of crystallization, and neutralizing with alkali to obtain the compound shown as the formula I.

In one embodiment of the present invention, a purification process is provided comprising the steps of:

1) adding D- (+) -dibenzoyl tartaric acid into a mixture of a compound shown as a formula I and a compound shown as a formula II to form a salt,

2) separating after one or more times of crystallization to obtain D- (+) -dibenzoyl tartrate of the compound shown in the formula I,

3) and neutralizing the obtained D- (+) -dibenzoyl tartrate of the compound shown in the formula I with alkali to obtain the compound shown in the formula I.

The invention also provides a salt shown as a formula III and a preparation method thereof, wherein the preparation method comprises the following steps:

1) in a mixed solvent of ethyl acetate and n-heptane, compound I and D- (+) -dibenzoyl tartaric acid are mixed to form salt with the molar ratio of 1:1,

2) crystallizing at 20 deg.C, filtering, drying,

the invention also provides a salt shown as a formula IV and a preparation method thereof, wherein the preparation method comprises the following steps:

1) in a mixed solvent of ethyl acetate and n-heptane, compound I and D- (+) -dibenzoyl tartaric acid are mixed to form salt with the molar ratio of 2:1,

2) crystallizing at 20 deg.C, filtering, drying,

the mixture of the compound of formula I and the compound of formula II is obtained by the preparation method described in chinese patent application 2012100041647.4.

Detailed Description

The present invention will be explained in detail below with reference to specific examples so that those skilled in the art can more fully understand the present invention, and the specific examples are only for illustrating the technical solutions of the present invention and do not limit the present invention in any way.

Example 1 (preparation of a mixture of compounds of formula I, II, from Chinese patent application 2012100041647.4)

Step 1:

trimethylchlorosilane (6.0g) was added dropwise to a solution of phenol (5g) and triethylamine (10.1mL) in dichloromethane (150mL) at 0 ℃ and, after completion of the addition, the reaction was stirred at 20 ℃ for 18 hours. The white solid was filtered off and the solid was washed with dichloromethane. The filtrates were combined and the solvent was distilled off to give 4.2g of phenoxytrimethylsilane as a colorless oil.

Step 2:

adding DMF (0.1mL) and thionyl chloride (0.73g) dropwise to a turbid solution of Tenofovir (1g, available from Heideson pharmaceutical science and technology Co., Ltd., Suzhou) in sulfolane (2.5 mL) at 70 ℃, heating to 100 ℃, continuing to react the mixture at 100 ℃ for 1.5 hours until the mixture is totally clear, rapidly adding phenoxytrimethylsilane (0.70g), reacting the mixture at 100 ℃ for 1.5 hours, evaporating the solvent under reduced pressure to obtain a viscous yellow oily liquid, dissolving methanol, adjusting the pH to 3 with a 45% potassium hydroxide aqueous solution, filtering, and drying to obtain a white powdery solid IIa0.7 g. MS (m/z) 363.96 (MH)⁺).

And step 3:

DMF (0.1mL) and thionyl chloride (343mg) were added to a sulfolane (1mL) mixture of IIa (600 mg) at 60 ℃ and the mixture was stirred at 60 ℃ for 30 minutes until it was clear. The above solution was added to a solution of the amino acid ester IIIa (750mg, purchased from Shanghai Dairy Fine Chemicals, Ltd.) and diisopropylamine (452mg) in dichloromethane (7mL) at 0 ℃. The reaction mixture was heated to 20 ℃ and reacted for 2 hours, and the reaction mixture was washed with a 5% sodium dihydrogenphosphate aqueous solution and a saturated sodium chloride aqueous solution in this order and then dried over anhydrous sodium sulfate. The solvent was evaporated to give a crude product as a yellow oil which was purified by column chromatography to give the product Ia150mg as an oily liquid (i.e. a mixture of compound I and compound II, compound I being 50.5% pure).

¹H-NMR(400MHz,CDCl₃):8.34(m,1H),8.05(m,1H),7.36～6.95(m,5H),6.49(b,2H),6.22～5.84(m,1H),5.01(m,1H),4.42(m,1H),4.40～3.60(m,3H),1.52～1.18(m,15H).MS(m/z):491.13(MH⁺).

Example 2

A mixture of Compound I and Compound II (1.0 g, 50.5% purity of Compound I) obtained by the method of example 1 was dissolved in ethyl acetate (10 mL) and n-heptane (2 mL), D- (+) -dibenzoyltartaric acid (0.35 g) was added at 20 degrees, the mixture was stirred at 20 degrees for 24 hours for crystallization, D- (+) -dibenzoyltartaric acid salt (0.8 g) was obtained by filtration, the obtained salt was added to methylene chloride (10 mL) and water (10 mL), 2mL of aqueous sodium hydroxide solution was added, the organic layer was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a product (0.42 g) having a purity of Compound I of 79.5%.

Example 3

A mixture of Compound I and Compound II (1.0 g, 79.5% purity of Compound I) obtained as in example 2 was dissolved in ethyl acetate (10 mL) and n-heptane (2 mL), D- (+) -dibenzoyltartaric acid (0.55 g) was added at 20 degrees, the mixture was stirred at 20 degrees for 24 hours for crystallization, D- (+) -dibenzoyltartaric acid salt (0.92 g) was obtained by filtration, the obtained salt was added to dichloromethane (10 mL) and water (10 mL), 2mL aqueous sodium hydroxide solution was added, the organic layer was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a product (0.42 g) with a purity of Compound I of 89.8%.

Example 4

A mixture of Compound I and Compound II (1.0 g, 79.5% pure Compound I) was dissolved in ethyl acetate (10 mL) and n-heptane (2 mL), D- (+) -dibenzoyltartaric acid (0.29 g) was added at 20 degrees, the mixture was stirred at 20 degrees for 24 hours for crystallization, D- (+) -dibenzoyltartaric acid salt (0.9 g) was obtained by filtration, the obtained salt was added to dichloromethane (10 mL) and water (10 mL), 2mL aqueous sodium hydroxide solution was added, the organic layer was separated, dried over anhydrous sodium sulfate, and the product (0.52 g) was obtained after concentration, with Compound I purity 91.6%.

Example 5

A mixture of compound I and compound II (1.0 g, purity of compound I: 79.5%) was dissolved in various solvents, D- (+) -dibenzoyltartaric acid (0.29 g) was added at 20 degrees, the mixture was stirred at 20 degrees for 24 hours for crystallization, D- (+) -dibenzoyltartaric acid salt was obtained by filtration, the obtained salt was added to methylene chloride (10 mL) and water (10 mL), 2mL of an aqueous sodium hydroxide solution was added, an organic layer was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a product, and the purity of compound I contained in the salt precipitated in various solvents was as shown in Table I.

TABLE 1 purification Effect of various solvents on Compound I

Example 6

Dissolving a mixture of compound I and compound II (10.0 g, compound I purity 79.5%) in ethyl acetate (100 mL) and n-heptane (20 mL), adding D- (+) -dibenzoyltartaric acid (2.9 g) at 20 degrees, stirring for 24 hours at 20 degrees for crystallization, and filtering to give D- (+) -dibenzoyltartaric acid salt (9.8 g, compound I purity 91.4%); dissolving the obtained salt in ethyl acetate (60 mL) at 50 ℃, cooling to 20 ℃ and stirring for 18 hours for crystallization to obtain D- (+) -dibenzoyl tartrate (6.9 g, the purity of the compound I is 97.1%); the resulting salt was dissolved in ethyl acetate (40 mL) at 50 degrees, cooled to 20 degrees and stirred for 18 hours to crystallize to give D- (+) -dibenzoyl tartrate (5.2 g), which was added to dichloromethane (50 mL) and water (50 mL), 10mL aqueous sodium hydroxide solution was added to separate the organic layer, dried over anhydrous sodium sulfate, and concentrated to give the product (3.6 g) of compound I with a purity of 99.1%.

Example 7

Dissolving the compound I (1.0 g) in ethyl acetate (10 mL) and n-heptane (2 mL), adding D- (+) -dibenzoyl tartaric acid (0.36 g), stirring at 20 ℃ for 18 hours, and crystallizing to obtain D- (+) -dibenzoyl tartrate (1.21 g) shown in formula III.

¹H-NMR：（400MHz，CDCl₃）8.18-8.15（m，6H），7.98（s，2H），7.52（t，2H），7.39（t，4H），7.26-7.20（m，4H），7.09-7.05（m，2H），6.98（d，4H），,6.09（s，2H），5.02（m，2H），4.34-4.30（dd，2H），4.16-4.10（q,2H）,4.01(d,2H)，3.94-3.97（m，4H），3.69-3.64（m，2H），1.52（s，6H），1.44（s，6H），1.26-1.20（m，18H）

Example 8

Dissolving the compound I (1.0 g) in ethyl acetate (10 mL) and n-heptane (2 mL), adding D- (+) -dibenzoyl tartaric acid (0.73g), and stirring at 20 ℃ for 18 hours to crystallize to obtain D- (+) -dibenzoyl tartrate (0.92 g) as shown in formula IV.

¹H-NMR：（400MHz，CDCl₃）9.52（brs，2H）,8.12-8.09（m，6H）,7.99（s，1H），7.51-7.47（t，2H），7.38-7.34（m，4H）,7.18(t，2H)，7.04（t，1H），6.97（d，2H），6.04（s，2H），4.98-4.95（m，1H），4.29-4.25（m，1H），4.12-4.07（m，1H），4.03（d，1H），3.92-3.87（m，2H），1.44（s，3H），1.38（s，3H），1.22-1.18（m，6H），1.16-1.14（m，3H）

Since the invention has been described in terms of specific embodiments thereof, certain modifications and equivalent variations will be apparent to those skilled in the art and are intended to be included within the scope of the invention.

Claims (7)

1. A method for purifying a compound shown as a formula I,

it is characterized in that D- (+) -dibenzoyl tartaric acid is added into a mixture of a compound shown in a formula I and a compound shown in a formula II for salification, the mixture is separated after one or more times of crystallization, and the compound shown in the formula I is obtained after alkali neutralization,

2. purification process according to claim 1, characterized in that it comprises the following steps:

1) adding D- (+) -dibenzoyl tartaric acid into a mixture of a compound shown as a formula I and a compound shown as a formula II to form a salt,

2) separating after one or more times of crystallization to obtain D- (+) -dibenzoyl tartrate of the compound shown in the formula I,

3) and neutralizing the obtained D- (+) -dibenzoyl tartrate of the compound shown in the formula I with alkali to obtain the compound shown in the formula I.

3. Purification process according to claim 1 or 2, characterized in that the salification step is carried out in a solvent selected from a mixed solvent of ethyl acetate/n-heptane, acetonitrile, 2-methyltetrahydrofuran, acetone/water.

4. The purification process according to claim 1 or 2, wherein the base is sodium hydroxide.

5. The purification process according to claim 1 or 2, characterized in that the step of neutralization with a base is carried out in a solvent which is a dichloromethane/water mixed solvent.

6. A salt of formula III:

7. a salt of formula IV: